Journal of Molecular and Cellular Cardiology 98 (2016) S1S85

Contents lists available at ScienceDirect

Journal of Molecular and Cellular Cardiology

journal homepage: www.elsevier.com/locate/yjmcc

Abstracts from the 2016 ISHR World Congress Annual Meeting,

April 18-21, 2016, Buenos Aires, Argentina

TU-002
PGE2 promotes biliary cholesterol excretion and attenuates dietinduced atherosclerosis by activation of EP3-mediated HNF4/
CYP7A1 pathway in liver
Shuai Yan, Juan Tang, Yuanyang Wang, Shengkai Zuo, Guilin Chen, Jian
Zhang, Di Chen, Ying Yu
Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences,
Graduate School of the Chinese Academy of Sciences, Chinese Academy of
Sciences, Shanghai, China
Objective: Inammation has been proposed to inuence multiple aspects of cholesterol metabolism. Prostaglandin E2 (PGE2) is
an important lipid mediator in inammation. However, whether
or how PGE 2 regulates hepatic cholesterol metabolism remains
unknown.
Methods: Plasma, hepatic cholesterol and bile acid levels were
assayed in western diet-fed mice. Bile acid composition in serum and
liver were analyzed by LC-MS. Bile acid related genes were determined
by RT-PCR and western blot.
Results: PGE2 receptor subtype 3 (EP3) expression was upregulated
in livers when exposed to a high-cholesterol diet. Deletion of EP3 receptor in liver resulted in hypercholesterolemia and augmented dietinduced atherosclerosis in mice by suppression of hepatic bile acid synthesis. CYP7A1, catalyzing the rst and rate-limiting step in the bile acid
synthetic pathway, was down-regulated in EP3-decient livers. Forced
expression of CYP7A1 in liver rescued the impaired biliary cholesterol
excretion in EP3 decient mice. Mechanistically, we found that EP3 regulates CYP7A1 expression via depressing PKA-dependent phosphorylation of nuclear receptor HNF4, which reduced its transcriptional
activity.
Conclusion: Our results demonstrated that EP3 receptor modulates
biliary cholesterol excretion in liver through PKA/HNF4/CYP7A1 pathway, also provided new evidence for a direct link between inammatory eicosanoid and cholesterol homeostasis.

TU-003
Protective Effect of Omega-3 Polyunsaturated Fatty Acid in
Myocardial Infarction in Mice A Metabolomics Based Study
Xuan Fang1, Xu Zhang2, Ding Ai2, Chun Jiong Wang2, Jin Long He2,
Yi Zhu1,2
1

Peking University Health Science Center, Beijing, China,

Tianjin Medical University, Tianjin, China

http://dx.doi.org/10.1016/j.yjmcc.2016.06.065
S0022-2828/ 2016 Published by Elsevier Ltd.

Objective: -3 polyunsaturated fatty acids (PUFA) have benecial

effects on many pathological processes especially the cardiovascular disease. PUFA comprise hundreds of bioactive molecules derived from complex metabolism network. -3 PUFA may protective against coronary
heart disease (CHD), in which their protective metabolites are thought
to play an important role. However, the underlying mechanisms by
which dietary PUFA protect against acute myocardial ischemia (AMI)
are largely unknown. This study aimed to investigate whether -3
PUFA metabolites play a role in the prevention and treatment of myocardial infarction in a mouse model and explore the possible mechanism.
Method: we established a mouse model of myocardial infarction to
test our hypothesis. After feeding with chaw diet or high -3 PUFA diet
for 3 weeks, the protective effects in Infarction size and heart function
were evaluated and the plasma samples were analyzed by a
metabolomic approach.
Result: The results revealed that -3 PUFA diet could signicantly reduce the size of infarction. A LC-MS/MS based eicosanoid metabolomic
method was developed, which could measure 32 arachidonic acid
(ARA) metabolites and 37 -3 PUFA derived products. Using this
metabolomic method, we subsequently quantied eicosanoids in
mouse plasma and heart with -3 PUFA supplementation and myocardial infarction model. Correlation network analysis on mouse plasma data
indicated a obvious change of metabolic proles amoung different
groups was observed. Further, we utilized a fat-1 transgenic mice, an experimental model to endogenously synthesize -3 PUFA to conrm our
nding with diet. In vitro, the effects of eicosapentaenoic acid (EPA) on
the response of neonatal rat cardiomyocytes to simulated ischemia (SI)
and hypoxia. Cardiomyocytes isolated from the hearts of new born
Wistar rats were cultured with or without EPA and exposed to 12 h hypoxia followed by detection of apoptosis and cell death. In this work, a systematic eicosanoid metabolomic analysis was performed in order to
study the impact of fed -3 PUFA diet and myocardial infarction model.
Conclusion: we proved a metabolic evidence that the role of -3
PUFA in the myocardial infarction. The work also proved the highlyspecic eicosanoid metabolomic method, which would be a powerful
tools for systematically analyze eicosanoid metabolism in diseases.

TU-004
A Comparative Study on High-Fat Diet Induced Metabolic Abnormalities in Male and Female C57BL/6 Mice
Mukesh Nandave, Anup Ramdhave
Dept. of Pharmacology SPP School of Pharmacy and Technology Management SVKM's NMIMS University, Mumbai, Maharashtra, India

S2

Abstracts

Background: With the rise in incidence of metabolic syndrome

(MetS) among pre- and post-menopausal women, female C57BL/6
mice with perturbed metabolic state can play a role model for
targeting MetS linked comorbidities.
Methods: In this study we compared metabolic, cardiac, hepatic,
pancreatic, and renal changes in male as well as female C57BL/6
mice fed with either high-fat diet (HFD) or low-fat diet (LFD) for
16 weeks.
Results: Within both the sexes, mice fed with HFD showed a significant gain in body weight, body mass index (BMI), energy intake, and
abdominal circumference. These changes were accompanied by compromised glucose and insulin tolerance, hyperinsulinemia, dyslipidemia, elevated plasma IL-6, and TNF- concentration. Histologically,
hepatocytes showed an elevated fat accumulation with mild focal
swelling, suggesting the initiation of non-alcoholic steatohepatitis
(NASH). This was also appended by an increase in plasma activities of
liver enzymes. The pancreas showed upsurge in number of -cells
with subsequent increase in size of islet implying its compromised
state. While the kidney showed mild tubulointerstitial brosis indicating initiation of kidney impairment, heart showed mild degenerative
changes in cardiac bres denoting absence of cardiac remodelling due
to HFD.
Conclusions: Male and female C57BL/6 mice showed variations in
physical development wherein, male mice had greater body weight,
BMI, central adiposity, and energy intake as compared to female mice.
Further, both male and female C57BL/6 mice fed with HFD developed
features of NASH, hyperinsulinemia, dyslipidemia, impaired glucose
and insulin tolerance but the magnitude of these abnormalities was
found to be less in female mice.

TU-005
Role of hyperhomocysteinemia in Alzheimers neurodegeneration
and the protections
Jianzhi Wang
Tongji Medical College, Wuhan, China

Background: Hyperhomocysteinemia (HHcy), beta-amyloid (Abeta)

overproduction and tau hyperphosphorylation are critical etiological
and pathological factors in Alzheimer disease, however, whether and
how HHcy affects Abeta production and tau phosphorylation are not
fully understood.
Methods: Intraperitoneal or the vena caudalis injection of homocysteine were used in rats to produce the model of HHcy; ELISA, immunohistochemistry and Western blotting with site-specic
phosphor-tau antibodies were employed for measurement of
Abeta, tau phosphorylation and the related protein kinases and protein phosphatases.
Results: We found that Intraperitoneal or the vena caudalis injection of homocysteine for two weeks remarkably increased the
plasma level of homocysteine. At mean time, the HHcy rats
showed the increased levels of Abeta, phosphorylated tau at multiple Alzheimer-associated sites, the activity of glycogen synthase
kinase-3 (GSK-3) with spatial learning and memory decits,
while the activity of protein phosphatase-2A (PP-2A) decreased.
Simultaneous supplementation of folate and vitamin-B12, betaine,
or a synthesized juxtaposition (named SCR1693) composed of an
acetylcholinesterase inhibitor (AChEI) and a calcium channel
blocker (CCB) attenuated the hyperhomocysteinemia-induced
Abeta overproduction, tau hyperphosphorylation and restored
the activity of GSK-3 and PP-2A with improvement of spatial
learning and memory capacities.
Conclusions: HHcy can induce Alzheimer-like histopathologies
and behavioural decits, and supplement of folate and vitamin-

B12, betaine, SCR1693 can efciently attenuate the toxic effects

of HHcy in rats.

TU-006
Bilirubin mediates heme oxygenase-1-induced vascular benets in
diabetic mice
Yu Huang, Jian Liu
Chinese University of Hong Kong, Hong Kong, China
Background: Heme oxygenase-1 (HO-1) exerts vasoprotective effects. Such benet in diabetic vasculopathy is not clear. We have demonstrated that bilirubin mediates HO-1-induced vascular benets in
diabetes (Liu et al., 2015, Diabetes 64:1564-75).
Methods: Diabetic db/db mice were treated with HO-1 inducer
hemin for 14 days and aortas were used for functional and molecular
studies. NO generation was measured in cultured endothelial cells.
Results: Hemin treatment augmented endothelium-dependent relaxations and elevated Akt and eNOS phosphorylation in diabetic mouse aortas, which were reversed by HO-1 inhibitor SnMP or HO-1 silencing.
Hemin administration increased serum bilirubin, and ex vivo bilirubin
treatment improved relaxations in diabetic mouse aortas. Biliverdin reductase silencing reduced the effect of hemin. Chronic bilirubin treatment
improved the relaxations in diabetic mouse aortas. Hemin and bilirubin
reversed high glucose-induced reductions in Akt and eNOS phosphorylation and NO generation. Biliverdin reductase silencing inhibited the effect
of hemin but not bilirubin. In addition, bilirubin augmented acetylcholineinduced relaxations in renal arteries from diabetic patients.
Conclusion: HO-1-induced recovery of endothelial function in diabetic mice is mediated mainly by bilirubin, which preserves NO bioavailability through the Akt/eNOS/NO pathway, indicating that bilirubin is a
potential therapeutic target for clinical intervention against diabetic vasculopathy (supported by CUHK2/CRF/12G and T12-402/13N).

TU-007
Inhibition of miR-92a Improves Endothelial Function in Diabetes
Lingshan Gou, Jiangyun Luo, Lei Zhao, Li Wang, Chi Wai Lau, Yu Huang
Chinese University of Hongkong, Hongkong, China

Rational: Cardiovascular disease is a major complication of diabetes

and the leading cause of morbidity and mortality. Endothelial dysfunction is the hallmark and also the trigger for the development of diabetic
cardiovascular diseases. MicroRNAs (miRNAs), critical regulators of
gene expression, possess a wide spectrum of biological functions including regulation of endothelial function. MiR-92a, abundant in endothelial
cells, has been reported to control endothelial function through regulating several target genes in cell studies. However, the pathophysiological
role of miR-92a in endothelial dysfunction in diabetic animals remains
unclear, and whether inhibition of miR-92a improves endothelial function in diabetes deserves investigation.
Objective: To investigate the effect of miR-92a inhibition and subsequent signaling in the improvement of endothelial function in diabetes.
Results: The expression of miR-92a is higher in the aortas of db/db
diabetic mice compared with those of non-diabetic db/m + mice, accompanied with impaired acetylcholine-induced endotheliumdependent relaxations. Inhibition of miR-92a restores the impaired relaxations, and normalizes the decreased phosphorylation of eNOS at
Ser1177 in db/db mouse aortae. Likewise, diabetic risk factor, advanced
glycation end products (AGEs) increases the miR-92a expression in
human umbilical vein endothelial cells (HUVECs), which is reversed
by NF-B inhibitor. In addition, inhibition of miR-92a recovers the diminished nitric oxide (NO) levels in AGEs-treated HUVECs.

Abstracts

Conclusion: The present study provides new evidence that miR-92a

inhibition improves endothelial function in diabetes, likely through increasing the expression and activity of eNOS/NO signaling in endothelial
cells.

TU-008
MicroRNA-18 suppresses LXR expression in human neuroblastoma
cells and hepatocytes
Dandan Shang, Xin xin, Mei Han
Hebei Medical University, Shijiazhuang, Hebei province, China
The liver X receptor (LXR, NR1H3) and LXR (NR1H2) are members of the nuclear hormone receptor superfamily. They play a critical
role in the transcriptional control of lipid metabolism. MicroRNAs
(miRs) are regarded as important negative regulators of gene expression. It has been reported that miR-1/miR-206 suppress LXR-induced
lipogenesis in hepatocytes. However, the regulation of LXR by
microRNAs hasnt been reported. In this study, we found that miR18 repressed LXR expression in both human neuroblastoma cells
and hepatocytes at both mRNA and protein levels. In addition, bioinformatics analysis predicted a same putative target-site for miR-18 located within the 3-untranslated region (3-UTR) of LXR mRNA. The
luciferase reporter gene assay in HEK293 cells revealed that miR-18a directly targeted the 3-UTR of LXR mRNA. Taken together, we for the
rst time demonstrated that miR-18 repressed LXR expression by
targeting the 3-UTR of LXR mRNA.

TU-009
Phosphodiesterase-5 inhibition protects against the development of
diabetic cardiomyopathy in type-2 diabetes mellitus
Tams Radovits1, Csaba Mtys1, Balzs Tams Nmeth1, Attila Olh1,
Mihly Ruppert1, Dalma Kellermayer1, Marianna Trk1, Lilla Szab1,
Alex Ali Sayour1, Gbor Szab2, Bla Merkely1
1

Heart and Vascular Center, Semmelweis University, Budapest, Hungary,

Department of Cardiac Surgery, University of Heidelberg, Heidelberg,
Germany
2

Background: Diabetes mellitus (DM) is associated with a special

heart disease, termed diabetic cardiomyopathy. The pathophysiological
role of cGMP signaling has been intensively investigated in DM. The second messenger cGMP, broken down by the phosphodiesterase-5 enzyme (PDE5), has been shown to exert cytoprotective effects. We
investigated the effect of chronic inhibition of PDE5 by vardenal in
type-2 DM related cardiomyopathy.
Methods: For type-2 DM Zucker Diabetic Fatty (ZDF) rats were used.
ZDF Lean (ZDFL) rats served as controls. Animals received either vehicle
(ZDFL, ZDF) or 10mg/kg BW vardenal per os (ZDFLVard, ZDFVard)
from 7 to 32 weeks of age. Cardiac morphology was followed by echocardiography. Left ventricular (LV) function was assessed using a
pressure-volume (P-V) conductance microcatheter system. Gene expression analysis of atrial natriuretic factor (ANF; qRT-PCR), cardiomyocyte diameter/tibia length (CD/TL) and Massons staining (brosis score
(FS)) were used to prove pathological myocardium hypertrophy.
Results: Cardiac hypertrophy (echocardiography: LV anterior wall
thickness in systole (LVAWs): 2.81 0.1mm; relative wall thickness
(RWT): 0.490.02; LVmass/TL: 0.300.01g/cm; CD/TL: 3.530.02m/
cm; ANF: 3.040.26 vs ZDFL (LVAWs: 2.530.04mm; RWT:
0.430.02; LVmass/TL: 0.230.004g/cm; CD/TL: 3.090.02m/cm;
ANF: 0.920.17); pb0.05) and brotic remodelling (FS: 1.050.09 vs
ZDFL (0.570.13); pb0.05) have been observed in ZDF. Drug treatment
signicantly decreased myocardial hypertrophy and brosis (LVAWs:
2.470.05mm; CD/TL: 3.150.02; ANF: 1.390.21; FS: 0.590.08 vs

S3

ZDF; pb 0.05) in DM. PV analysis showed impaired diastolic function

and increased cardiac stiffness (time constant of LV pressure decay ():
9.170.25ms; slope of end-diastolic P-V relationship (EDPVR):
0.0780.002mmHg/l vs ZDFL (: 8.180.13ms; EDPVR:
0.0450.003mmHg/l); pb 0.05) while contractility parameters and
blood pressure remained unchanged in ZDF. Vardenal improved diastolic parameters (: 8.620.34ms, EDPVR: 0.0620.006mmHg/l vs ZDF;
pb 0.05). Vardenal did not have any effects in ZDFL.
Conclusions: We reported that chronic administration of vardenal
prevents DM associated myocardial complications. PDE5 inhibition
might be an important target to improve cardiovascular outcome in diabetic patients in the future.

TU-010
Central Body Fat Distribution Attenuates Heart Rate Recovery after
Maximal Exercise in Young Healthy Obese Women
Wanda R P Lopes-Vicente1, Felipe X Cepeda2, Maria F Hussid1, Katia De
Angelis1, Simone Dal Corso1, Fernanda C Lanza1, Fernanda M ConsolimColombo1,2, Ivani C Trombetta1,2
1

Universidade Nove de Julho, So Paulo, So Paulo, Brazil,

Heart Institute, University of Sao Paulo, So Paulo, So Paulo, Brazil

Background: Obesity causes negative changes in the hemodynamic

and autonomic control, what have an adverse effect on the cardiovascular
risk. The attenuation of the decline in heart rate recovery after maximal
exercise test (HRR) reects a vagal dysfunction, what is an independent
predictor of mortality. Thus, the aim of this study was to test the hypothesis that obese women with central body fat distribution had lower HRR
compared with obese women with peripheral body fat distribution.
Methods: Fifty-one healthy young obese women with waist circumference (WC) N 88 cm, were divided into two groups: With central fat distribution (CF), dened as waist-to-hip ratio (WHR) N0.85 (n= 24,
33.51.4 y), and with peripheral fat distribution (PF) with WHR 0.85
(n =27, 32.31.3 y). All volunteers were submitted to maximal cardiopulmonary exercise test.
Results: CF and PF showed similar body mass index (33.20 0.51 vs.
33.56 0.50 kg/m2, respectively, p=0.612). As expected, CF had higher
WHR (0.910.01 vs. 0.800.01, p b0.001) and WC (108.171.42
vs.102.691.42, p=0.009). Interestingly, despite similar BMI, CF had attenuated HRR at rst minute compared with PF (13.41.5 vs. 18.31.5
beats, p=0.026). In addition, HRR was associated with WHR (r=-0.31,
p=0.025) and WC (r=-0.38, p=0.006).
Conclusion: Our data suggest that in young obese women, WHR is a
better risk related marker of central body fat distribution that impairs
the vagal autonomic function, characterized by attenuated of 1st min of
heart rate recovery after maximal exercise test.
RESULTS:
n
Systemic
characteristics
Blood glucose (mM)
Final bodyweight (g)
Cardiac
inammation and
remodelling
LV macrophage
content (AU)
-MHC:18s
mRNA(fold)
CTGF:18s mRNA (fold)
Cardiac collagen
content (AU)
Cardiac function

ANX-A1+/+
non-diabetic diabetes
10
9

ANX-A1-/non-diabetic diabetes
8
6

8.70.2
33.40.6

30.31.0*
28.71.0*

9.40.4
31.10.3

30.61.4*
25.80.6*

64.82.0

64.12.2*

61.93.6

92.15.0*#

1.00.3

5.11.4*

1.0 0.5

11.15.3*

1.000.23
4.10.4

2.020.16* 0.920.19
8.80.4*
4.20.4

1.500.36*
11.70.1*#

(continued on next page)

S4

Abstracts

(continued)
LV E:A ratio (AU)
LV-dP/dt (mmHg/s)
LV+dP/dt (mmHg/s)

1.950.08
9340560
12100 652

1.500.08* 1.810.10
8070284 8710514
9940553 9100444

1.430.11*
7181890
78601020

*p b 0.05 genotype non-diabetic counterparts; #p b 0.05 vs diabetic

ANX-A1+/+ (2-way ANOVA, followed by Tukeys post-hoc test). MHC, -myosin heavy chain; CTGF, connective tissue growth factor.

TU-011
Deciency of Annexin-A1 Exaggerates Diabetic Cardiomyopathy in a
Mouse Model of Type 1 Diabetes
Cheng Xue Qin1,2, Sarah Rosli1,3, Helen Kiriazis1, Minh Deo1, Eric F
Morand4, Yuan H Yang4, Xiao-Jun Du1, Rebecca H Ritchie1,4
1

Baker IDI Heart and Diabetes Institute, Melbourne, Australia

Department of Pharmacology, University of Melbourne, Melbourne,
Australia
3
Department of Medicine (Central Clinical School), Monash University,
Melbourne, Australia
4
Centre of Inammatory Diseases, Monash University, Clayton, Australia
2

Background: Diabetes is a chronic metabolic disease associated

with low-grade inammation and increased risk of heart failure. We
have recently shown that deciency of anti-inammatory protein
annexin-A1 (ANX-A1) exaggerates myocardial infarction; its impact
on other cardiac pathologies has not been investigated. The aim of this
study was to test the hypothesis that deciency of ANX-A1 exaggerates
diabetic cardiomyopathy in Type 1 diabetic (T1D) mice.
Methods: T1D was induced in 6-week-old ANX-A1+/+ and ANX-A1-/male mice via streptozotocin (55mg/kg/day i.p. for 5-days), and mice
followed for 16wks. At study end, cardiomyocyte hypertrophy, cardiac
inammation, remodelling, and dysfunction were assessed. Blood glucose and body weight were monitored fortnightly.
Results: T1D signicantly increased blood glucose levels, with cardiac
inammation and remodelling; cardiac function was also impaired (See
Table). Interestingly, cardiac inammation and remodelling (but not
diastolic dysfunction) were further exaggerated in ANX-A1-/- T1D mice.
Conclusion: This study was the rst to demonstrate the deciency of
ANX-A1 exacerbates diabetes-induced cardiomyopathy in T1D. ANX-A1
may thus represent a therapeutic target for the management of
diabetes-induced heart failure.

TU-012
-adrenergic and AMPK signaling regulates cardiomyocyte glycogen
autophagy in metabolic stress settings.
Kimberley Mellor1,2, Vicky Benson1, Upasna Varma2, Ellie Stevens1,
Lea Delbridge2
1

University of Auckland, Auckland, New Zealand

University of Melbourne, Victoria, Australia

Autophagy disturbance and glycogen mishandling have been observed in the diabetic heart. We have recently demonstrated that an autophagy process specic for glycogen (glycophagy) is modulated by
metabolic stress and is an important regulator of glycogen content in
the heart. The aim of this study was to investigate the upstream
glycophagy signaling mechanisms.
Excised hearts from type 1 (STZ rat) and type 2 (db/db mouse) diabetic rodents were analyzed for glycogen content, and expression of
glycogen regulatory enzymes. Fixed heart tissue was processed and imaged by electron microscopy. -adrenergic signaling activation by 106M isoproterenol perfusion of isolated rat hearts was used to determine
-adrenergic involvement in glycophagy response in non-diabetic
hearts. A role for AMPK signaling was investigated using 1mM AICAR

treatment (AMPK activator) of neonatal rat ventricular myocytes

(NRVMs) cultured in 5mM or 30mM glucose.
In vivo cardiac glycogen was elevated in type 1 and type 2 diabetic
rodent models (3.9-fold and 1.9-fold respectively), and this was not associated with changes in glycogen synthase and phosphorylase activation. Glycophagy involvement was evidenced by accumulation of
glycogen in phagosome double-membrane structures visualized by
electron microscopy. Ex vivo isoproterenol-induced adrenergic activation markedly increased expression of glycophagy markers,
GABARAPL1 and acid -glucosidase (3 fold and 1.5-fold respectively,
p b0.05) coincident with depletion of glycogen content (71% lower
after 5min, fully depleted after 60min, p b0.05). In vitro, activation of
AMPK attenuated high glucose-induced glycogen accumulation in
NRVMs with no change in phosphorylase activation, suggesting a role
for AMPK stimulated glycophagy-breakdown of glycogen.
This is the rst study to show that diabetes-induced cardiac glycogen
accumulation is linked with induction of glycophagy. Furthermore,
these ndings suggest that adrenergic and AMPK signaling positively
regulate glycophagy. Glycophagy may be an important new target for
rescue of diabetic cardiomyopathy and further mechanistic interrogation of these signaling pathways is warranted.

TU-013
Aromatase expression in the myocardium and pericardial adipose
a potential arrhythmogenic modulator?
Gabriel Bernasochi1, James Bell1, Wendy Ip1, Wah Chin Boon2, Salvatore
Pepe3, Jonathan Kalman4, Stephen Harrap1, Lea Delbridge1
1

Department of Physiology, University of Melbourne, Melbourne, Victoria,

Australia
2
The Florey Institute of Neuroscience, University of Melbourne, Melbourne,
Victoria, Australia
3
Murdoch Childrens Research Institute, Department of Paediatrics, University of Melbourne, Melbourne, Victoria, Australia
4
Department of Cardiology, Royal Melbourne Hospital and the Department
of Medicine, University of Melbourne, Melbourne, Victoria, Australia
In obesity, increased pericardial adipose deposition is associated
with elevated incidence of atrial brillation. Estrogen-only hormone
supplementation therapy also increases risk of atrial brillation. Adipose is a major endocrine/paracrine tissue capable of sex steroid synthesis, converting testosterone to estrogen via aromatase action. Thus links
between adipose, aromatase, estrogen and arrhythmia are postulated.
The aim of this study was to identify a potential role for pericardial
fat-derived aromatase in regulating local cardiac sex steroid balance
and arrhythmia propensity.
Atrial appendage tissues were obtained from coronary artery bypass
patients. In parallel studies, myocardium and pericardial adipose were
excised from anaesthetized male/female Sprague Dawley rats (SpD),
from Hypertrophic Heart Rats (HHR) and control Normal Heart Rats
(NHR). Using Western immunoblotting, aromatase was detected in
both human and rat myocardium and pericardial adipose. In SpD, aromatase expression was greater in female myocardium (female vs.
male, arb.units; 1.30 vs. 0.97, pb 0.05) and pericardial adipose (1.68 vs.
0.75, pb 0.05). In both male and female SpD, aromatase levels were approximately 30-fold greater at 50wks (aged) vs 8wks young adult controls (50wk vs. 8wk, arb.units; female 2.139 vs. 0.044, p b0.05; male
1.022 vs. 0.033, pb 0.05). Aromatase expression was increased in both
male/female hearts with an underlying pathological hypertrophy
(HHR vs. NHR, arb.units; female 1.158 vs. 2.139, p b0.05; male 1.218
vs. 1.022, p=ns) compared with NHR controls.
This is the rst study to show aromatase expression in atrial and pericardial adipose tissue. Data indicate the pathophysiological importance of
aromatase is modulated according to sex, age and hypertrophic status.
These ndings suggest that increased pericardial adipose deposition (ie

Abstracts

in aging & obesity) provides capacity for augmented steroid conversion

through elevated levels of aromatase. The paracrine actions of locally synthesized estrogens in the heart may exert important inuence on
myocyte contractility and viability, and on arrhythmia vulnerability.

TU-014
Effects of perindopril on cardiovascular function in middle- aged
diet-induced rat models of the metabolic syndrome
Andrew Fenning, Kylie Connolly, Fiona Coulson
CQUniversity, North Rockhampton, Qld, Australia
RAAS blockade remains a mainstay of cardiovascular pharmacology
for the treatment of hypertension, heart failure, left ventricular hypertrophy, vascular dysfunction, diabetes and renal disease yet its role in
modulating body mass and weight loss following the metabolic syndrome is yet to be fully established. This study aimed to assess the effect
of perindopril (P) on preventing cardiovascular dysfunction in animal
models of metabolic syndrome with diet induced obesity and hypertension. Sixteen week old male WKY and SHR rats were randomly assigned
to one of eight treatment groups; WKY, WKY + P WKY-HFHC, WKYHFHC +P, SHR, SHR+ P, SHR-HFHC, and SHR-HFHC +P. Rats in HFHC
groups were fed a high fat high carbohydrate diet for a period of 20
weeks, while control rats were fed standard chow. Treatment with
perindopril (1mg/kg/day) was administered to rats for 12 weeks commencing at week 8 of the 20 week treatment period. Perindopril treatment had signicant impacts on body weight and fat mass (WKYHFHC - 31 1*mg/g bwt; WKY-HFHC + P 19 3**mg/g bwt) in
HFHC fed animals, preventing obesity-induced cardiovascular dysfunction in these animals. Perindopril treatment also prevented the development of hypertension in normotensive HFHC fed rats (WKY-HFHC 1623*mmHg; WKY-HFHC+ P - 1364**mmHg). Improvements in
a number of metabolic parameters were also noted. Decreased oxidative
stress, improved lipid proles and vascular function, in addition to prevention of cardiac brosis and electrical dysfunction were observed in
obese rats with and without genetic hypertension. It was also found
that perindopril had very little anti-hyperglycaemic effect in these rats
indicating that the benecial effects observed in this study occurred independently of any blood glucose lowering activity. Perindoprils antihypertensive effects have been extensively studied in various
hypertensive disease contexts; however this study has provided some
insight into perindoprils effects in obesity and the metabolic syndrome,
intervening at both primary and secondary end points.

TU-015
Protein content of serum exosomes are correlated to atherosclerosis
Jing Quan1, Mei Jiang2, Sifeng Chen1

S5

inammation, immune reaction and proteinase activity. In the mean

times, proteins responsible for metabolism and transportation of lipid
and cholesterol as well as for proteinase inhibition were decreased. The
changes of the proteins were proportional to the size of atherosclerosis
plaque. Thus, increased bad exosomes in serum may be an etiological factor of atherosclerosis. Further identication of the source of bad exosomes
may reveal new mechanisms and risk factors of atherosclerosis.

TU-016
Common Variation in WNK1 and Blood Pressure Responses to Dietary Sodium or Potassium Interventions: A FamilyBased Association Study
Jianjun Mu, Fuqiang Liu, Chao Chu, Tongshuai Guo, Zuyi Yuan
Cardiovascular Department, First Afliated Hospital of Xian Jiaotong University, Xian, China
Objects: WNK1(With No-lysine Kinase 1) could regulate numerous
sodium or potassium transport related ion channels involved in sodium
or potassium transport in the kidney, and involve in blood pressure. Common variations in WNK1 were associated with hypertension and sodium
or potassium homoeostasis. However, because of interference between
gene and environment interactions, it is difcult to fully detect genetic
contribution of WNK1 gene polymorphism to BP variability. Our aim
was to detect the effect of common WNK1 variants on the shift of blood
pressure under strict dietary intervention of salt or potassium intake.
Methods: 342 subjects from 126 families were selected from a rural
community of Northern China. They were sequentially maintained on
normal diet for 3 days at baseline, a low-salt diet for 7 days (3 g/day,
NaCl), then a high-salt diet for 7 days (18 g/day), and high-salt diet
with potassium supplementation for another 7 days (4.5 g/day, KCl).
Five single nucleotide polymorphisms were selected from WNK1
gene. Single marker and haplotype analyses were conducted using the
Family Based Association Test program.
Results: The data shown that rs880054 and rs12828016 were
associated with DBP response during low-sodium or high-sodium
intervention, and rs2301880 was signicantly associated with SBP,
DBP and MAP responses to high-sodium intervention ( all P b0.05
). Regretful, no associations for WNK1 SNPs and the constructed
haplotype blocks of WNK1 with blood pressure responses to highsalt-and-potassium supplement intervention reached nominal statistical signicance.
Conclusions: Our data support the hypothesis that the WNK1 gene
might be mechanistically involved in the variation in blood pressure response to dietary sodium and potassium intake among individuals , and
these genetic variants might contribute to the variation of this complex
phenotype.
Keywords: blood pressure; gene polymorphism; potassium; sodium; WNK1

Dept. of Physiology and Pathophysiology, College of Basic Medical Sciences,

Fudan University, Shanghai, China,
2
Dept. of Neurology, Gongli Hospital, Shanghai, China
Cell-derived exosomes have been demonstrated to be efcient carriers
to transfer proteins and other cellular contents to surrounding or distant
cells. An exosome can be benecial or harmful, depending on the cell it
comes from. Atherosclerosis is one of the main reasons of coronary
heart disease. Since arteries expose to serum constantly, we believe the
proteins in serum exosomes are closely related to atherosclerosis.
Exosomes were isolated from sera of age-match healthy and atherosclerosis patients using a method combining commercial kit and ultracentrifuge. The will identify by electron microscope, Nanosize and biomarkers.
The protein contents of the exosomes were analysed by protein mass
spectrometry. We found that in exosomes isolated from the sera of atherosclerosis patients contained signicantly more proteins that promote

TU-017
High Salt Intake Fail to Enhance Plasma Adiponectin in Normotensive SaltSensitive Subjects
Jianjun Mu, Fuqiang Liu, Tongshuai Guo, Chao Chu, Zuyi Yuan
Department of Cardiology, First Afliated Hospital of Xian Jiaotong University, Xian, China
Objects: Evidences show that salt could modulate adiponectin and
inammation level in normal individuals. Therefore, we hypothesized
that abnormalities of adiponectin and inammation may be the potential mechanism of salt sensitivity. Aims of the study were to investigate
whether different alteration of adiponectin and inammation level in

S6

Abstracts

response of high salt were exhibited between normotensive salt sensitive and salt resistant subjects.
Methods 30 normotensive subjects (aged 25 to 50 years) were
selected from a rural community of Northern China. All of the people were sequentially maintained on 3 days baseline investigate, a
low-salt diet for 7 days (3 g/day, NaCl), then a high-salt diet for 7
days (18 g/day).
Results: Salt-sensitivity was diagnosed in 10 subjects who exhibited a response of the increase in mean BP by 10% from lowsalt period to high-salt period. Plasma adiponectin higher signicantly in high salt intake than low salt diet(6.1 1.3vs7.1 1.7g/
ml, P = 0.047) in normotensive salt resistant subjects but not in
normotensive salt sensitive subjects (6.4 2vs5.9 2.1g/ml,
P = 0.481). High salt intake increased markedly plasma TNF-( P
b0.0001 ) and MCP-1(P b0.0001) in normotensive salt sensitive
subjects as well as normotensive salt resistant subjects. No signicant change of plasma hs-CRP was observed.
Conclusions: Our data indicates that the disturbance of adiponectin
exists in normotensive salt sensitive subjects during high salt diet,
which may be a novel underlying mechanism of salt sensitivity.
Keywords: sodium-dependent, adiponectin, inammation,
normotensive

TU-018
Effects of renin-angiotensin system inhibitors on renal expression of
renalase in Sprague-Dawley rats fed with high salt diet
Jianjun Mu, Yang Wang, Wenling Zheng, Yongbo Lv, Yumeng Cao,
Jiawen Hu, Tongshuai GUO, Chao Chu
Department of Cardiology, First Afliated Hospital of Xian Jiaotong University,
Xian, China
Objects: To investigate the effect of a high salt diet on renal expression of renalase and the potential role of local renin-angiotensin system
(RAS) in this process.
Methods: Sprague-Dawley (SD) rats were divided into normal-salt
(NS), high-salt diet (HS), high-salt intake with hydralazine group
(HS + H), high-salt diet with enalapril group (HS + E) and high-salt
diet with valsartan group (HS+V), for 4 weeks. Systolic blood pressure
(SBP) was monitored. Blood and urine samples were collected at the
end of intervention. Renin activity, angiotensin II (Ang II) and Ang II
type 1 receptor (AT1R) were detected by real-time PCR. Renalase
mRNA and protein were measured by real-time PCR, western blot and
immunohistochemistry.
Results: After 4 weeks, SBP and proteinuria were signicantly
increased in HS versus NS group. Dietary salt intake caused a dramatic decrease in expression of renalase in kidney. Renal cortex
renin, Ang II and AT1R increased signicantly in HS and HS + H.
Urinary protein was positively correlated with renal renin, Ang II
and AT1R. In addition, in HS + E and HS + V, enalapril or valsartan
failed to inuence renal expression of renalase but abolished the
increase of proteinuria, renal cortex renin, Ang II and AT1R when
compared with HS.
Conclusion: The present study indicates that a high salt intake reduces the renal expression of renalase, and renal RAS may be not involved in the regulation of renalase in SD rats fed with high salt.
Keywords: renin-angiotensin system; renalase; salt; proteinuria

TU-019
Efcacy and safety of losartan/amlodipine single pill versus free
combination at the same dose in hypertensive patients with
metabolic syndrome
Aniskhon Alyavi2, Jamol Uzokov1, Bekzod Karimov1, Akmal
Khudoykulov1, Gulnoza Sultonova1, Manzura Uzoqova1

Tashkent Medical Academy, Tashkent, Uzbekistan

JSC Republican specialized scientic-practical medical center of therapy
and medical rehabilitation, Tashkent, Uzbekistan

Background: The blockage of the RAS through ARB aids in slowing

down the processes of endothelial dysfunction and subsequent atherosclerosis. This results in reduced oxidative stress, improved vasodilation
and improved endothelial function. The renin-angiotensin system (RAS)
is a common link between hypertension and comorbidities of obesity
and metabolic syndrome (MS). CCBs inhibit the ow of extracellular calcium through ion-specic channels that span the cell wall. This causes
vascular smooth muscle cells to relax and thereby results in vasodilation,
blood pressure lowering and reduced peripheral arterial resistance.
Aim: of this work to estimate the effects of losartan 50 mg/
amlodipine 5 mg in single pill versus free combination of losartan
50 mg + amlodipine 5 mg.
Material and method: 82 patients with MS who have second or
third stage hypertension were enrolled in this study (aged 48-65
years old (mean: 538). Anthropometric and laboratory data obtained
at baseline and at the 4rd, 8th, and 12th months of follow-up were compared in the two groups.
Results: After 1 month of the treatment BP was well controlled in
both treatment groups, however, patients under single pill combination
tended to show a better positive response to the treatment than patients under free combination (87.4% vs. 81.2%; Pb0.05) and higher percentage of controlled patients (88.3% vs. 77.9%; P b 0.05). At week 12,
ofce SBP (22.4 12.9 vs. 21.1 13.8; P b0.002), and DBP (16.2 8.4
vs. 13.4 8.2; P b0.02) decreases were still in favor of the single pill
leading to high levels of response to the treatment (88.4% vs. 86.2%;
Pb0.05) and BP control (82.2% vs. 81.2%; Pb0.02). All treatments were
well tolerated.
Conclusions: Losartan 50 mg /Amlodipine 5 mg in single pill tend to
show better positive response and higher percentage of controlled patients already after one month of treatment compared to a free combination in patients not controlled by previous antihypertensive therapy
in patients with metabolic syndrome.

TU-020
Cardiogenetics Mapping of Cardiovascular Diseases and Using Those
Variants as a Biomarker
Mahmut Cerkez Ergoren1, Esra Ozerkman3, Sehime G. Temel2, etin
Lt Baydar4, Cenk Conkbayr5, Gamze Mocan1
1

Near East University, Faculty of Medicine, Department of Medical Biology,

Nicosia, Cyprus
2
Near East University, Faculty of Medicine, Department of Embryology and
Histology, Nicosia, Cyprus
3
Near East University Hospital,Medical Genetics Laboratory, Nicosia,
Cyprus
4
Near East University, Faculty of Medicine, Department of Forensic Medicine, Nicosia, Cyprus
5
Near East University, Faculty of Medicine, Department of Cardiology,
Nicosia, Cyprus
Genetic variation is a rich source of knowledge for cardiovascular
disease because many, if not all, cardiovascular disorders are highly
heritable. Genetic risk scores are a useful tool for examining the cumulative predictive ability of genetic variation on cardiovascular
diseases (CVDs). Important considerations for creating genetic risk
scores include the choice of genetic variants, biochemical parameters, and ethnicities.
The questions still remain about the ultimate clinical utility of the genetic risk score, further investigation in high-risk populations and new
ways to combine genetic risk scores with traditional risk factors may
prove to be fruitful.

Abstracts

To investigate the CVD genetic risk score prole, we compared 144

subjects with a cardiac problem and 180 without; we based on HapMap,
1000 genome and dbSNP datas and picked previously identied 36 different SNPs on 24 different genes that are suggested to have association
with CVDs for different populations. This study is the rst analysis of the
highest SNP coverage that shown the association of genetic variants
with CVDs in North Cyprus.
Our data is the rst data shown the association of all 24 gene and 36
polymorphism to CVD and thus these data are demonstrating the
cardio-genetic prole of North Cyprus. North Cyprus has a unique mixture of allele distribution for each SNP to the other close by country
neighbors. Thus, SNP-SNP interactions and also their relation with biochemical pathways might play critical role for developing genetic related diseases like CVD, metabolic syndromes etc. To conclude, this study
will help for understanding the genetic prole of CVDs in the Island
and also will be great source and useful tool for prevention of CVDs.

TU-021
Enhanced CD34 expression was an potential independent prognostic
factor for breast cancer
SHENHUA Xu, ZHANHONG CHEN, WEIZHEN XU, ZHIQIANG LING, GU
ZHANG, LEI LEI, XIYING SHAO, XIAOJIA WANG
Zhejiang Cancer Hospital, Hangzhou, China
The aim of the present study was to investigate the immunohistochemical expression of cluster of differentiation (CD)34 and vascular endothelial growth factor (VEGF) in breast cancer tissue, and their
prognostic signicance. High CD34 expression levels (microvessel density, N 15/HPV) were identied in 27.3% (12/44) of cases, exhibiting no signicant correlation with the clinicopathological characteristics of the
patients. However, Kaplan-Merer analysis demonstrated that the survival
time of patients with high CD34 expression was signicantly shorter than
that of patientow CD34 expression (50.0% vs.90.6%; P=0.003) Samples
with high VEGF expression levels (++or+++) accouunted for 63.6%
(28/44) of the total number of cases. High VEGF expression was signicantly prevalent in patients aged 50 years conmpared with patients
aged b50 years (78.6% vs.37.5%;P=0.006). Furthermore, all patients
with vascular invasion exhibited high VEGF expression levels; thus, patients with vascular invasion presented with signicantly higher VEGF expression rates conmpared with patients with no vascular invasion(100%
vs.55.6%;P=0.018). However, Kaplan-Merer analysis demonstrated that
high VEGF expression was not correlated with the overal survival of the
patients (P=0.366). By contrast, Cox multivariate analysis identied
that clinical stage, triple-negative subtype and age were independent
prognostic factors for patients with breast cancer (P=0.005, P=0.006
and P=0.032, respectively), and that CD34 expression was a potential independent prognostic factor (P=0.055).Therefore, the present study determined that for patients with breast cancer, a high level of CD34
expression may be a potential indicator of a poor prognosis.

TU-022
High fat diet increases the activity of cardiac ryanodine receptors in
lipid bilayers
Luis Montecinos, Jose Finkelstein, Genaro Barrientos, Jaime Riquelme,
Paola Llanos, Gina Sanchez, Ricardo Bull, Paulina Donoso
Instituto de Ciencias Biomedicas. Facultad de Medicina. Universidad de
Chile, Santiago, Chile
Mice fed with high fat diet become obese in a few weeks and develop
cardiac hypertrophy after 4 month. Intracellular calcium plays a key role
in cardiac physiology and pathology but calcium handling proteins in
the heart of obese animals has not been characterized. Activity of

S7

ryanodine receptors (RyR2), the calcium release channels of the sarcoplasmic reticulum (SR), is redox dependent. Since obesity induces oxidative stress, we hypothesized that a redox dependent change in RyR2
activity occurs in obese mice. Therefore we investigated single channel
activity of RyR2 incorporated in planar bilayers.
Single RyR2 channels present in SR vesicles obtained from mice hearts
can be classied, according to their response to cytoplasmic calcium, into
low, moderate or high activity. Channels from hearts of animals fed with
control diet exhibit moderate activity with higher frequency (15 out of 21
channels) and low or high activity with lower frequency (3 out of 21
channels in each case). In mice fed with high fat diet, 10 out of 19 RyR2
channels recorded, displayed high activity while 8 showed moderate
and only 1 channel showed low activity. Therefore, high-fat diet induced
a marked change in the distribution of RyR2 responses increasing the fraction of high activity channels from 14 % to 53 %, and reducing the fraction
of moderate activity channels from 71 % to 42 % and that of low activity
channels from 14 % to 5 %. Addition of apocynin to the diet had no effect
on channel activity in control mice, but prevented the change induced by
the high fat diet. Therefore, high fat diet increases the sensitivity of RyR2
channels to calcium, favoring calcium-induced calcium release, probably
via a redox dependent mechanism.
Funded by Fondecyt 1130407

TU-023
Tetrahydroxystilbene glucoside inhibits excessive autophagy and
improves microvascular endothelial dysfunction in prehypertensive
spontaneously hypertensive rats
Qianqian Dong, Siwang Wang, Haifeng Zhang
Fourth Military Medical University, Xi'an, China
Aims: Autophagy exists in vascular endothelial cells, but the relationship between autophagy and vascular dysfunction in hypertension
remains elusive. This study aimed to investigate role of autophagy in
vascular endothelial dysfunction in prehypertension and hypertension,
and underlying mechanisms. Furthermore, we determined if and how
tetrahydroxystilbene glucoside (TSG), the active ingredient of Polygonum multiorum Thunb with cardiovascular protective properties in Chinese medicine, inuences vascular endothelial function.
Methods: Age-matched male spontaneously hypertensive rats
(SHRs) and Wistar Kyoto rats (WKY) aged 4 weeks and 12 weeks
were randomized into 4 groups and treated for fortnight by gavage
with a) vehicle (normal saline), b) TSG (100 mg/kg/day),
c) rapamycin (i.p., 1 mg/kg/day), or d) TSG + rapamycin, and the vascular function of their isolated aorta and mesenteric artery was assessed
in vitro. HUVECs were incubated serum-starved to induce excessive autophagy, and then incubated with DMEM and treated with a) 10 nmol/L
insulin-like growth factor 1 (IGF-1), b)100 mol/L TSG, c) pre-treated
with rapamycin for 1 h and further incubated with TSG.
Results: Compared with WKY, young and adult SHRs showed endothelial dysfunction of the aorta and mesenteric artery, along with decreased pAkt, pmTOR, and autophagic marker protein p62 and
increased LC3 II/I in microvascular but not aortic tissues. TSG administration for fortnight signicantly improved mesenteric vascular endothelial
function, increased levels of pAkt and pmTOR, and decreased autophagy.
Pretreatment of young SHRs with the mTOR inhibitor rapamycin blocked
the antiautophagic and vasodilative effects of TSG. Moreover, TSG signicantly activated Akt-mTOR signaling in HUVECs and reduced the autophagic levels in vitro, which were almost completely blocked by rapamycin.
Conclusions: Microvascular endothelial dysfunction in prehypertensive SHRs is attributable to excessive autophagy in vascular tissues.
TSG partly restores microvascular endothelial dysfunction through activating Akt/mTOR pathway and consequently suppressing autophagy.
Keywords: Autophagy; Prehypertension; Vascular endothelial dysfunction; Mesenteric arteries.

S8

Abstracts

TU-024
The effects of epicatechin on vascular smooth muscle cells in an
animal model of obesity.
Kirsty MacRae, Rebecca Vella, Andrew Fenning
Central Queensland University, Rockhampton, Australia
Background: Metabolic syndrome (MetS) is a signicant publichealth challenge worldwide leading to CVD and cardiovascular dysfunction. Flavonoids, such as epicatechin have been shown to prevent the
development and progression of cardiovascular disease associated
with obesity, however the precise mechanisms remain unknown.
Therefore, the aim of this study was to assess the vascular response of
epicatechin in tissues from an animal model of obesity.
Methods: 18 male Wistar rat were randomly divided in two groups
(Control (n=10) or High-Fat High-Calorie (HFHC) (n=8)). HFHC animals were treated for a period of 20 weeks, after which assessment of
biometrics, organ weight and vascular function were made.
Results: HFHC treated animals demonstrated a signicant increase
in body weight (C 658.81 11.64; HFHC 771.42 23.4*g), fat
mass, serum glucose (C 8.880.87; HFHC 11.59*mmol/L), cholesterol and triglycerides and left ventricular organ mass and a signicant
decrease in serum nitric oxide levels. HFHC mesenteric arteries demonstrated no change to sodium nitroprusside or noradrenaline but exhibited a reduced relaxation to acetylcholine. Concentration-response
curves revealed epicatechin alone did not alter vasoreactivity in either
control or HFHC animals. In pre-contracted arteries, epicatechin induced a signicant relaxation in control animals that was reduced in
HFHC animals. In contrast, epicatechin alone induced a signicant contraction in aortas from HFHC animals whilst no change was observed
in control tissues. In pre-contracted aortas, epicatechin caused a significant relaxation in control animals that was reduced in HFHC animals.
Conclusion: Results suggest a diet high in fat and carbohydrates
may contribute to the development of metabolic syndrome and its associated cardiovascular complications. In healthy animals, epicatechin
may improve cardiovascular function by inducing nitric oxide dependent vasorelaxation in conduit and resistant arteries, suggesting a diet
rich in avonoids may improve cardiovascular health. However, in endothelium compromised individual, consumption of epicatechin will
achieve minimum cardioprotective effects.

TU-025
The Lack of Toll Like Receptor 4 Did Not Prevent the DiabetesInduced Cardiac Electrical Changes
Maria Micaela Lopez Alarcon, Maria Julieta Fernadez Ruocco, Gustavo
Monerrat-Calhi, Emiliano Medei
Instituto de Biofsica Carlos Chagas Filho, Universidade Federal do Rio de
Janeiro, Brazil
Background and aims: Different studies have shown the important
role of inammation in Diabetes Mellitus (DM). In the last decade the
presence and function of cardiac Toll Like Receptors 4 (TLR4), which
were typically associated tothe innate immune system, has been studied. Several groups demonstrated that the lack of this receptor can prevent distinct cardiac diseases, such as cardiac hypertrophy. In the
present work we investigated whether the lack of TLR4 could prevent
the DM-induced cardiac and renaldysfunction.
Method: male wild type and TLR4-/- mice were used. In order to induce diabetes both groups were treated with streptozotocin (STZ:
50mg/kg/day/i.p for 5 days). ECG was recorded 8 weeks after DM induction, when all animals were euthanized. Intracellular microelectrodes
were usedfor ventricular action potential recordings. Urea and creatinine in serum was measured by colorimetric tests. qRT-PCR was used
to assess vimentine mRNA expression.

Results: Cardiac electrical remodeling was observed in wild type

diabetic mice. This remodeling resulted in longer QT and QTc interval and a corresponding delay in repolarization (phase 3) of the cardiac action potential. The lack of TLR4 did not prevent/improve these
cardiac electrical changes. In contrast,while the DM-mice presented
impaired renal function the TLR4-/- diabetic mice showed conserved
kidney function. Similar urea and creatinine levels and comparable
vimentin mRNA expression in the TLR4-/- diabetic mice when compared to either wild type or TLR4 -/- non-diabetic mice were observed.
Conclusions: even though TLR4has been reported as an important key molecule in cardiac diseases, such as cardiac hypertrophy,
in DM model this receptor is not involved in cardiac electric remodeling. However, the presence of TLR4 appears important in the pathogenesis of DM-induced renal diseases.

TU-026
Carbonic anhydrase and ion transporters in diabetic
cardiomyopathy
Carolina Jaquenod De Giusti1, Paula G. Blanco2, Juan M. Lofeudo1,
Bernardo V. Alvarez1
1

Centro de Investigaciones Cardiovasculares, CONICET Facultad de Ciencias

Mdicas, Universidad Nacional de La Plata, La Plata, Bs As, Argentina
2
Facultad de Ciencias Mdicas, Universidad Nacional de La Plata, La Plata,
Bs As, Argentina
Diabetic cardiomyopathy (DC) describes diabetes-associated
changes in the structure and function of the myocardium which
commonly leads to heart failure. Myocardial intracellular pH (pHi)
in the heart is regulated by acid/base transporters (ABT) such as
the NHE1 Na + /H+ exchanger, the NBC Na +/HCO-3 cotransporter,
and the AE Cl-/HCO-3 exchanger, among others. pHi alterations lead
to changes in heart function and changes in the activity/expression
of NHE1, NBC, and AE have been associated with cardiac disorders.
Conversely, carbonic anhydrases (CAs) enzymes are widely distributed in all organs and tissues, catalyzing the reversible conversion
of CO2/HCO -3. Functional/physical interaction between CA and the
AE, NBC, and NHE ABT occurs in cardiac muscle cells, maximizing
ion uxes, creating a membrane transport metabolon (MTM). Herein, we study the role of the MTM in cardiac dysfunction linked to obesity and DC. We characterized the expression and function of CA, and
NHE1 and NBC ABT, using an obese mice model (C57BL6 ob-/- mice).
So far our results showed DC features in the heart of ob-/- female mice
starting as soon as 8 weeks old, characterized by an increased septum thickness and posterior wall thickness, and increased left ventricular diameter. Furthermore, ob-/- mice had increased left
ventricular mass and left ventricular mass index, indicating cardiac
hypertrophy. To study the role of NHE1 in the ob-/- and wild type
(ob+/+) mice, isolated cardiomyocytes were loaded with the BCECFAM uorescent dye and the NHE1-dependent pHi recovery measured
in myocytes subjected to NH4Cl-induce acid loading, monitored by
epiuorescence. Preliminary results showed increased NHE1 activity
in the hypertrophic myocardium of ob-/- compared to ob+/+ mice,
0.30 0.02 vs. 0.20 0.02 pH units.min-1.100, respectively (n = 5,
pb 0.01), measured in isolated cardiomyocytes. We conclude that activation of NHE1 is a component that may prompt and/or accentuate
NHE1-induced myocardial pathology in the DC.

TU-027
High intensity exercise reduces brosis and hypertrophy but not
oxidative stress in diabetic cardiomyopathy
Ulises Novoa, Diego Arauna, Carmen Zambrano, Madelaine Nuez,
Daniel Gonzalez

Abstracts

Universidad de Talca, Talca, Chile

Diabetic cardiomyopathy refers to the cardiac manifestations observed in the heart as a result of altered glucose homeostasis that is
reected as brosis, cellular hypertrophy, increased sources of oxidative
stress, such as the NADPH oxidases (NOX), apoptosis, and nally systolic
and diastolic dysfunction. Exercise is known to exert salutary effects on
cardiovascular function, mainly through the increase in the expression
of nitric oxide synthase, particularly eNOS.
Aims: We tested the hypothesis that chronic exercise could reverse
the cardiac maladaptations and oxidative stress that are produced by
diabetes.
Methods. Diabetes was induced in Sprague-Dawley rats by a single
dose of alloxan (200/mg kg, i.p). Diabetic rats were randomly assigned
to a sedentary group (n=5) or submitted to a program of exercise on
a motor-driven treadmill (80% of maximal aerobic capacity) 5 days/
week, for 4 weeks (n = 5). Another group of normoglycemic rats was
used as control (n = 5). Cardiac brosis was evaluated by Sirius red
staining, hypertrophy was estimated measuring the perimeter and
cross sectional area of cardiac myocytes in haematoxylin & eosin stained
sections. The levels of NOX and NOS enzymes were evaluated by realtime PCR and Western Blotting. Cardiac levels of tetrahydrobiopterin
were analyzed by HPLC.
Results. Chronic exercise reduced cardiac brosis: 4.43 0.9 % control, 8.68 0.7 % diabetic and 5.72 0.7 % diabetic + exercise, pb 0.05,
ANOVA. Cellular hypertrophy was also reduced in diabetic rats by exercise: myocyte perimeter 297 17 m2 in control group, 446 26 diabetic group and 363 14 diabetic + exercise; myocyte perimeter: 73 7
m in control group, 89.5 4.3 diabetic group, 78.7 2.7 diabetic + exercise, pb 0.05. Biochemically, exercise increased the levels of the NADPH
oxidases NOX2 and NOX4 mRNA levels (pb0.05, ANOVA). Neither diabetes nor exercise induced changes in the levels of cardiac eNOS
(p=0.4139). On the contrary, diabetes increased the level of uncoupled
eNOS, evaluated as the ratio of eNOS dimer/monomer: 1.3 0.36 in control group, 0.38 0.04 diabetic group and 0.26 diabetic + exercise,
pb 0.05. Furthermore, exercise was unable to restore the intracardiac
levels of tetrahydrobiopterin, an essential cofactor for NOS activity, that
were reduced in diabetic rats: 2.69 1.3 nmol/L in control group, 0.31
0.04 diabetic group and 0, 36 0.06 in diabetic + exercise, pb 0.05.
Conclusions. These results suggest that chronic exercise is able to
reverse cardiac remodelling in the diabetic heart, but is unable to restore
the nitroso-redox imbalance imposed by oxidative stress. This later
could by restored by pharmacological manipulations.

TU-029
The impact of diabetes mellitus on miR expression of patients with
or without heart failure
Raiana Barbosa1, Bruna Farjun1, Alexandre Siciliano2, Adriana Carvalho1
1

Federal University of Rio de Janeiro, Rio de Janeiro/RJ, Brazil

National Institute of Cardiology, Rio de Janeiro/RJ, Brazil

Diabetes mellitus (DM2) is an important risk factor for coronary artery disease (CAD). However, the direct involvement of DM2 in the
pathogenesis of heart failure (HF) is still under investigation. The objective of this work was to assess changes in miR expression in diabetic patients with or without HF and to look for possible targets of these miRs.
Based on their clinical proles, patients were divided into 4 groups: CAD
(n = 9), CAD + DM2 (n = 11), both with normal cardiac function, HF
(n = 13) and HF + DM2 (n = 7). Right atrium samples were obtained
from these patients during CABG and the relative quantication of 20
miRs was analyzed by qRT-PCR. The groups analyzed showed no differences in gender, body mass index, number of patients with hypertension
or dyslipidemia. Ejection fraction (EF) and cavity diameters were preserved in all patients of CAD and CAD+ DM2 groups, while in HF and

S9

HF+DM2 groups, EF was 40.5 7.2% and 37.6 11.0% respectively.

It was veried the DM2 factor signicantly downregulated the expression of miR-15a, -29a and -499 in CAD+DM2 group when compared
to the CAD group. DM2 also upregulated let-7b expression in HF+DM
group compared to HF group. MiR-1, -7, -9, -15b, -16, -21, -34a, -126,
-133a, -145, -185, -192, -200a, -208a, -208b and -210 were not altered
by DM2. Then, we used the database TargetScan to select possible target
mRNAs, such as ATP2A2, SCN5A, KCNJ2 and HCN4 transcripts, whose
deregulation is associated with arrhythmias and atrial brillation. By
qRT-PCR, we conrmed an increased expression of KCNIP2, a predicted
target of miR-29a, in CAD+DM2 group. Moreover, the transfection of
pluripotent stem cell derived-cardiomyocytes with miR-29a inhibitor
induced an upregulation of KCNIP2, indicating a possible mechanism
by which diabetes promotes electrical changes in the heart.

TU-030
Nitric oxide bioavailability in rats with metabolic syndrome: effect
of ()-epicatechin in the heart
Barbara Piotrkowski1, Valeria Calabr1, Laura Fischerman1, Marcela
Vazquez-Prieto2, Monica Galleano1, Cesar Fraga1
1

Physical chemistry-IBIMOL, University of Buenos Aires-CONICET, Buenos

Aires, Argentina
2
Dept of Pathology-IMBECU, University of Cuyo-CONICET, Mendoza,
Argentina
Fructose overload promotes functional and metabolic changes in
humans and animal experimental models. Evidence suggests that dietary
avonoids can prevent or attenuate the development of metabolic diseases. In this study we investigated the effects of ()-epicatechin on the
modications induced by fructose overload in rats heart in terms of nitric
oxide and superoxide metabolism. Male Sprague Dawley rats were divided in three groups that received for 8 weeks: i) water and rat chow diet
(C group), ii) 10% (w/v) fructose in the drinking water (F group); iii)
10% (w/v) fructose in the drinking water with ()-epicatechin (20
mg/kg body weight/day) in the rat chow diet (FEC group). These conditions of fructose overload did not lead to heart hypertrophy or tissue remodeling. However, biochemical and molecular changes were observed
and could represent the onset of functional alterations. In this line, an increase in nitric oxide synthase (NOS) activity was observed in FEC with
respect to C and F (pb0.001 vs. C and pb0.05 vs F. These results were correlated with a higher level of eNOS phosphorylation and changes in the
pattern of expression of iNOS and nNOS in the three groups studied. Regarding superoxide anion metabolism, a higher production of this oxidant was found in F group with respect to C and FEC (p b 0.05),
associated with a higher expression of p47 subunit and NOX4.
Superoxide dismutase and glutathione peroxidase activities were
lower in F group compared to C and FEC (p b 0.05). The higher oxidized/reduced glutathione ratio observed in F, was prevented by
()-epicatechin.
In summary, ()-epicatechin was able to ameliorate fructose induced biochemical modications in the heart through modulating the
expression and/or activity of specic proteins. Thus, resulting in a controlled oxidant metabolism favoring NO bioavailability in rats heart.

TU-031
Characterization of the CYP2C19*2 allelic variant distribution in
Chilean coronary disease patients.
JENNY RUEDLINGER1, YALENA PRADO1, NICOLS SAAVEDRA1,
FERNANDO LANAS1, BRAULIO BOBADILLA1, LUIS PEREZ2, LUIS A.
SALAZAR1
1

Universidad de la Frontera, Temuco, Chile

Universidad de Concepcin, Concepcin, Chile

S10

Abstracts

Background: Clopidogrel is a widely used antiplatelet drug by patients undergoing percutaneous coronary interventions (PCI), being
metabolized by the Cytochrome P450 2C (CYP2C) subfamily of enzymes. It has been reported that single nucleotide variants of CYP2C19
gene, the hepatic enzyme involved in biotransformation of clopidogrel
to its active metabolite, can affect the metabolism and anti-platelet response of this drug and the use of an alternative antiplatelet medication
has been recommended.
Objectives: The aim of this study was to assess the prevalence of the
loss-of function allele CYP2C19*2 in a group of Chilean coronary disease
patients.
Methods: 147 patients with history of coronary artery disease who
underwent PCI were included. Clinical and demographic variables
were registered. Single nucleotide polymorphism CYP2C19*2
(rs4244285) was genotyped by real-time PCR using a TaqMan Drug
Metabolism Genotyping Assay.
Results: General characteristics of the analysed population included:
male sex 75.5%, age 63.7 10 years, Diabetes mellitus 31.3%, smokers
19%, body mass index 28 4 kg/m2, systolic blood pressure 134.5
25 mmHg, total cholesterol 179.8 132 mg/dL, and glycaemia 122.2
53 mg/dL. The CYP2C19*2 genotype frequency for GG, AG and AA
was 83%, 16.3% and 0.7% respectively, and the A allele presented a frequency of 8.8%. We found no signicant differences in genotype frequency between men and women (p= 0.12) nor between patients divided by
age (under 65 years and equal or older than 65 years, p= 0.28).
Conclusion: Our ndings indicate the existence of a lower frequency
of the CYP2C19*2 variant in Chilean patients with coronary artery disease, when compared to what has been reported for other populations.
These results bring more information about metabolic phenotypes regarding the use of this drug in Chilean population. Fondecyt 1141292.

TU-032
Inhibition of phosphoinositide 3-kinase promotes cardiac
mitophagy and prevents anthracycline-related cardiomyopathy
Alessandra Ghigo1, Mingchuan Li1, Maria Chiara De Santis1, Nicola
Pianca2, Irene Franco1, Sebastiano Sciarretta3, Fulvio Morello4, Marco
Sandri2, Tania Zaglia2, Marco Mongillo2, Emilio Hirsch1
1

Department of Molecular Biotechnology and Health Sciences, Molecular

Biotechnology Center, University of Torino, Torino, Italy
2
Department of Biomedical Sciences and Venetian Institute of Molecular
Medicine, University of Padova, Padova, Italy
3
Department of Medical and Surgical Sciences and Biotechnologies, Univer
sity of Rome Sapienza, Latina, Italy
4
S.C. Medicina d'Urgenza A.O. Citt della Salute e della Scienza di Torino,
Torino, Italy
Purpose: Anthracycline-induced cardiomyopathy has become a
leading cause of morbidity and mortality among cancer survivors, but
little is known about the underlying mechanisms. We demonstrated
previously that phosphoinositide 3-kinase (PI3K) promotes maladaptive cardiac remodeling and its inhibition prevents pressure
overload-induced heart failure. Here we intend to investigate whether
PI3K inhibition is benecial in a preclinical model of anthracyclineinduced cardiomyopathy.
Methods and results: Mice expressing a kinase inactive PI3K
(PI3K kinase-dead; KD) and wild-type controls (WT) were injected
with a cumulative dose of 12 mg/kg doxorubicin (DOX) via 3 weekly injections. DOX-induced systolic dysfunction was completely prevented
in KD animals as compared to WT controls (% Fractional shortening:
WT DOX 20.5 vs KD DOX 36.6). Accordingly, cardiac atrophy, cardiomyocyte apoptosis and collagen deposition were signicantly lower in
KD than in WT hearts. Mechanistically, PI3K was found to serve as a
negative regulator of cardiac mitophagy via a P-Akt/mTOR/Ulk-1 signaling axis. DOX-induced mitophagy was more pronounced in KD hearts

and cardiomyocytes than in WT counterparts, as evidenced by increased

expression of LC3II in mitochondrial fractions as well as accumulation of
GFP-LC3 puncta, both well-established markers of auto-phagosome formation. This was paralleled by ultrastructural preservation of KD
cardiomyocytes, while WT hearts displayed marked mitochondrial
damage and vacuolization after exposure to DOX. Intriguingly, pharmacological inhibition of PI3K with AS-605240 promoted cardiac
mitophagy, prevented DOX-mediated contractile impairment and delayed tumor growth in Her-2/NeuT transgenic mice, a model of spontaneous mammary tumor growth.
Conclusion: Overall, these data suggest that PI3K inhibitors may
concomitantly prevent anthracycline-induced cardiomyopathy and
tumor progression, by favoring cardiac mitohormesis and likely limiting
a tumor-supportive inammatory response.

TU-033
Oxidative Activation of cAMP-dependent
Protein Kinase by Nitroxyl modulates
Myolament Protein Phosphorylation
Simon Diering1, Mara Goetz1, Sophie Schobesberger1, Sebastian Pasch2,
Sonia Donzelli1, Konstantina Stathopoulou1, Angelika Piasecki1, Bruce
King3, Viacheslav Nikolaev4, Susanne Lutz2, Philip Eaton5, Friederike
Cuello1
1
Department of Experimental Pharmacology and Toxicology, University
Medical Center Hamburg-Eppendorf; Cardiovascular Research Center;
DZHK partner site Hamburg/Lbeck/Kiel, Hamburg, Germany
2
Institute of Pharmacology, University Medical Center Gttingen, GeorgAugust University Goettingen, Gttingen, Germany
3
Department of Chemistry, Wake Forest University, Winston-Salem, North
Carolina, USA
4
Institute of Experimental Cardiovascular Research , University Medical
Center Hamburg-Eppendorf, Hamburg, Germany
5
Kings College London, Cardiovascular Division, The British Heart Foundation Centre of Excellence, The Rayne Institute, St Thomas Hospital, London,
SE1 7EH, UK

Background: Heart failure is a severe disease, which is dened by

the hearts inability to maintain sufcient blood ow, accompanied by
reduced force development and -adrenergic desensitisation. Nitroxyl
(HNO), released by donors such as 1-Nitrosocyclohexylacetate (NCA),
shows positive inotropic and lusitropic properties, which are maintained under failing conditions. However, how NCA exerts these benecial actions remains elusive. In healthy hearts -adrenergic stimulation
and subsequent cAMP-dependent protein kinase (PKA) activation is the
major pathway for adjustments concerning cardiac performance. PKA, a
heterotetrameric enzyme consisting of two regulatory (PKAreg) and two
catalytic (PKAcat) subunits, phosphorylates proteins involved in
excitation-contraction coupling, leading to increased cardiac output.
Notably, besides typical receptor-mediated activation, PKA can be activated directly by oxidants, causing dimerisation of PKAreg subunits by
interdisulde formation.
Hypothesis: The HNO donor NCA leads to oxidative activation of
PKA and subsequent phosphorylation of myolament proteins.
Results: Western Blot analysis of NCA-treated adult rat ventricular
myocytes (ARVMs) showed an increased phosphorylation of the PKA
substrate cardiac myosin-binding protein C (cMyBP-C). Frster resonance energy transfer (FRET) measurements of cells expressing an Akinase-activity reporter (AKAR3) conrmed NCA-mediated PKA activation. To observe oxidative modications of PKA, ARVMs were harvested
under non-reducing conditions after incubation with NCA. Interestingly,
increased PKAreg dimer formation in these samples was detected by
Western Blotting. Furthermore, we could show an intradisulde bond
forming within the PKAcat. In vitro kinase assays with PKAcat again
allowed detection of this intradisulde formation within PKAcat after

Abstracts

exposure to NCA, which was accompanied by decreased kinase activity.

Preincubation of PKAcat with ATP prior to NCA treatment restrained this
inhibitory effect.
Conclusion: HNO released by NCA leads to oxidation and thus activation of PKA which phosphorylates the sarcomeric protein cMyBP-C.
Resultant protein phosphorylation is a net product of inhibitory
intradisulde formation within PKAcat and activatory interdisulde formation of PKAreg subunits.

TU-034
Testosterone activates MEF2 through CaMKII and androgen receptor
to induce cardiomyocyte hypertrophy
Javier Duran, Daniel Lagos, Manuel Estrada

S11

the roles of other post-translational modications of SERCA2a are unknown. Here, we show that the activity of SERCA2a is impeded by acetylation at lysine 492 (K492), and that this inhibitory event can be
reversed by SIRT1, a NAD+-dependent class III histone deacetylase.
SIRT1 interacted directly with and deacetylated SERCA2a in vitro, and
downregulation of SIRT1 increased SERCA2a acetylation and decreased
its enzymatic activity in vitro and in vivo. Concomitant with reductions
in its enzymatic activity, an increase in SERCA2a acetylation was observed in failing hearts, and these defects were restored by lapachone (-lap), a metabolic activator of SIRT1. Structural modeling
analyses suggested that acetylation at K492 may prevent ATP accessing
its binding pocket in SERCA2a. These results indicate that acetylation is a
critical post-translational modication of SERCA2a that is implicated in
reduced function of this calcium pump, and that SIRT1 can restore the
contractile dysfunction of failing hearts via deacetylation of SERCA2a.

Universidad de Chile, Santiago, RM, Chile

Ca2+/Calmodulin-dependent protein kinase (CaMKII) and androgen
receptor are involved in cardiomyocyte hypertrophy. CaMKII regulates
myocyte-enhancer factor 2 (MEF2) that plays a key role in controlling
cardiomyocyte growth. However, whether CaMKII/MEF2C signaling
pathway is involved in testosterone-induced cardiomyocyte hypertrophy remains unknown. The aim this work was to investigate the testosterone effects on the CaMKII-MEF2C pathway in hypertrophy. Our
results showed that testosterone (100 nM) increased the phosphorylation of both CaMKII (Thr286) and phospholamban (Thr17) in neonatal
rat cardiomyocytes. Moreover, testosterone stimulated the nuclear
translocation of MEF2C and MEF2-luc activity. These effects were
prevented in cardiomyocytes pretreated with AIP (a CaMKII inhibitor)
or bicalutamide (an androgen receptor inhibitor) and also by use of
siRNA to MEF2C and CaMKII. Transfection of cardiomyocytes with a
constitutively active isoform of CaMKII (CaMKII-T286D) results in an increased MEF2-luc activity. Testosterone enhances MEF2-luc activity in
T286D cardiomyocytes and it was suppressed by bicalutamide suggesting that MEF2C activation involve both canonical androgen receptor as
well as Ca2 +-mediated pathways. Cardiomyocyte hypertrophy was
assessed by increases in -myosin heavy chain and skeletal -actin proteins, aminoacid incorporation and cell size. All these parameters were
increased by testosterone and prevented by AIP, siRNA-CaMKII and
siRNA-MEF2C. Collectively, these evidences suggest that testosterone
activate CaMKII/MEF2C signaling pathway to induce cardiomyocyte
hypertrophy.

TU-035
Acetylation of SERCA2a inhibits its function and is modulated by
SIRT1
Changwon Kho 1, Dongtak Jeong1 , Ahyoung Lee1 , Seung Pil Jang2 ,
Dong Kwon Yang1, Przemek Gorski1, Jae Gyun Oh1, Woo Jin Park2,
Roger Hajjar1
1

Cardiovascular Research Center, Icahn School of Medicine at Mount Sinai,

New York, NY, USA
2
Gwangju Institute of Science and Technology, Gwangju, Republic of Korea

During the diastole of heart pumping, calcium ions in the cytosol are
re-sequestered into the sarcoplasmic reticulum (SR) by the cardiac SR
Ca2+-ATPase pump (SERCA2a). Reduced levels and activity of SERCA2a
are hallmarks of heart failure. Restoration of SERCA2a expression level
via a gene transfer improves cardiac function, energetics, and survival
in rodent and porcine models of heart failure. In addition, phase 1 and
2 human trials, in which the SERCA2a gene was delivered to the myocardium of patients with advanced heart failure, have conrmed
SERCA2a as an effective therapeutic target. We showed recently that
the activity of SERCA2a is enhanced by conjugation of small ubiquitinrelated modier 1 (SUMO1) at two specic lysine residues. However,

TU-036
Contribution of serotonergic 5-HT2B receptors to the mobilization of
bone marrow endothelial progenitors in cardiac valve degeneration
Roland LAWSON1, Estelle AYME-DIETRICH1, Houda BOUHADJA1,
Claudia De TAPIA1, Helne ROUILLARD2, Jordane STOLTZ2, Sophie
BANAS3, Bernard GASSER2, Jean-Phillipe MAZZUCOTELLI4, Luc
MAROTEAUX3, Laurent MONASSIER1
1

Laboratory of Neurobiology and Cardiovascular Pharmacology (Faculty of

Medicine EA 7296), Strasbourg, France
2
Laboratoire de Pathologie (Centre Hospitalier Emile Muller), Mulhouse,
France
3
Institut du Fer Moulin (Inserm UMR S-839), Paris, France
4
Service de chirurgie cardiaque (Centre Hospitalier de Strasbourg), Strasbourg, France
Valvular heart disease (VHD) is one of the most frequent cardiovascular pathology in industrialized countries. Chronic use of anorexigens,
amphetamine or ergot derivatives targeting the serotonin system has
been associated with VHD.
The rst aim of this study was to characterize the pattern of serotonergic expression in various human VHD. In a second part of the work,
we investigated the contribution of serotonin (5-HT) effectors in a
model of valve degeneration induced by nordexfenuramine the main
metabolite of the anorexigens dexfenuramine and benuorex.
Surprisingly, we found that valve lesions were made by numerous
non-proliferative CD34+ endothelial progenitors both in humans and
mice VHD. Chronically activated 5-HT2B receptors by nordexfenuramine
in mice mimicked early steps of mitral valve remodeling attested by increased valve thickness and cell density. Lesions were totally prevented
by blocking 5-HT2B receptors (SB206553 or Htr-/2B mice) and both 5-HT2A
and 5-HT2B receptors (ritanserin or Htr-/2A/2B) but not 5-HT2A receptors
alone (Htr-/2A).
We observed that valve lesion associated endothelial progenitors
originated from bone marrow that shared 5-HT2B receptor expression
and were mobilized by serotonergic 5-HT2B receptor stimulation, revealing crucial contribution of bone marrow derived endothelial progenitor cells in valve tissue homeostasis and remodelling.

TU-038
Chronic inammation inhibits myobroblast activation through
macrophage Ccl12 secretion
Kristine DeLeon-Pennell1, Rugmani Padmanabhan Iyer1, Courtney
Cates1, Elizabeth Flynn1, Yonggang Ma1, Presley Cannon1, De'Aries
Shannon1, Michael Garrett2, William Buchanan3, Merry Lindsey1,4
1

Mississippi Center for Heart Research, Department of Physiology and Biophysics, University of Mississippi Medical Center, Jackson, USA

S12

Abstracts

2
Department of Pharmacology, University of Mississippi Medical Center,
Jackson, USA
3
Department of Periodontics and Preventative Science, University of Mississippi Medical Center, Jackson, USA
4
Research Service, G.V. (Sonny) Montgomery Veterans Affairs Medical Center, Jackson, USA

Background: Chronic inammation is a risk factor for adverse remodeling post-myocardial infarction (MI). Cross-talk between the inammatory and brotic response is needed for inammation resolution and
stable scar formation, and the macrophage is a prime intermediary cell.
Previously, we showed chronic lipopolysaccharide (LPS) accelerated macrophage inltration at day 1, resulting in increased cardiac rupture postMI. We hypothesized that chronic inammation would exacerbate macrophage secretion of pro-inammatory cytokines to subsequently decrease activation of the reparative broblast.
Methods: We infused C57BL/6J mice (5 months old; n 6/sex/
group) with subseptic levels of LPS (0.8 ug/g/day) for 28 days to simulate chronic inammation. Coronary artery ligation was performed
and macrophage phenotype, broblast activation and proliferation,
and extracellular matrix (ECM) deposition were evaluated at day 7
post-MI. Stimulation of resident cardiac broblasts with macrophage
conditioned media, with and without Ccl12 blocking antibody, was performed to dissect signaling mechanisms of action.
Results: Macrophage associated pro-inammatory cytokine genes
were elevated in the infarct tissue of the LPS mice at day 7 post-MI,
with Ccl12 demonstrating the largest expression change (pb0.05). By
immunouorescence, markers of reparative broblast activation ( smooth muscle actin and F-actin) were decreased in day 7 post-MI cardiac broblasts from LPS exposed mice compared to controls (p0.05).
By in vivo BrdU labeling, post-MI broblasts isolated from LPS exposed
mice were 3-fold more proliferative than non-exposed broblasts
(p0.05). Collagen III, bronectin, and lysyl oxidase were at least 2-fold
lower in the infarcts of LPS mice at day 7 post-MI (all p0.05). Stimulation of resident cardiac broblasts with macrophage conditioned
media from LPS mice decreased ECM expression, differentiation, and increased proliferation compared to controls; selective Ccl12 inhibition
reversed the secretome effect (p0.05).
Conclusion: Our study revealed for the rst time that chronic inammation increases Ccl12 production in macrophages to stimulate broblast dysfunction and adverse cardiac wound healing.

TU-039
HnRNPA1 regulates neointima formation through modulating
vascular smooth muscle cell functions
Qishan Chen1, Yuan Huang1, Guanmei Wen2, Mei Yang1, Bing Dai1, Le
Luong2, Jianhua Zhu1, Qingzhong Xiao2, Li Zhang1
1
First Afliated Hospital of Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
2
Queen Mary, University of London, London, UK

Background: Our previous study reported that hnRNPA1 regulates

vascular smooth muscle cell (VSMC) differentiation from stem cells
in vitro and in vivo. However, little is known about the functional involvements of hnRNPA1 in VSMC functions and neointima formation.
In the current study, we aimed to investigate the functional roles of
hnRNPA1 in the contexts of VSMC functions, injury-induced intimal hyperplasia, and human neointima lesions.
Methods and results: Primary mouse aorta VSMCs were isolated and
showed that hnRNPA1 expressions were consistently regulated in VSMCs
upon various pathological stimuli. hnRNPA1 over-expression in VSMCs
signicantly reduced VSMC proliferation and migration, while decreased
hnRNPA1 promoted VSMC proliferation and migration, respectively.
Moreover, hnRNPA1 exerted its effects on VSMCs via regulating IQ motif

containing GTPase activating protein 1 (IQGAP1), a well-reported

VSMC function modulator. Our data suggested that IQGAP1 expression
was negatively regulated by hnRNPA1 through two ways. Firstly,
hnRNPA1 protein directly bound to IQGAP1 mRNA AU-rich elements
and consequently decreased IQGAP1 mRNA stability. Secondly,
hnRNPA1 was involved in miR-124 biogenesis and up-regulated miR124 expression which then post-transcriptionally reduced IQGAP1
mRNA and protein expression levels. Furthermore, hnRNPA1 expression
was dramatically down-regulated during wire-injury induced neointima
formation. In accordance, peri-vascular ectopic over-expression of
hnRNPA1 markedly inhibited VSMC proliferation and attenuated wireinjury induced neointimal hyperplasia. Importantly, decreased expression
of hnRNPA1 was observed in human atherosclerotic neointima lesion.
Conclusions Our data have demonstrated that hnRNPA1 is a critical
regulator in VSMC functions and neointima formation, suggesting its
potential therapeutic application for vascular diseases.

TU-040
Bach1 represses Wnt/-catenin signaling and angiogenesis
Dan Meng1, Li Jiang1, Xiangxiang Wei1, Junxu Liu1, Cong Niu1, Xie Xu1,
Jianyi Zhang2, Sifeng Chen0
1

Fudan University, Shanghai, China

University of Minnesota Medical School, Minneapolis, USA

Background: Wnt/-catenin signaling has an important role in the

angiogenic activity of endothelial cells (ECs). Bach1 is a transcription
factor and is expressed in ECs, but whether Bach1 regulates angiogenesis is unknown. Objective: This study evaluated the role of Bach1 in angiogenesis and Wnt/-catenin signaling.
Methods and results: Hind-limb ischemia (HLI) was surgically induced in Bach1/ mice and their WT littermates and in C57BL/6J mice
treated with adenoviruses coding for Bach1 or GFP. Lack of Bach1 expression was associated with signicant increases in perfusion and vascular
density and in the expression of pro-angiogenic cytokines in the ischemic
hindlimb of mice, with enhancement of the angiogenic activity of ECs
(e.g., tube formation, migration, and proliferation). Bach1 overexpression
impaired angiogenesis in mice with HLI, and inhibited Wnt3a-stimulated
angiogenic response and the expression of Wnt/-catenin target genes,
such as interleukin 8 (IL-8) and VEGF, in human umbilical vein endothelial cells (HUVECs). IL-8 and VEGF were responsible for the antiangiogenic response of Bach1. Immunoprecipitation and GST pull-down
assessments indicated that Bach1 binds directly to TCF4 and reduces the
interaction of -catenin with TCF4. Bach1 overexpression reduces the interaction between p300/CBP and -catenin, as well as -catenin acetylation, and chromatin immunoprecipitation experiments conrmed that
Bach1 occupies the TCF4-binding site of the IL-8 promoter and recruits
histone deacetylase 1 (HDAC1) to the IL-8 promoter in HUVECs.
Conclusion: Bach1 suppresses angiogenesis after ischemic injury and
impairs Wnt/-catenin signaling by disrupting the interaction between
-catenin and TCF4 and by recruiting HDAC1 to the promoter of TCF4targeted genes.

TU-041
Kruppel-like Factor 2 Mediates the Suppressive Effect of Statin on
BMP4-Smad Signaling
Jiang-Yun Luo, hongsong Zhang, lingshan Gou, Chi Wai Lau, Yu Huang
The Chinese University of Hong Kong, Hong Kong, China
Rationale: Bone morphogenic protein 4 (BMP4) is a proinammatory and oxidative protein in vascular endothelial cells (ECs).
Statins, the HMG-CoA reductase inhibitors, exert anti-inammatory
and anti-oxidant effects by upregulation of Kruppel-like factor 2

Abstracts

(KLF2) in ECs. Whether and how statins modulate BMP4 signaling in

ECs is largely unknown.
Objective: We aim to investigate the effects of statins on BMP4triggered signaling and function in ECs. Moreover, we also study the
role of KLF2 induced by statins in modulation of BMP4-Smad signaling.
Results: Ex vivo treatment of mouse aortic rings with statins restored
BMP4-induced impairment of endothelium-dependent relaxations (EDR)
and this benecial effect was abolished by Ad-KLF2-shRNA transduction.
Oscillatory shear stress (OSS) induced BMP4-Smad activation was also attenuated by statin treatment and is dependent on KLF2 levels. Statin
treatment of human umbilical vein endothelial cells (HUVECs) for 24
hours suppressed the expression of BMP4 and Smad1 at both mRNA
and protein levels, which was abolished in KLF2-silenced HUVECs. Statins
inhibited BMP4-induced expression of pro-inammtory genes such as
ICAM-1 and COX-2, phosphorylation of Smad1/5 and Smad1/5mediated gene transcription, which are also abrogated in HUVECs with
KLF2 knockdown. KLF2 overexpression by Ad-KLF2 in HUVECs showed
that KLF2 directly suppresses the expression of BMP4 and Smad1 and
BMP4-initiated Smad phosphorylation, indicating the negative regulatory
effect of BMP4-Smad signaling by KLF2. Moreover, luciferase assay
showed that KLF2 inhibited the promoter activity of BMP4 and Smad1.
Conclusion: Statins have suppressive effects on BMP4-Smad signaling through upregulation of KLF2, which negatively regulates BMP4 and
Smad1 expression at transcription levels. (This study is supported RGC
CRF)

TU-042
A role for antioxidants in reversing Tiotropium induced
cardiotoxicity
Shabana Cassambai, Sadie Dean, Christopher J Mee, Katherine L Harvey,
Afthab Hussain
Coventry University, Coventry, West Midlands, UK
Tiotropium bromide is a long-acting muscarinic receptor antagonist
(LAMA) used in the treatment of chronic obstructive pulmonary disease
(COPD); a progressive inammatory condition of the airways. LAMAs
target muscarinic receptors to result in dilation of airway smooth muscle. Recently, clinical studies have correlated the use of anti-muscarinics
with cardiovascular events, including stroke and myocardial infarction.
Cardiac damage is often associated with reactive oxygen species (ROS)
production and calcium overload. However, ROS also function as second
messengers and are known to result in the activation of Akt. Although
Akt is associated with promoting cell survival, constitutive activation
of Akt can itself result in cell death.
The aim of this study was to assess the cardiotoxicity and associated intracellular mechanisms of Tiotropium using a whole heart
model. Langendorff hearts were subjected to a stabilisation period
(20 minutes), followed by reperfusion (155 minutes) Tiotropium
bromide (10nM-0.1nM) and the anti-oxidant, Resveratrol (10M)
alone or combined with Tiotropium (1nM). Following reperfusion,
hearts underwent triphenyl-tetrazolium chloride staining to assess
infarct/risk ratio (%) or were snap-frozen for western blot analysis
of p-Akt (Ser473) expression.
Tiotropium (10nM-0.1nM) administration during reperfusion, significantly increased infarct/risk ratio (%) compared with normoxic controls
in a concentration dependent manner. Administration of Resveratrol
(10M) showed no signicant difference with respect to controls
(12.281.5% vs. 10.271.94%), however co-administration of Resveratrol with Tiotropium (1nM) attenuated infarct development
(11.991.71% vs. 18.691.79%, pb0.0002, n=3/4). Western blot analysis showed signicant increase in p-Akt (Ser473) expression in
Tiotropium treated groups compared to time-matched control
(79.1020.04% vs. 26.862.70%, pb0.01 at 1nM), which was abrogated
by Resveratrol administration 79.1020.04% vs. 32.051.62%, pb0.05).

S13

This is the rst pre-clinical study to suggest that Tiotropium increases infarct/risk ratio in a whole heart model, which may account
for adverse cardiac side-effects seen clinically. This also proposes a
role for Resveratrol in reducing Tiotropium mediated cardiotoxicity.

TU-043
MicroRNA-26a Inhibits Vascular Smooth Muscle Cell Proliferation
and Neointimal Hyperplasia by Targeting MAPK6
Tan Juanjuan1, Yang Liguo2, Liu Cuicui3,4, Yan Zhiqiang3,4
1

School of Life Sciences and Biotechnology,Shanghai Jiao Tong university,

Shanghai, China
2
Southern Medical University, Guangzhou, China
3
Fengxian Hospital Afliated to Southern Medical University, Shanghai,
China
4
Sixth People's Hospital South Campus Afliated to Shanghai Jiao Tong University, Shanghai, China
RationaleSaphenous vein graft disease is a timely problem in coronary artery bypass grafting. Long term patency of vein grafts is limited
due to neointimal formation caused by vascular smooth muscle cell
(VSMC) migration and proliferation in the intima. Therefore,identifying
novel strategies to prevent neointimal thickening is important. Understanding the role of microRNA provides a opportunity to identify both
functional drivers of VSMC proliferation and possible therapies of vascular pathology.
ObjectiveBecause microRNA-26a (miR-26a) is involved in regulation of functions of various cells, we investigated the effect of miR-26a
on the proliferation and migration of VSMC and the development of
neointimal hyperplasia after autogenous vein graft.
Methods and Results Using quantitative reverse-transcription polymerase chain reaction, we identied that miR-26a was one of the
most signicantly down-regulated microRNAs in jugular veins which
were interposed in the rat carotid artery. miR-26a was also markedly
down-regulated in VSMCs from rat jugular vein stimulated with
Platelet-derived growth factor-BB (PDGF-BB). Overexpression of miR26a inhibited VSMC cell proliferation and migration. MAPK6 was predicted as one of the top targets of miR-26a by using several computational
miRNA target prediction tools, and was negatively regulated by miR26a in VSMCs. Luciferase assay showed miR-26a substantially repressed
wild type MAPK6-3'-UTR-luciferase activity in VSMCs, but not mutant
MAPK6-3'-UTR-luciferease reporter. Furthermore, Knocking-down of
MAPK6 reduced cell proliferation and migration, whereas overexpression of MAPK6 enhanced VSMCs proliferation and migration, which
consisted in the activation of Akt and Erk. Data from co-transfection experiments also revealed that miR-26a inhibited VSMC proliferation and
migration through modulating MAPK6 gene expression levels.
Conclusions.These results have demonstrated that miR-26a is an
important regulator in VSMC functions and neointima hyperplasia, suggesting its potential therapeutic application for saphenous vein graft
disease (This projector found by NSFC 11172176).

TU-044
New insights into adrenergic regulation of cardiac remodelling
Youyi Zhang
Institue of Vascular Medicine,Peking University Third Hospital, Beijing, China
Beijing Key Laboratory of Cardiovascular Receptors Research, Beijing, China
Key Laboratory of Cardiovascular Molecular Biology and Regulatory
Peptides, Ministry of Health, China
Background: Heart failure is characterized by enhanced sympathetic
nervous activity and subsequent activation of adrenergic receptors (ARs)
through release of stress hormones (catecholamine). Thus, these exist

S14

Abstracts

unwanted signalling via ARs activation leading to cardiac remodelling and

dysfunction, constituting causal mechanism in heart failure initiation and
progression. Whilst the responsible AR signal mechanisms remain undened. The conventional view of GPCR signalling holds that the receptor
couples with its G protein only at the cell membrane. Recent studies, including those from our group, have shown that several events are thought
to terminate this signalling pathway.
Methods: Western blot, real time PCR, single molecule image, mice
models were used.
Results: (1) Binding of the -arrestins to the receptor are involved in 2-AR-mediated p38 MAPK earlier activation. (2) Internalization of the receptor through the process of endocytosis is
associated in 1A-AR induced ERK activation, which is independent
of Gq/PLC/PKC signalling. (3) 1-AR induces STAT3 activation mainly through transactivation of Epidermal Growth Factor Receptor
(EGFR) in neonatal rat cardiomyocytes, which plays an essential
role in 1-AR-induced cardiac hypertrophy.
Conclusions: The new insights of adrenergic receptor signalling
is one receptor and four signalling pathways. The signalling pathways and core molecules can be claried in the cardiac remodelling
induced by adrenergic activation, providing clues and theory basis
for develop more specic treating target for cardiac remodelling.

TU-045
ER Stress mediates cardiac ion channel changes in heart failure
Man Liu, Guangbin Shi, Anyu Zhou, Samuel C. Dudley
Rhode Islan Hospital and Brown University, Providence, RI, USA
Introduction: Heart failure (HF) is associated with endoplasmic reticulum (ER) stress and activation of the unfolded protein response
(UPR). UPR inhibits protein translation. Ion channel downregulation is
associated with arrhythmic risk. We hypothesized that UPR could be
contributing to electrical remodelling in HF.
Methods: Hypertensive HF was induced in C57BL/6 mice by unilateral nephrectomy, deoxycorticosterone acetate pellet implantation, and
salt water substitution. Sham operated mice were used as controls. After
6-7 weeks, isolated ventricular myocytes were utilized for whole-cell
patch clamp recording, and heart tissue was used for mRNA measurements. GSK2606414, a specic inhibitor of protein kinase R like ER kinase (PERK), was applied to myocytes at 4 nM for 20 h (30 min
pretreatment when coapplied with tunicamycin, 10 g/ml, 20 h).
Results: HF myocytes showed classical electrical remodelling with
action potential duration prolongation (APD90: 203 26 vs.
108 16 ms of sham, P b 0.05) and DADs. PERK activation in HF
myocytes was indicated by elevated mRNA and/or protein levels of
Grp78, phospho-PERK, phospho-eIF2, ATF4, and CHOP. Peak INa and
three types of K+ currents (Ito, IK1, and IKslow) were decreased signicantly in HF group, while L-type Ca2+ current and some other types of
K+ currents were not affected. These changes were similar to those observed in myocytes treated with the classic UPR inducer, tunicamycin.
An inhibitor of the PERK branch of the UPR, GSK2606414, restored INa
and IK,slow, and shortened the APD of the DOCA myocytes.
Conclusions: UPR appears to be responsible for reductions of INa, Ito,
IK1 and IK,slow in heart failure. Nav1.5 and Kv1.5 were downregulated by
the PERK branch of UPR. Inhibiting the UPR may be a novel antiarrhythmic strategy.

TU-046
The neuro-cardiac interaction denes an extracellular microdomain
required for neurotrophic signaling
Mauro Franzoso1,2, Tania Zaglia1,2, Nicola Pianca1,2, Libero Vitiello3,
Marco Mongillo1,2

Venetian Institute of Molecular Medicine, Padova, Italy

Department of Biomedical Sciences, University of Padova, Padova, Italy
3
Department of Biology, University of Padova, Padova, Italy
2

Purpose: Cardiac activity is tuned by sympathetic neurons (SNs),

whose survival depends on limiting amounts of neurotrophins released
by the myocardium. This study aims i) to determine whether specic cellular structures are present at the SN/cardiomyocyte (CM) contact site, ii)
to investigate the role of SN/CM interaction in NGF-mediated signaling.
Methods and results: Electron microscopy and immunouorescence on mouse heart slices and rat SN/CM co-cultures showed close association between SNs and CMs and enrichment of the NGF receptor
(TrkA) at the contact site. These data support that specialized and locally organized signaling domains exist (neuro-cardiac junction, NCJ).
We tested the functional role of the NCJ in sustaining neuronal survival. Silencing of NGF expression by CMs in co-cultures led to 66% decrease of neuronal density, supporting that SN viability depends on
NGF released by CMs. SNs cultured on NGF-silenced CMs showed 20%
decrease in the NCJ area when compared to those on wild type CMs of
the same culture. Moreover, NGF uptake was observed only in processes
contacting NGF-overexpressing CMs, supporting that the NCJ is central
to neurotrophin-mediated signaling. Consistently, cultured SNs in contact with CMs survived NGF withdrawal, whereas neurons alone treated
with CM-conditioned medium did not survive because of the very low
NGF concentration (1.61 pg/mL). Conversely, NGF concentration at the
contact site was estimated by using the TrkA inhibitor K252a and resulted about 1000-fold higher (1.75 ng/mL), supporting that the NCJ allows
amplication of intercellular NGF signaling.
Dystrophin accumulation on CM membrane contacted by SNs was
observed in mouse cardiac slices. Consistently, hearts from mdx mice
showed 74.36% decrease of innervation, with no signicant changes of
NGF expression, supporting that ablation of dystrophin impairs cardiac
SNs.
Conclusions: Taken together, our results suggest that NGFdependent signaling to the neurons requires a direct and specialized interaction with myocytes.

TU-047
Chronic lead exposure impairs vascular reactivity through oxidative
stress dependent mechanism: MAPKs pathway activation
Maylla Simes1, Bruna Azevedo1, Jonaina Fiorim1, Cindy Toscano1,
Mercedes Salaices3, Dalton Vassallo1,2
1

Universidade Federal do Esprito Santo, Vitria, ES, Brazil

EMESCAM-Escola Superior de Cincias da Santa Casa de Misericrdia de
Vitria, Vitria, ES, Brazil
3
Universidad Autnoma Madrid, Madrid, Spain
2

Introduction: Chronic exposure to lead (Pb) alter cardiovascular parameters. Aim: To investigate chronic exposure to Pb low concentrations in aorta and VSMC function, assessing oxidative stress and
MAPKs pathway.
Methods: Rats were treated with lead for 30 days (1st dose 10mg/
100g, subsequent doses 0.125mg/100g/day im) and controls, salineim. Vascular reactivity to phenylephrine (Phe) was measured in the
presence and absence of endothelium and after 30 min of incubation
with L-NAME and apocynin (APO). Isolated aortas were processed to
obtain primary cultures of VCMS. Pb (20 g/dL) was used to stimulate
the cells for 48h.
Statistical analysis: mean SEM; One way ANOVA or Student t-test.
p b 0,05. Ethics Committee (UFES-063/2011) and (UAM-CEI-22-488).
Results: The treatment produced blood lead concentration of
21,7 g/dL and increased vascular reactivity to Phe. Removal of the endothelium and incubation with L-NAME increased reactivity with lower proportion compared to the Pb group. APO reduced vascular reactivity to Phe

Abstracts

in both groups, but with greater magnitude in Pb group. Pb increased

gp91phox, Cu/Zn-SOD and Mn-SOD protein expression. In VSMC,
NADPH oxidase activity and superoxide anion production were also enhanced by Pb and normalized by Celecoxib, Rofecoxib, ML171, Tempol
and Mito-TEMPO. Pb augmented NOX1, NOX4, Mn-SOD and EC-SOD expression. Celecoxib reversed the upregulation of NOX1 and NOX4. Pb also
induced the activation of ERK1/2 and p38MAPK signaling pathways that
augmented NOX1 and NOX4 expression but did not induce proliferation
or migration of VCMS.
Conclusion: Our ndings suggest that treatment with low doses of
lead, below the reference values, increased BP, promoted vascular dysfunction and activate the MAPKs signaling pathways, which are associated with the NADPH oxidase activation. Since lead is reported to be
involved in hypertension development, its exposure should be considered
an environmental risk factor for cardiovascular disease.

TU-048
mTORC1 and mTORC2 preserve cardiac function by regulating metabolism and contractility
Lifen Xu1, Pankaj Shende1, Christian Morandi1, Thierry Pedrazzini2,
Laura Pentassuglia1, Sonia Lebboukh1, Michael Hall1, Markus A.
Regg1, Marijke Brink1
1

University of Basel, Basel, Switzerland

University of Lausanne Medical School, Lausanne, Switzerland

The mammalian target of rapamycin (mTOR), an evolutionary conserved serine/threonine kinase of the phosphatidylinositol-3-kinaserelated kinase family, integrates intracellular and environmental cues
such as amino acid availability, growth factors, energy status and stress.
In response to these stimuli, mTOR regulates metabolic mechanisms including protein turnover, nucleotide synthesis and lipid synthesis, to ultimately control cellular growth. To exert its best-characterized function
of protein synthesis, mTOR must be assembled in the multiprotein complex mTORC1. We have previously shown that cardiomyocyte-specic
deletion of raptor, an essential and specic component of mTORC1,
leads to cardiac dysfunction and death under basal conditions and that
functional deterioration is accelerated in pressure-overloaded hearts.
The dysfunction was related not only to reduced protein synthesis and
the consequent lack of adaptive hypertrophy, but also to reduced mitochondrial content and a change in energy substrate use.
In contrast, cardiac rictor-decient mice (rictor encodes an essential
and specic component of mTORC2) had no phenotype during growth
or adulthood under basal laboratory conditions up to 54 wks of age. However, aortic constriction-induced pressure overload signicantly increased
rictor protein levels along with PKCII and PKC phoshorylation in control
mice, but not in the cardiac rictor knockout mice. Pressure overload resulted in hypertrophy with maintained ventricular function in controls,
but led to systolic dysfunction in the rictor-decient hearts, without having any effects on cardiac weight, hypertrophy markers, or brosis. Our
data suggest that mTORC2 regulates metabolism and contractility of the
heart via PKCII and PKC. As several compounds inhibiting both mTOR
complexes are in clinical trials for the treatment of cancer, special attention should be paid in these studies to patients with concurrent cardiovascular disease such as hypertension or valve disease. On the other hand,
our novel insights into cardiac mTORC2 signaling may also open up new
avenues for the treatment of cardiac disease.

TU-050
Uncovering novel signaling components for DCM development - a
phospho-proteomics approach
Stephan Lange1, Lauren Waller1, Nancy Dalton1, Erika Alvarez1, Kirk
Peterson1, Ju Chen1, Elisabeth Ehler2, Majid Ghassemian1

S15

UC San Diego, La Jolla, CA, USA

King's College London, London, UK

Background: The muscle-lim protein (MLP/CSRP3) knockout

mouse has long been used as a model to investigate dilated cardiomyopathy (DCM) disease mechanisms. Known pathway components that
are involved in the development of the disease include PKCalpha, phospholamban, and proteins associated with beta-adrenergic signaling.
In addition, we identied CARP1/Ankrd1 as essential for DCM development. Hearts of double knockout mice do not develop the disease,
and present with normal cardiac morphology and physiology. As a molecular mechanism we showed that CARP1 is required for the pathological activity of PKCalpha in MLP knockout hearts.
Methods We probed for novel pathway components implicated in the
development of DCM by using cardiac extracts of MLP knockouts, and
CARP1-MLP double-knockouts. Because of the role that posttranslational
modications by PKCalpha play in DCM pathology, we utilized stable isotope dimethyl-labeling in a differential phospho-proteomics approach to
identify candidate proteins with disease associated changes to their phosphorylation pattern. Biochemical and cell-biological assays were used to
further characterize their involvement in DCM development.
Results We identied 13000 differentially labeled phospho-peptides
from approx. 600 proteins that are representative of every cell compartment. One of the proteins identied is AHNAK-1, which is known to take
part in cardiac signaling pathways, including PKC, and in addition is
thought to regulate cardiac calcium channel activity. Along the AHNAK1 polypeptide chain, we identied several novel phosphorylation sites
that are differentially phosphorylated between diseased MLP hearts and
healthy CARP1-MLP double knockout hearts. Intriguingly, a C-terminal
mutation in AHNAK-1 known to cause cardiomyopathy in humans affects
one of these novel phosphorylation sites in in-vitro kinase assays.
Conclusions Further analysis of AHNAK-1, and other identied candidate proteins will give better insights into DCM mechanisms, and may
reveal novel targets for a treatment of this disease.

TU-051
1-Adrenergic stimulation induces HDAC5 nuclear accumulation by
B55-PP2A-mediated dephosphorylation
Kate Weeks, Antonella Ranieri, Chris Molenaar, Metin Avkiran
King's College London, London, UK
When localized to the nucleus, histone deacetylase 5 (HDAC5) prevents cardiomyocyte hypertrophy by repressing MEF2 transcription factors. Stimulation of Gq protein-coupled receptors induces HDAC5
nuclear export via its phosphorylation at S259/S498. In contrast, stimulation of -adrenergic receptors has been proposed to induce both
phosphorylation-independent nuclear export and protein kinase A
(PKA)-dependent nuclear accumulation through S279 phosphorylation.
We aimed to: (1) denitively determine the impact of -adrenergic signaling on the phosphorylation, localization and function of HDAC5 in
adult rat ventricular myocytes (ARVM); (2) delineate the relative importance of altered phosphorylation at S259/S498 and S279 in regulating
HDAC5 localization in this cell type. Towards these aims, we established
a new confocal microscopy method to objectively quantify the wholecell nuclear/cytoplasmic distribution of GFP-tagged HDAC5 in living
ARVM. Isoprenaline (ISO; 10 nM) induced HDAC5 dephosphorylation at
all three sites and HDAC5 nuclear accumulation, which was blocked by
PKA inhibition. Mutation of S259/S498 to non-phosphorylatable alanine
promoted nuclear accumulation and MEF2 inhibition, whereas ablation
of the S279 phosphorylation site had no effect on these parameters and
did not block ISO-induced nuclear accumulation. HDAC5 dephosphorylation was sensitive to PP2A inhibition with okadaic acid. Furthermore, coimmunoprecipitation experiments revealed a specic interaction of
HDAC5 with the PP2A regulatory/targeting subunit isoform B55, as

S16

Abstracts

well as PP2A catalytic and scaffolding subunits, and these interactions increased N 3-fold with ISO stimulation. We conclude that -adrenergic
stimulation induces HDAC5 nuclear accumulation by a mechanism that
is PKA-dependent but requires B55-PP2A-mediated dephosphorylation
of S259/S498 rather than PKA-mediated phosphorylation of S279.

TU-052
Identication of a high afnity, high efcacy adenosine A2B receptor
agonist with potent anti-brotic activity
Elizabeth Vecchio1, Chung Chuo1,2, Peter Scammells1, Arthur
Christopoulos1, Bing Wang2, Henry Krum2, Paul White1, Lauren May1
1

Monash Institute of Pharmacy and Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia
2
Centre of Cardiovascular Research and Education in Therapeutics, School
of Public Health and Preventive Medicine, Monash University, Melbourne,
Victoria, Australia

Background. The adenosine A2B receptor (A2BAR) has been therapeutically implicated in the heart with key roles in ischemiareperfusion injury, inammation and brosis. However to date, effective
modulation of A2BAR signalling has been limited by a lack of potent agonists. Recent screening of an adenosine receptor bitopic agonist,
VCP746, revealed signicant and previously unappreciated A2BAR activity. VCP746 is a hybrid ligand comprised of an orthosteric agonist moiety (VCP900) and an adenosine A1 receptor (A1AR) allosteric
modulator moiety (VCP171). This study aimed to rigorously characterise the binding and function of VCP746 at the A2BAR and examine its
anti-brotic activity in cardiac broblasts.
Methods. The afnity and efcacy of VCP746 was examined in
FlpInCHO cells stably expressing the human A2BAR. Agonist concentration response curves were generated across multiple functional pathways and compared to conventional A2BAR agonists NECA and BAY606583. The ability of VCP746 to inhibit TGF- or angiotensin II- mediated
collagen synthesis was measured by [3H]-proline incorporation in isolated neonatal rat cardiac broblasts (NCFs).
Results. VCP746 had a signicantly higher afnity and potency than
NECA or BAY60-6583 at the A2BAR. Functional assays demonstrated
VCP746 stimulated robust increases in cAMP accumulation, ERK1/2
phosphorylation, IP1 accumulation and Ca2+. In primary NCFs, VCP746
stimulated potent inhibition of both TGF- and angiotensin II- mediated
collagen synthesis in NCFs (pIC50 7.6 0.4 and 7.8 0.4, respectively;
n=4-6). The inuence of VCP746 on collagen synthesis was selectively
reversed in the presence of an A2BAR antagonist, demonstrating that
these effects were mediated through A2BARs endogenously expressed
in NCFs.
Discussion. VCP746 was found to be the highest afnity and highest
efcacy A2BAR agonist identied to date. Furthermore, VCP746
displayed potent anti-brotic effects in NCFs, thus we believe that
VCP746 will provide a novel tool to further investigate the role of the
A2BAR in cardiac (patho)physiology.

TU-053
Catestatin modulates adrenergic signaling and reverses the hypertrophic effects of norepinephrine in H9c2 cardiac myoblasts
Md. Jahangir Alam1, Nitish R Mahapatra2, Shyamal K Goswami1
1
School of Life Sciences, Jawaharlal Nehru University, New Delhi, Delhi,
India
2
Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, Indian Institute of Technology, Chennai, Madras, India

Background: Upon treatment with 2 and 100M Norepinephrine (NE), H9c2 cardiac myoblasts elicit hypertrophic and

apoptotic responses respectively. The two respective pathways

are distinguished by the induction of distinctive redox-kinase signaling pathways. In mammalian heart, NE is co-released with
Catestatin (CST), a catecholamine release inhibitory peptide derived from chromogranin A. CST plays an important role in the
regulation of cardiovascular functions and associated diseases including hypertension, cardiomyopathy, myocardial infarction and
heart failure; but the mechanisms of its actions are not known.
Here, we aim to explore its mechanism of action; its downstream
signaling pathway and target genes involved in the induction of
cardiac hypertrophy.
Results and conclusion: We demonstrate that CST reverses the
induction of fetal genes in H9c2 cardiac myoblasts by norepinephrine (NE). CST attenuates the ROS generated by NE treatment as evidenced by redox sensitive uorescent probes DCFH-DA, HPF, DHE
and Amprex red. Luciferase and gel shift assay shows that it modulates the redox responsive transcription factors AP-1 and Nrf2, either alone or in presence of NE. Expression of fosB and AP-1
promoter reporter constructs is also modulated by CST alone or in
association with NE, though it has preference for the - rather
than -AR signaling. However, it does not prevent apoptosis induced by a higher dose of NE. Effects of CST on reporter gene expression suggest that it acts through multiple signaling pathways. Taken
together, this study suggests that CST modulates the adrenergic signaling by suppressing RO/NS generation and differentially modulating activities of AP1, FosB, Fra1 and Nrf2.

TU-054
Protective effect of Aronia melanocarpa on cardiovascular system in
L-NAME-induced hypertension
Martina Cebova, Jana Klimentova, Andrej Barta, Zuzana Matuskova,
Radoslava Rehakova, Michaela Kosutova, Olga Pechanova
Institute of Normal and Pathological Physiology Slovak Academy of Sciences, Bratislava, Slovakia
Background: Polyphenols are a class of natural products exhibiting
multiple health benets beyond their antioxidant potential. Aronia
melanocarpa (black chokeberry) has attracted scientic interest due
to its dense contents of polyphenols, especially anthocyanins. The aim
of the present study was to analyze effects of non-alcoholic concentrate
from aronia melanocarpa (AM) on blood pressure (BP), total NOS activity and cytokine level in the left ventricle of L-NAME-induced hypertensive rats.
Methods: 12-week-old male WKY rats were assigned to control
group, group treated with L-NAME (40mg/kg/day), group treated
with AM concentrate (1ml/kg/day), and group treated with combination of L-NAME (40mg/kg/day) and AM concentrate (1ml/kg/
day) in tap water. The experiment lasted 3 weeks. BP was measured by the tail-cuff-plethysmography. NOS activity was determined by conversion of 3[H] Arginine to 3[H] Citrulline in the left
ventricle (LV). Cytokine levels were investigated using the BioPlex Pro Cytokine kit in the plasma.
Results: After 3 weeks of L-NAME treatment BP was increased by
28% than the control group. AM reduced BP by 21% in L-NAME + AM
group in comparison to L-NAME group. Moreover, AM inhibited TNF and IL-6 production in the plasma in L-NAME + AM group in comparison to L-NAME group. NOS activity of LV in L-NAME group was decreased by 40%, on the other hand AM was able to increase NOS
activity on 90% of control level.
Conclusion: The results of our study show that active substances from Aronia melanocarpa may have positive effect on
blood pressure, cytokine level and NOS activity in L-NAME induced hypertension.
Supported by VEGA 2/0165/15, 2/0144/14, APVV-14-0932.

Abstracts

TU-055
Role of NADPH Oxidase-2 under adrenergic stimulation in
cardiomyocytes
Nikhat Saleem, Shyamal K Goswami
School of Life Sciences, Jawaharlal Nehru University, New Delhi, Delhi, India
Background: Reactive oxygen species is involved in the pathogenesis of cardiovascular diseases, including atherosclerosis, hypertension,
cardiac hypertrophy and heart failure. Recent studies have emphasized
on the role of NADPH oxidases (NOXs) in cardiac hypertrophy induced
by pressure overload, angiotensin II and phenylephrine. However, the
role of specic NOX isoforms, site of ROS generation and underlying
mechanism under adrenergic stimulation induced cardiac hypertrophy
has not been explored.
Aim: In this study, we aim to investigate the spatial localization of
ROS generation and involvement of specic NOX isoform under adrenergic stress leading to downstream signalling events.
Methods and results: H9c2 cardiac myoblasts were treated with
2M norepinephrine (NE) inducing ROS generation that was inhibited
by NOX2 specic peptide inhibitor gp91ds-tat. Organelle specic hydrogen peroxide-sensitive GFP was used for monitoring ROS generation in
cytosol, mitochondria, and ER of which cytosolic Hyper-GFP was primarily positive. Induction of cardiac hypertrophy marker genes (MHC, ANP) with 2M NE treatment was restored by the NOX2 inhibition as measured by real-time PCR. Enhanced promoter activity of
some of the stress induced transcription factors (AP-1, FosB) was also
attenuated by NOX2 inhibition as estimated by promoter reporter
assay. We hypothesize that under pathological condition, perturbation
of this AR-NOX2 cross-talk cause -AR malfunction. To understand the
role of NADPH oxidase in vivo, we intraperitoneally injected rats with
apocyanin, an inhibitor of NOXs, for two weeks and concomitantly, subcutaneous injection of isoproterenol was given to induce cardiac hypertrophy. Our data suggested partial reversal of cardiac hypertrophy
marker proteins and genes with the inhibition of NOX by apocyanin.

TU-056
Nitro-oleic acid, a component of the mediterranean diet, prevents
MKK3- p38MAPK; dimer formation by steric obstruction of
redox-sensitive cysteines.
Rekha Bassi, Joseph Burgoyne, Gian de Nicola, Olena Rudyk, Vittorio de
Santis, Rebecca Charles, Philip Eaton, Michael Marber
King's College London, London, UK
Abstract:
Background: p38-MAPK (p38), a serine-threonine kinase plays a
pivotal role in a variety of biological processes and is thus activated by
diverse stimuli including oxidant stress. This activation is achieved by
its archetypal upstream kinase, MKK3, phosphorylating two key residues within the activation segment. Our purpose was to determine if
such activation is dependent on redox-sensing cysteines within p38.
Methods and results: Following the exposure of rat cardiomyocytes
or whole hearts to H2O2 (50 M) p38 was activated and formed a heterodimer with MKK3 that was sensitive to reduction by mercaptoethanol.
The abundance of this heterodimer was enhanced by co-administration of
Auranon (2 M) suggesting redox cycling occurs in vivo. We predicted
that Cys119 and/or Cys162, both close to the known MKK3 docking domain, could act as electron donors and form a disulphide bridge with
MKK3. Dimer formation was reduced with p38 Cys119Ser and increased
with p38 Cys162Ser suggesting these residues act as vicinal thiols.
p38Cys119Ser/Cys162Ser was incapable of sensing H2O2. Similarly,
heterodimer formation was abolished in H9C2 cells (rat heart embryonic
myoblast cell line) expressing MKK3 and p38Cys119Ser/Cys162Ser
following simulated ischaemia and reperfusion. p38. The anti-

S17

inammatory agents, 15d PGJ2, a naturally occurring end product of

prostaglandin D2 metabolism and 10-nitro-oleic acid, a component of
the Mediterranean diet, reduced p38 activation and covalently modied Cys119/Cys162, likely obstructing MKK3 access.
Conclusion: Our novel ndings suggest that cysteines within p38
act as redox sensors dynamically regulating p38 activation.

TU-061
Pluripotent stem cell microRNA-294 as a mediator of cardiac proliferative response in the heart after myocardial infarction
Mohsin Khan1, Brandon Booth1, Constantine Troupes2, Emily Nickoloff1,
Sadia Mohsin2, Cynthia Benedict1, Steven Houser2, Walter Koch1,
Raj Kishore1
1

Center for Translational Medicine, Lewis Katz School of Medicine, Temple

University, Philadelphia, PA, USA
2
Cardiovascular Research Institute, Lewis Katz School of Medicine, Temple
University, Philadelphia, PA, USA
Rationale: Embryonic heart is characteristic of rapidly dividing
cardiomyocytes required to build a working myocardium. In contrast,
cardiomyocytes retain some proliferative capacity in the neonates but
lose most of it in adulthood. Embryonic stem cell cycle (ESCC) miRs
are a class of microRNAs regulating the unique cell cycle of ESCs and
their characteristic pluripotency. Nevertheless, expression of miR-294,
a member of the ESCC miRs is lost during developmental transitions
from the ESCs to mature cells. Effect of miR-294 to induce cardiac proliferation and heart function has not been previously studied.
Objective: To determine whether miR-294 drives cardiac proliferative response leading to augmentation of cardiac function after myocardial infarction.
Methods and Results: miR expression analysis in the heart during development revealed elevated levels of miR-294 in the prenatal stages
while the expression was lost in the neonates and adults as conrmed
by qRT-PCR. Neonatal ventricular cardiomyocytes (NRVMs) treated
with miR-294 mimic to showed elevated mRNA levels of cell cycle
markers (E2F family and cyclins) concurrent with increased expression
of p-histone 3, Ki67 and Aurora B kinase (G2/M) as conrmed by immunocytochemistry compared to control cells. Cardiac progenitor cells
(CPCs) engineered with miR-294 lentivirus led to increased proliferation and metabolic activity. AAV-9 carrying miR-294 was administered
in mice subjected to myocardial infarction augmented cardiac function
8 weeks after injury. Increase myocyte proliferation was observed in
the heart after miR-294 treatment as analyzed by BrdU uptake, pHistone 3 and Aurora B expression by immunostaining. Concurrently,
a decrease in infarct size along with decreased apoptosis was observed
in the miR-294 hearts compared to the control. Furthermore, increased
c-kit+ CPCs activation and proliferation was observed in the miR-294
receiving hearts.
Conclusion: Ectopic expression of miR-294 recapitulates embryonic
signaling and enhances cardiomyocyte ability to proliferate together
with CPC activation and expansion leading to augmented cardiac function in mice after myocardial infarction.

TU-062
Proliferation of the cardiac precursor cells expressing the Stem Cell
Antigen-1 is modulated by activation of the Natriuretic Peptide
Receptors.
Stphanie Rignault-Clerc1, Christelle Bielmann1, Lucas Liaudet2, Bernard
Waeber1, Franois Feihl1, Nathalie Rosenblatt-Velin1
1

Departement de Physiopathologie Clinique Centre Hospitalier Universitaire

Vaudois and University of Lausanne, Lausanne, Switzerland
2
Service de Mdecine Intensive Adulte CHUV, Lausanne, Switzerland

S18

Abstracts

Introduction: A part of the cardioprotective role of the Brain Natriuretic Peptide (BNP) in mouse hearts is due to its effect on the cardiac
precursor cell (CPC) proliferation and differentiation. Thus, in this
study we identied the CPC subset able to respond to BNP as well as
the signaling pathway involved.
Methods and results: We demonstrated by immunohistochemistry
and by ow cytometry analysis that the c-kit+ and the Sca-1+ cell subsets
in neonatal and adult murine hearts express the NPR-A and NPR-B receptors and are thus able to be stimulated by BNP. In vitro, BNP only stimulated the proliferation of the Sca-1+ cells and not of the c-kit+ cells. Among
Sca-1+ cells, BNP treatment led to increased number of Sca-1+ Nkx2.5+
cells, which were able to differentiate into cardiomyocytes.
To determine by which receptor BNP acts on Sca-1+ cells to stimulate their proliferation, cells were isolated from neonatal hearts of
mice decient for the NPR-A (NPRA-KO) or NPR-B receptor. BNP stimulated the proliferation of the Sca-1+ NPR-A KO cells but not of the Sca1+ cells lacking the NPR-B receptor, demonstrating that Sca-1+ cell proliferation is linked to NPR-B activation. This was conrmed by stimulating the Sca-1+ cells by the C-Natriuretic Peptide able also to activate the
NPR-B receptor.
BNP binding to NPR-B receptor led in Sca-1+ cells to Protein Kinase G activation and increased phosphorylation of phospholamban
and p38. Reducing PKG activation inhibited BNP-induced-Sca-1+ cell
proliferation, whereas reducing p38 phosphorylation increased Sca1 + cell proliferation after BNP treatment. Phosphorylation of p38
was not mediated by BNP binding to NPR-B receptor but by its binding to NPR-A.
Conclusion: In this work, we identied the Sca-1+ cells as being the
targets of BNP in vitro and in vivo. BNP via NPR-B binding and PKG activation clearly stimulated the proliferation of the CPCs expressing Sca-1.
Interestingly, is the dual role of the NPR-A and NPR-B receptors which
control Sca-1+ cell proliferation.

TU-064
Enzymatic degradation of 7-Ketocholesterol (7-KC), a new strategy
for the treatment of Atherosclerosis.
Irum Perveen
Quaid-i-Azam University, Islamabad, Pakistan
Background
7-ketocholesterol (7KC), an oxidized derivative of cholesterol, has
been implicated in a variety of chronic diseases including atherosclerosis, Alzheimers disease, Parkinsons disease, cancer and age-related
macular degeneration.
It is formed by the autooxidation of cholesterol and especially
cholesterol-fatty acid esters found in lipoprotein deposits, its elevated
concentrations are associated with disruption of cellular homeostasis,
decreased cell viability, and increased cell death.
Enzymatic cleavage of 7-KC can serve as a key solution for the cure of
a number of chronic diseases directly associated with its accumulation.
Methods Isolation of potential 7KC degraders was done from a diverse environmental samples. Molecular identication was done and
HPLC analysis was carried out.
Results Alcanivorax jadensis IP4 (accession number KP309836), isolated from sea water and sediment sample, Streptomyces auratus IP2
(accession number KP309837),Serratiamarcescens IP3 (accession number KP309838) isolated from soil, and ThermobidafuscaIP1 (accession
number KM677184), isolated from manure piles was found to effectively degrade 7-KC. All the isolates were capable of utilizing 7KC as the sole
organic substrate, resulting in its mineralisation.
Further characterization of microbial genes and ultimately the enzymes involved in 7KC catabolism can lead to the development of a single potential therapeutic enzyme preparation to target number of above
mentioned chronic diseases.

TU-065
Restoration of prostaglandin E2 levels in the mesenchymal stem
cells prevents their rejection in the ischemic heart and preserves
ventricular function
Niketa Sareen, Ejlal Abu-El Rub, Glen Lester Sequiera, Meenal Moudgil,
Sanjiv Dhingra
Institute of Cardiovascular Sciences, St. Boniface Hospital Research Centre,
University of Manitoba, Winnipeg, Canada
Introduction: Allogeneic mesenchymal stem cells (MSCs) from
young healthy donors are immunoprivileged. The initial phase I and II
clinical trials with allogeneic MSCs suggested that transplanted cells
were safe and improvement in the heart function was observed. However, the long term fate of the transplanted cells in these trials was not
determined. We recently reported that MSCs lost their
immunoprivilege late after implantation in the ischemic heart and
were rejected, resulting in progressive deterioration of heart function.
The present study reveals the mechanisms responsible for this posttransplantation immune switch in MSCs.
Methods/Results: MSC immunoprivilege was found to be mediated
by prostaglandin E2 (PGE2), the levels of this soluble factor decreased in
rat MSCs after exposure to hypoxia/ischemic conditions which was associated with loss of immunoprivilege. We observed increased cytotoxicity in hypoxic MSCs caused by allogeneic T cells in the in vitro coculture. Furthermore, blocking PGE2 biosynthesis in normoxic MSCs increased the immunogenicity of MSCs. MSCs immunoprivilege is reported to be established by the absence of major histocompatibility complex
class II (MHC-II) molecules. Our data suggests that MHC-II expression
increased in MSCs after exposure to hypoxia. PGE2 treatment of hypoxic
MSCs decreased MHC-II expression and preserved their
immunoprivilege. In a rat myocardial infarction (MI) model, allogeneic
MSCs (3 106 cells/rat), with or without a biodegradable hydrogel
that slowly released PGE2, were injected into the infarct region. Five
weeks later, MSCs were rejected in the control group (no PGE2), but
in the PGE2-treated group, signicant number of the transplanted
cells survived and heart function were signicantly improved.
Conclusions: Immunoprivilege of allogeneic MSCs was maintained
by PGE2 mediated regulation of MHC-II levels, exposure to hypoxia/ischemia decreased PGE2 and increased MHC-II levels that was associated
with loss of immunoprivilege and rejection of MSCs. Maintaining PGE2
levels preserved immunoprivilege and restored cardiac function after
an MI.

TU-066
Upconversion Nanoparticle-mediated Photodynamic Therapy
Induces THP-1 Macrophage Apoptosis and THP-1 Macrophagederived Foam Cell Autophagy via ROS Burst
Liming Yang1, Zhaoyu Zhong1, Xing Zhu1, Jiayuan Kou1, Xuesong Li1, Ye
Tian1,2
1

Department of Pathophysiology, Harbin Medical University, Harbin, Heilongjiang Province, China

2
Division of Cardiology, the First Afliated Hospital, Harbin Medical University, Harbin, Heilongjiang Province, China
Background: AS is a chronic disease characterized by accumulation
of lipid and inltration of inammatory cells, which is the major cause
of acute cardiovascular events. Of several contributing cell types, macrophages play a vital role of atherosclerotic plaque progression. Photodynamic therapy (PDT) has emerged as a useful therapeutic naturopathy
not only in the treatment of cancer but also in the treatment of AS.
Here we investigated the molecular mechanisms based on PDT, using
mesoporous-silica-coated upconversion uorescent nanoparticles encapsulating chlorin e6 (UCNPs-Ce6) in the induction of apoptosis in

Abstracts

THP-1 macrophages and autophagy in THP-1 macrophage-derived

foam cells.
Results: Firstly, the induction of reactive oxygen species (ROS) and
regulation of mitochondrial permeability transition pore (MPTP) to depolarize mitochondrial membrane potential (MMP) were observed in in
THP-1 macrophages via UCNPs-Ce6-mediated PDT. Both Bax translocation and the release of cytochrome C were examined using immunouorescence and Western blotting. Our results indicated that the levels of
ROS were signicantly increased in the PDT group, resulting in both
MPTP opening and MMP depolarization, which led to apoptosis. In addition, immunouorescence and Western blotting revealed that PDT induced both Bax translocation and the release of cytochrome C, as well
as upregulation of cleaved caspase-9, cleaved caspase-3, and cleaved
poly(ADP-ribose) polymerase. Moreover, we found that UCNPs-Ce6mediated PDT could induce autophagy in THP-1 macrophage-derived
foam cells, which showed the LC3-II/LC3-I conversion, increased expression of Beclin 1 and decreased expression of P62, and the formation
of acidic vesicular organelles (AVOs). Assuredly, autophagy was induced by ROS and could be blocked by pretreatment with ROS inhibitor
N-acetyl cysteine (NAC). Furthermore, The UCNPs-Ce6-mediated PDT
induced autophagy was activated through PI3K/AKT/mTOR pathway.
Conclusion: In summary, we demonstrated that ROS as vital intracellular mediators, produce by UCNPs-Ce6-mediated PDT can induce
apoptosis in THP-1 macrophages and autophagy in THP-1
macrophage-derived foam cells.

TU-067
Bone marrow mesenchymal stem cells reprogrammed into cardiac
progenitor cells by nano-protein transfection bio-unit
Lin Jiang1, Xiaohong Li1, Yueheng Wu1, Yuliang Feng1,2, Xi-Yong Yu1,2
1

Medical Research Center of Guangdong General Hospital, Guangzhou

510080, Guangdong, China
2
Guangzhou Medical University, Guangzhou 511436, Guangdong, China

Aim: Bone marrow mesenchymal stem cells (BMSCs) are stem cells
from mesoderm period with potential of self-renewing, multiple differentiation, and are popular in clinical application because of the low immunogenicity. However, most of recent study only focus on their paracrine
function, the ability of directly differentiating into cardiomyocyte has always been controversial, hence there is no doubt that direct BMSCs transplantation will fail to develop its full potential. Cardiac progenitor cells
(CPCs) is a kind of specic stem cells from heart tissue, besides the basic
characteristics of normal stem cells, they can differentiate into three
heart spectrum directly (including cardiomyocyte, endothelial cells and
smooth muscle cells in the heart), acting as a good stemness source.
Therefore, this study will focus on the differentiation efciency of how
protein induced BMSCs reprogramming to generate CPCs.
Methods: The nanometer reagents are set up in advance, then begin
to transfect different kinds of proteins (green uorescent protein GFP,
Tbx5, Hand2, Mef2c and Gata4 transcription factor) into BMSCs.
Lipofectamine-2000 was used as positive control. After inducting for 1
d, 3 d, 8 d and 15 d, the cells morphological changes were observed respectively, total RNA and protein were extracted for detecting the expression levels of myocardial progenitor markers. Protein-induced
CPCs (piCPCs) transplanted into rat hearts after myocardial infarction
to observe if the cardiac function was improved.
Results: Four transcription factors, Gata4, Hand2, Mef2c and Tbx5 all
can entirely be targeted and led into the hBMSCs nucleus when modied by nano-protein transfection bio-unit. Fluorescence microscope observation revealed a near 100% efciency of transfection. The whole
process of transfection took about more than 15 d to differentiate into
myocardial line. With regard to the expression of protein, we found
the expression of makers of all three cell lines of cardiac progenitor
cell: endothelial cell line (such as CD31), cardiomyocyte line (such as

S19

Nkx2.5) and smooth muscle cell line (such as sm_MHC and -SMA)
all had expressions at the 15d after transfection. After reprogramming,
H3K4me3 and H3K9ac increased on the -10kb enhancer region of
Nkx2.5. In rats, the hearts undergoing piCPC transplantation showed decreased brosis compared with those treated with vehicle at 4 weeks
after myocardial infarction.
Conclusion: Nano-protein transfection bio-unit can control the gene
expression of the host cells, leading to complete transformation of the
parental phenotype using a method that is virus free and does not introduce any foreign genetic material into the recipients system. This protein reprogramming strategy lays the foundation for future
renements and might provide a good source of CPCs for regenerative
approaches.
* This work was supported by NSFC key programs(Nos.
81120108003 and 81330007).
TU-068
Beta 2 adrenegic receptor expression and activation of endogenous
progenitor cells
Amanda Finan, Morgane Guisiano, Patrice Bideaux, Marie Demion,
Jerome Thireau, Sylvain Richard
INSERM U1046, Montpellier, France
Background: Endogenous progenitor cells may participate in cardiac repair after a myocardial infarction. The beta adrenergic pathway has been proposed to induce proliferation and migration of
progenitor cells. However, the mechanisms have not yet been
claried.
Methods and results: The mechanism underlying beta adrenergic
signaling on endogenous c-kit +/CD45- cardiac cells was investigated by inducing myocardial infarction in adult mice. Hearts were dissociated and ow cytometry analysis demonstrated that one week
after ligation, the percentage of c-kit +/CD45- cells expressing beta
1 or beta 2 adrenergic receptor was signicantly increased
(88.1 3% and 106.8 36.5% increase compared to sham respectively). Flow cytometry studies on cultured cardiac c-kit +/CD45- cells
conrmed increased beta 1 and 2 adrenergic receptor expression in
response to stress conditions, specically hypoxia (5%) or serum
starvation. Interestingly, stress conditions altered localization of
the beta 2 adrenergic receptor by increasing membrane expression.
The beta 2 adrenergic receptor signaling pathway was stimulated in
adult sham mice with the agonist fenoterol (0.25 mg/kg/day) administered in drinking water. Seven days after treatment the mice and nontreated controls were sacriced and progenitor cells were measured by
ow cytometry in the heart and blood. Fenoterol increased the proliferation and percentage of c-kit+/CD45- cells in the heart (123.386.2% and
70.944.6% increase compared to control respectively). Fenoterol treatment also elevated levels of circulating endothelial progenitor cells
(158.587.9% compared to control) and c-kit+/CD45- cells
(70.633.5% increase) in the peripheral blood.
Conclusion: Beta adrenergic receptor expression in cardiac c-kit+/
CD45- cells is increased after coronary ligation in vivo and in stress conditions in vitro. A beta 2 adrenergic receptor agonist may be used to improve endogenous cardiac repair through the activation of progenitor
cells.

TU-069
Adult ovine cardiomyocytes express the cell cycle-inhibiting gene
Meis1. A potential target for cardiac regeneration based on cardiomyocyte division
Paola Locatelli1, Carlos Sebastin Gimnez1, Fernanda Daniela Olea1,
Anna Hnatiuk1, Alberto Crottogini1, Daniel Ghiringhelli2, Mariano
Nicolas Belaich2

S20

Abstracts

Favaloro University, Buenos Aires, Argentina

Quilmes National University, Bernal, Buenos Aires, Argentina

Introduction: Meis1 is a transcription factor known to regulate adult

cardiomyocyte cell cycle. In mice it keeps cell cycle arrest through interactions with cell cycle inhibiting proteins (p15, p16, p19, p21), and its decreased expression is a mitogenic stimulus for postnatal cardiomyocytes.
Meis1 inhibition may therefore be a potential means to promote adult
cardiomyocytes division and this, in turn, could represent a potential cardiac regenerative therapy. We thus aimed at searching for Meis1 and other
cell cycle regulatory genes in adult sheep cardiomyocytes.
Methods: Primers were designed targeting meis 1, cdkn1a, cdkn2aip,
cdk2, cdk4, cdk6, ciclin E1, ciclin D2 and gapdh genes using the information of exon junctions to detect only mRNAs. End point RT-PCRs were
performed from adult healthy sheep myocardium RNA. PCR products
were recovered and molecularly cloned into a generic plasmid. The
plasmids, each containing the target sequences of one gene, were sequenced to conrm that the insert corresponded to the desired fragment. These plasmids were used as calibrators of real-time-PCR with
SyBrGreen to obtain efciency and dynamic range parameters.
Results: The genes meis1, cdkn1a, cdkn2aip, cdk2, cdk4, cdk6, ciclin E1
and ciclin D2 were expressed in ovine myocardium. Real time PCR was
optimized employing the calibrator plasmids, obtaining a dynamic
range of 102108 for all genes, and adequeate efciencies for quantitative estimations. The ratio target gene/GAPDH for the analyzed genes
was: Meis1: 2.7, cdk4: 2.5, cdk6: 2.8, Ciclin D2: 3.6.
Conclusion: Meis 1 is expressed in adult sheep myocardium, thus
being a potential target for silencing strategies aimed at fostering
adult cardiomyocyte cell cycle reentry and divison.

TU-070
High doses of high mobility group box-1 (HMGB1) protein increase
capillary and arteriolar densities and induce overexpression of
genes involved in angiogenesis and cell proliferation in ovine infarct
border zone
Fernanda Daniela Olea 1, Maria del Rosario Bauz1, Paola Locatelli1,
Carlos Sebastin Gimnez1, Anna Hnatiuk1, Leonardo Sganga2, Luis
Cuniberti1, Alberto Crottogini1
1

Favaloro University, Buenos Aires, Argentina

Leloir Institute, Buenos Aires, Argentina

Introduction: In mice with AMI, administration of the proinammatory protein HMGB-1 improved heart function and induced
angiogenesis due to VEGF overexpression. CKit+ cells were also detected, suggesting cardiomyogenesis. However, neither the effects nor the
optimal dose of HMGB-1 in large mammalian models of AMI have
been addressed, this being important to develop translational therapies
for humans. We thus assessed the effect of two doses of HMGB-1 on microvascular neoformation and the expression of genes involved in angiogenesis and cell proliferation in the infarct border zone of sheep with AMI.
Methods: Twenty-one sheep with AMI received, 4 hours after coronary ligation, a total of 10 g (high dose, n=7) or 1 g (low dose, n=7)
of HMGB1 in 10 intramyocardial injections in the peri-infarct zone. Placebo animals (n = 7) received PBS. One week later, animals were
sacriced to quantify capillary and arteriolar densities (anti- lectin and
smooth muscle actin immunohistochemistry, respectively) and expression of vegf, ckit, gata 4 and nkx2.5 genes (RT-qPCR).
Results: Arteriolar density was higher than placebo in the high-dose
group (39.411 vs. 23.24 arterioles/mm2, pb 0.05, XSD, ANOVABonferroni) and in low-dose group (3914, p=NS) although not statistically signicant. Capillary density was higher than placebo in highdose group (2828 511 capillaries/mm2 p b 0.05) vs placebo
(1711 194 cap/mm2, p b 0.01) but not in the low dose group (2341
379 capillaries/mm2, p=NS). Vegf, ckit, and nkx2.5 expression was

signicantly higher than placebo only in the high-dose group. Gata4

was signicantly higher than placebo in both high and low-dose groups.
Conclusions: In a large mammalian model of AMI, high, but not low,
dose of HMGB1 injected in the peri-infarct zone induced overexpression
of angiogenic and cell proliferation genes and microvascular proliferation.
Studies addressing whether high-dose HMGB1 is in fact cardioprotective
in terms of infarct size limitation and cardiac function improvement in
the long term are ongoing.

TU-071
P2Y2 nucleotide receptor prompts human cardiac progenitor cell
activation
Farid Elsayed, Steven Greene, Jonathan Nguyen, Mark Sussman
San Diego State University, San Diego, California, USA
Heart failure is a leading cause of death in the US due to the limited
capability of adult mammalian heart to regenerate following injury. Autologous stem cell therapy holds promise for regeneration of injured
myocardium after myocardial infarction. However, stem cells derived
from diseased organs exhibit impaired proliferative and migratory capabilities and increased susceptibility to cell death. Empowering stem cells
from diverse origins, including cardiac progenitor cells (CPCs), with prosurvival genes has been attempted. Despite the well-established roles of
purinergic signaling mediated by extracellular nucleotides in regulating
diverse cellular responses in cardiovascular diseases, it has not been
well-dened in CPCs. This study shows, for the rst time, that the majority of P2 purinergic receptors are expressed and exhibit functional responses to ATP and UTP in mouse and human CPCs. The G proteincoupled P2Y2 receptor (P2Y2R) is a pivotal stress detector that senses
ATP and UTP accumulated in extracellular space following injury/stress
and responds with the proper regenerative responses. P2Y2R induces
cardioprotective responses in animal models as well as human
cardiomyocytes and regulates a wide range of signaling pathways that
are crucial to tissue repair in various experimental models and in stem
cells from diverse origins. Hence, we aimed to determine whether
P2Y2R plays similar roles in CPCs. P2Y2R stimulation with UTP enhances
human CPC (hCPC) proliferation and migration. UTP-induced proliferation in hCPCs involves activation of the canonical ERK1/2 pathway. UTP
also induces YAP activation revealing a novel link between extracellular
nucleotides released during cardiac ischemia and extracellular matrix
sensing and Hippo signaling that have been recently implicated in cardiac regeneration. Interestingly, hCPCs that exhibit relatively slower
growth kinetics and higher levels of senescence markers show dramatic
decreases in P2Y2R expression compared to fast-growing hCPCs consistent with our hypothesis that overexpressing P2Y2R participates in rejuvenating hCPCs and improving their regenerative potential.

TU-072
Polylactic acid sheets seeded with genetically modied ovine
diaphragmatic myoblasts for myocardial regeneration
Carlos Sebastian Gimnez1, Fernanda Daniela Olea1, Paola Locatelli1,
Anna Hnatiuk1, Milagros Pena2, Ricardo Dewey2, Florencia Montini
Ballarin3, Gustavo Abraham3, Alejandro Orlowski4, Luis Cuniberti1,
Alberto Crottogini1
1

Favaloro University, Buenos Aires, Argentina

IIB- INTECH-UNSAM-CONICET, Chascomus, Buenos Aires, Argentina,
3
INTEMA-UNMDP-CONICET, Mar del Plata, Buenos Aires, Argentina,
4
CIC-UNLP-CONICET, La Plata, Buenos Aires, Argentina
2

Our aim was to isolate, culture and characterize ovine diaphragmatic

myoblasts (DMs), transduce them with connexin 43 gene (Cx43) to induce connection between cells, and nally grow them on scaffolds

Abstracts

made from different materials to generate DMs-carrying sheets for later

application on infarcted areas of sheep hearts with coronary artery ligation. Sheep diaphragm biopsies were digested with collagenase. The extracted DMs were cultured on a feeder layer of autologous activated
macrophages (MFD) and characterized with antibodies against desmin,
sarcomeric -actin, SERCA-2 ATPase and Ki-67. To promote inter- cell
connections, DMs were transduced with a lentivirus encoding
connexin-43 after testing transduction efciency of diverse multiplicities
of infection (MOI) using lentivirus-GFP. DMs were satisfactorily grown on
MFD, were positive for all antibodies and were able to differentiate into
myotubes expressing myo-D and myosin heavy chain. With MOI=100,
transduction efciency was 70.8% and Cx43 was extensively expressed.
Finally, in order to select the most adequate material to build up DMscarrying sheets, we seeded DMs on scaffolds made from ovine pericardium (OP, n =8), pig bladder extracellular matrix (ECM, n=8) and
polylactic acid (PLA, n=8), and tested DMs conuence (C) at 4 days
using a score in which 0=0% C, 1=1-20% C, 2=21-40% C, 3=41-60%
C, 4=61-80% C, and 5 =81-100% C. C was 0 with PB, 2.61.1 with
ECM and 4.750.5 with PLA (pb 0.0001 vs. PB and ECM; XSD,
ANOVA-Bonferroni). Conclusion: MDs were successfully isolated, cultured and transduced. PLLA was the most appropriate material to generate DMs-carrying sheets. These results are the rst step towards testing
therapeutic efcacy of DMs in sheep models of myocardial infarction.

TU-073
Rapid Stabilisation of Atherosclerotic Plaque with 5-Aminolevulinic
Acid-Mediated Sonodynamic Therapy
Ye Tian
Division of Cardiology, the First Afliated Hospital of Harbin Medical
University, Harbin, China
Division of Pathophysiology, Harbin Medical University, Harbin, China
Background: 5-Aminolevulinic acid-mediated sonodynamic therapy (ALA-SDT) effectively induces the apoptosis of atherogenic macrophages, but whether it can stabilise atherosclerotic plaque in vivo is
unclear. Here, we used an animal model to evaluate the effects of ALASDT on plaque stabilisation.
Methods: Sixty rabbits were induced atherosclerotic plaques in the
femoral artery with a combination of silastic tube placement with atherogenic diet, and randomly assigned into control (n = 12) and SDT
(n = 48) groups. In the SDT group, after intravenous injected with
ALA (60 mg/kg) animals underwent the treatment of ultrasound with
intensities of 0.75, 1.00, 1.50 and 2.00 W/cm2 (n = 12 for each intensity). Seven days after the treatment, the plaque disruption assay was
performed to test plaque stability.
Results: We found that ALA-SDT with ultrasound intensity of 1.5 W/
cm2 showed the strongest efcacy to stabilise plaques. Under this condition, the frequency of plaque disruption decreased by 88 % (p b 0.01), positive area of macrophages reduced by 94 % (p b 0.001) and percentage
content of lipids dropped by 60 % (p b 0.001), while percentage content
of collagens increased by 127 % (p b 0.001). We also found that the plaque
stabilisation by ALA-SDT was associated with increased macrophage apoptosis and apoptotic cell clearance. Moreover, ALA-SDT decreased the
contents and activities of matrix metalloproteinase-2,9 and increased
the levels of tissue inhibitors of matrix metalloproteinase-1,2 in plaques.
Conclusion: Our studies demonstrate that ALA-SDT promotes
plaque stabilisation by inducing macrophage elimination and inhibiting
matrix degradation. This method might be a promising regimen for atherosclerosis therapy.

TU-074
Cellular mechanisms of osteogenic differentiation in the development of aortic valve calcication

S21

Mariia Bogdanova1,2, Katarina Zihlavnikova Enayati1, Anna

Malashicheva2, Jarle Vaage3,4, Kre-Olav Stenslkken1, Arkady
Rutkovskiy1,3
1

Div. of Physiology, Institute of Basic Medical Sciences, University of Oslo,

Oslo, Norway
2
Almazov Federal Heart Centre, Saint Petersburg, Russia
3
Dept. of Emergency and Critical care, Oslo University Hospital, Oslo,
Norway
4
Institute of Clinical Medicine, University of Oslo, Oslo, Norway
Background: Interstitial cells of the aortic valve (VICs) may transform into osteoblast-like cells causing calcication and valve stenosis.
The mechanism of this process is unclear. The process occurs exclusively
on the aortic side of the valve leaets, and not on the ventricular side.
We sought to investigate the side-specic role of inammation and mechanical stretch and to study if valvular endothelial cells (VECs) may
have a role in osteogenic differentiation of VICs.
Methods: VICs were isolated from human aortic valves with or without calcication harvested during surgery. 1. VICs were cultured with or
without osteogenic medium for 21 days. 2. VICs were cultured on collagen or elastin pre-coated plates (to simulate the aortic and ventricular
side of the valve leaets respectively) and subjected to 10% stretch at
1 Hz (FlexCell bioreactor), or lipopolysaccharide (LPS) 0,1g/ml, or
both. Calcication was assessed by Alizarin Red staining with quantication. 3. VECs were seeded over a 3D culture of VICs embedded in 2
mg/ml collagen gel and cultured for 21 days in osteogenic medium.
The expression of mRNA of osteogenic markers (bone morphogenetic
protein 2 (BMP2), beta-catenin (BCAT) and runt-related protein-2
(RUNX2) was evaluated by RQ-PCR.
Results: VICs from calcied valves cultured with osteogenic medium
showed higher calcium accumulation and expression of osteogenic
markers then from non-calcied. LPS triggered calcication in a culture
of VICs on collagen, but not on elastin (Alizarin red staining and mRNA
expression of osteogenic markers). Mechanical stretch of broblasts cultured on collagen augmented the effect of LPS. VECs obtained from calcied valves stimulated osteogenic differentiation of non-calcied VICs.
Conclusion: Cell culture models of osteogenic differentiation and
valve calcication were established. LPS-induced inammation and
stretch contribute to calcication of VICs on the collagen-coated surface
in contrast to the elastin-coated. VECs may stimulate valve calcication
by cross-talk with VICs.

TU-075
Cortical bone stem cells derived exosomes can promote cardiac
repair mechanisms after myocardial injury
Sadia Mohsin, Constantine Troupes, Mohsin Khan, Timothy Starosta,
Hajime Kubo, Remus Berretta, Raj Kishore, Steven Houser
Temple University, Philadelphia, USA
Rationale: Adoptive transfer of stem cells into failing human hearts
has been shown to be safe, but leads to modest improvements due to
low cell retention and diminished viability of cells after transplantation
in the ischemic environment. Recently we have shown in a mouse
model that cortical bone derived stem cells (CBSCs) possess enhanced
ability to improve cardiac function after MI mainly via secretion of paracrine factors. Since exosomes represent the active component of released
factors whether CBSC derived exosomes have the potential to repair
heart after injury in a cell autonomous manner is presently unknown.
Objective: Determine the therapeutic value of CBSC exosomes and
their contents for myocardial repair.
Methods and results: Exosomes were isolated from murine CBSCs
by ultracentrifugation. The puried fraction of exosomes was analyzed

S22

Abstracts

for size by dynamic light scattering measurement and transmission

electron microscopy and showed typical size range of exosomes from
30-100nm. CBSCs derived exosomes showed increased cardiac protection in vitro in NRVMs after hypoxic challenge as measured by TUNEL
staining. To determine myocardial repair ability, CBSC exosomes
(60g) were injected in mice after myocardial infarction. Improved cardiac function was observed in CBSC exosomes injected mice compared
to saline controls 6 weeks after MI. Importantly, CBSC exosomes treated
animals showed increased myocyte survival and angiogenesis. The underlying mechanism for benecial effects was tied to increased packaging of cardioprotective miRNAs in the exosomes compared to the parent
cells as conrmed by MiRNA array analysis.
Conclusion: Exosomes derived from CBSCs provides a cell free system that uses the reparative power of CBSC to augment cardiac function
after myocardial injury recapitulating our earlier ndings with CBSCs.
Increased packaging of cardioprotective miRs compared to the parents
cells highlighting a potential new insight into the salutary effects of
exosome therapy.

TU-076
Detection of serum vascular endothelial growth factor
and its clinical signicance in lymphoma patients
Qian Lijuan1, Zhang Meng2, Zhang Qingyun2
1

Zhejiang Cancer Hospital, Hangzhou Zhejiang China, China

Peking University School of Oncology, Beijing Cancer Hospital, Haidian
District, Beijing, China

cardiovascular applications. Genetic simplicity, possibility of generating

high-titer vector preparations, lack of inammatory response and, most
notably, capacity to deliver genes into postmitotic cells are among their
most notable characteristics. Over the last few years, using AAV9, the
most cardiotropic serotype, we have obtained extensive and persistent
transduction of the myocardium in both small (rodent) and large
(dogs, pigs) animals and taken advantage of this property to assess
function of both protein coding genes and small RNA molecules.
Notwithstanding their favorable characteristics, several unknowns still hamper a broader and more effective use of these
vectors. These limitations are mostly related to our still limited understanding of their interaction with the host cell proteins. To tackle
this issue in a systematic manner, we performed an unbiased high
throughput RNAi screening (18,120 human target genes) and identied 1,483 genes affecting vector efciency more than 4-fold and up
to 50-fold, either negatively or positively. Most of the identied factors have never previously been associated to AAV infection. The
most effective siRNAs we identied were independent from the
virus serotype or the cell type used and were equally evident for
single-stranded and self-complementary AAV vectors. A common
characteristic of the most effective siRNAs was the induction of cellular DNA damage and the activation of a cell cycle checkpoint. These
characteristics appear important to explain the specic tropism of
these vectors for post-mitotic cells, including cardiomyocytes, in
which DNA damage recognition occurs through molecular pathways
differing from those active in replicating cells.

Abstract :
Objective: To explore the correlations of serum vascular endothelial
growth factor ( VEGF ) levels with clinical tumor size in lymphoma patients and to evaluate the value of VEGF in the diagnosis of lymphoma.
Methods: Serum VEGF levels were detected by ELISA in 53 patients
with lymphoma and 42 healthy controls.
Results: The serum VEGF levels in patients with lymphoma
were (263.11 23.13) pg/ml, and the serum VEGF levels in healthy
controls were (93.45 13.23) pg/ml. So, the serum VEGF levels in
lymphoma patients compared to that in healthy controls was almost three-folds. It is signicantly higher than their healthy controls (t =-3.810 , P 0.05 ). Serum VEGF level in lymphoma
patients was signicantly related to tumor size (t =-2.520 ,
P 0.05). The sensitivity of VEGF for the diagnosis of lymphoma
were 90.0%, and was signicantly higher than other common
serum tumor markers and the specicity was 80.6%.
Conclusion: The level of serum VEGF signicantly increased in lymphoma patients and was also associated with tumor size. which may
have great clinical signicance for the screening and diagnosis of
lymphoma.
Key words: vascular endothelial growth factor ; tumor markers ;
lymphoma.

TU-077
Genome-wide siRNA screening identies cellular genes regulating
AAV transduction in the cardiovascular system
Lorena Zentilin1, Miguel Mano1,2, Edoardo Schneider1, Serena
Zacchigna1, Mauro Giacca1
1

International Centre for Genetic Engineering and Biotechnology (ICGEB),

Trieste, Italy
2
Center for Neuroscience and Cell Biology (CNC) University of Coimbra,
Cantanhede, Portugal
Recombinant adeno-associated viral vectors (AAVs) are currently
considered as the vectors of choice for in vivo gene transfer for

TU-079
Role of miRNA-33a in Dilated Cardiomyopathy
Anupam Mittal1,6, Santanu Rana4, Rajni Sharma2, Vikas Arige5, Sanskriti
Khanna2, Nitish Mahapatra5, Sagartirtha Sarkar4, Uma Nahar3, Ajay
Bahl1, Shyamal Goswami6, Madhu Khullar2

Department of Cardiology, Postgraduate Institute of Medical Education

and Research, Chandigarh, India
2
Department of Experimental Medicine and Biotechnology, Postgrduate Institute of Medical Education and Research, Chandigarh, India
3
Department of Histopathology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
4
Deprtment of Zoology University of Calcutta, Kolkata, India
5
Department of Biotechnology, Indian Institute of Technology, Chennai,
India
6
School of Life Sciences, Jawahar Lal Nehru University, New Delhi, India
Background: Dilated cardiomyopathy (DCM) accounts for approximately 1/3rd of total cases of heart failure (HF) and is a leading indication for cardiac transplantation. Myocardin (MYOCD), a potent
transcriptional co-activator of smooth muscle (SM) and cardiac genes,
is upregulated in failing myocardium in animal models and human
end-stage heart failure (HF). microRNAs (miRNAs) are 20-22 nucleotide
long non-coding RNAs regulate gene expression. However, the role of
miRNAs regulating MYOCD expression in heart failure remains unknown. The goal of this study was to identify the miRNAs regulating
the cardiac MYOCD and to study the molecular and functional consequences of cardiac modulation of MYOCD specic miRNA in an animal
model of HF/DCM.
Method and results: Our study design included identication and
validation of miRNA targeting MYOCD using in silico approach and 3UTR luciferase reporter assay and to study its cardiac expression in idiopathic DCM (IDCM) endomyocardial biopsies, renal artery ligation
(RAL) rat model of HF/DCM. We identied and validated miRNA-33a
as a putative regulator of MYOCD expression in cardiomyocytes. Cardiac
miRNA-33a expression was signicantly decreased in IDCM and in RAL.
We studied the role of miRNA-33a in two important processes of cardiac
remodelling that is cardiac hypertrophy and brosis. We also

Abstracts

investigated if cardiac specic augmentation of miRNA-33a expression

using a homing peptide conjugated siRNA could potentially modulate
the cardiac remodelling and outcome in RAL. We observed that targeted
modulation of miRNA-33a attenuated cardiac hypertrophy and brosis,
decreased expression of hypertrophy and brotic genes and ameliorated the impaired diastolic dysfunction in RAL model of cardiomyopathy.
Conclusion/Signicance: This data provide the rst evidence that
miRNA-33a is involved in regulating cardiac MYOCD expression as
well as regulation of cardiac remodelling process and cardiac specic
augmentation of miRNA-33a offers a putative therapeutic target in
DCM.
TU-082
Increased Expression of Calreticulin in the Heart: Cardiac Fibrosis
and Heart Failure
Jody Groenendyk
University of Alberta, Edmonton, Alberta, Canada
McGill University, Montreal, Quebec, Canada

One detrimental aspect of cardiac failure is an increase in brosis

with surplus deposition of extracellular matrix proteins. This can reduce
cardiac function but the underlying mechanism of why this happens is
still unclear. Increased abundance of calreticulin in adult heart has
been associated with dilated cardiomyopathy and heart failure. Here,
we discovered that increased expression of calreticulin in the adult
mouse heart leads to severe cardiac brosis. To investigate the mechanism behind calreticulin-dependent increase in cardiac brosis, we utilized microarray hybridization and monitored global gene expression in
calreticulin transgenic hearts with impaired ER homeostasis. We observed signicantly enhanced expression of TGF-1, a pleiotropic cytokine, as well as brillar collagens when compared with control hearts.
Validation of protein expression showed that TGF-1 expression and secretion into the circulatory system was signicantly increased as well as
receptor-regulated Smad2/3 expression, also activated in calreticulin
transgenic hearts. Several pro-inammatory factors and markers of brosis, including NF B p65, and pro-inammatory cytokines, TNF , IL1, and IL-6, were noticeably up-regulated. The expression and localization of periostin, a ligand for integrins that supports cellular adhesion
and migration, was increased in calreticulin transgenic hearts. Furthermore, ER stress was increased as measured by XBP1 splicing analysis
(IRE1 activity), due to the overexpression of calreticulin in the heart.
However, cardiac brosis triggered by calreticulin overexpression was
effectively reduced by administration of tauroursodeoxycholic acid
(TUDCA), possibly due to TUDCAs inhibitory effects on ER stress. We
concluded that the mechanism leading to cardiac brosis in adult hearts
overexpressing calreticulin may involve impaired ER homeostasis triggering activation of ER stress coping responses, activation the TGF-1/
Smad2/3 signaling pathway which may lead to cardiac brosis with
this pathogenesis suppressed by TUDCA treatment.
Supported by grants from Canadian Institutes of Health Research.

TU-083
TRPV2 regulates the development of myocyte hypertrophy
Sheryl Koch, Samuel Slone, Min Jiang, Michael Tranter, Jack Rubinstein
University of Cincinnati, Cincinnati, OH, USA
Background: Transient Receptor Potential Vanilloid (TRPV2) channels function as stretch mediated regulators of calcium homeostasis in
various cell types. We have demonstrated that TRPV2 channels are fundamental in contractility and calcium handling in the cardiomyocyte.
Herein, we tested the hypothesis that TRPV2 channels mediate the

S23

hypertrophic response of cardiomyocytes in vitro as well as with clinically relevant mouse models of hypertrophy via genetic ablation of the
channel and with a TRPV2 blocker (tranilast).
Methods: Isolated ventricular myocytes were obtained from wild
type (WT) mice, while WT and TRPV2-/- mice were used for in vivo experiments. Isolated myocytes were exposed to phenylephrine (PE) as a
hypertrophic stimulus and their calcium transients were measured via
FURO-4. In-vivo mice were exposed to various hypertrophic stimuli including transverse aortic constriction (TAC), isoproterenol infusion and
angiotensin II (Ang-II) infusion. Cardiac function was measured in vivo
via echocardiography weekly and via invasive catherization at the terminal endpoint. Post mortem molecular markers of hypertrophy and
failure as well as brosis and myocyte size were measured.
Results: We report that TRPV2 is upregulated in response to increased hypertrophic stimuli such as PE, TAC and Ang-II but not directly
via adrenergic stimulation in vivo. The genetic deletion and pharmacologic blockade of TRPV2 inhibited the hypertrophic response as noted
via echocardiography, histology and molecular markers of hypertrophy,
but did not result in cardiovascular collapse as noted via echocardiography, invasive catherization or markers of failure. Interestingly, both
TRPV2 deletion and blockade resulted in signicantly reduced myocardial brosis.
Conclusions: We conclude that TRPV2, as a stretch mediated channel, modulates the development of cardiomyocyte hypertrophy and
may be a target for the prevention of left ventricular hypertrophy in patients at risk for heart failure with preserved ejection fraction.

TU-084
Normalization of Cardiac Energy Metabolism and Left Ventricular
Hypertrophy Precede Functional Recovery in the Regression of
Heart Failure
Nikole J Byrne1, Jody Levasseur1, Miranda M Sung1, Grant Masson1,
Jamie Boisvenue1, Martin E Young2, Jason RB Dyck1
1

University of Alberta, Edmonton, Alberta, Canada

University of Alabama at Birmingham, Birmingham, Alabama, USA

Aims: Impaired cardiac substrate metabolism plays a key role in

heart failure (HF) pathogenesis. Since many of these metabolic changes
occur at the transcriptional level of metabolic enzymes, it is possible
that this loss of metabolic exibility is permanent and thus contributes
to worsening cardiac function and/or prevents full regression of HF
upon treatment. However, despite the importance of cardiac energetics
in HF, it remains unclear whether these metabolic changes can be normalized. In the current study, we investigated whether reversal of an elevated aortic afterload in mice with severe HF would result in recovery
of cardiac function, substrate metabolism and transcriptional
reprogramming, as well as determine the temporal relationship of
these changes.
Methods and results: Male C57Bl/6 mice were subjected to either
sham or transverse aortic constriction (TAC) surgery to induce HF.
After HF development, mice with severe HF (% ejection fraction b 30)
underwent a second surgery to remove the aortic constriction
(debanding). Three weeks following debanding, there was a near complete recovery of systolic and diastolic function, and gene expression of
several markers for hypertrophy/HF were returned to values observed
in healthy controls. Interestingly, pressure overload-induced left ventricular hypertrophy (LVH) and cardiac substrate metabolism were restored at 1 week post-debanding, which preceded functional recovery.
Conclusions: Regression of severe HF is associated with early and
dramatic improvements in cardiac energy metabolism and LVH normalization that precede restored cardiac function, suggesting that metabolic and structural improvements may be critical determinants for
functional recovery.

S24

Abstracts

TU-085
Genetic background does not affect progression to heart failure in a
mouse model with genetic ablation of RyR2-S2808 phosphorylation
Francisco J. Alvarado, Hector H. Valdivia

phosphorylated pRb. Taken together, our results reveal that derepression of

CDK6 and activation of Rb pathway contribute to the effect of attenuation
of miR-1 on provoking cardiomyocyte hypertrophy.

University of Michigan, Ann Arbor, MI, USA

TU-087
The interplay between genetic background and sexual dimorphism
of doxorubicin-induced cardiotoxicity
Beshay Zordoky1, Judith Radin2, Lois Heller1, Anthony Tobias1, Ilze
Matise1, Fred Apple1, Sylvia McCune3, Leslie Sharkey1

Background. The pathophysiological relevance of cardiac ryanodine

receptor (RyR2) phosphorylation has been of great interest for over 20
years. This eld was boosted when Marks et al. proposed that S2808
hyperphosphorylation is a critical mediator of heart failure (HF) progression. Many laboratories, however, have been unable to reproduce
key elements of this hypothesis, including a better outcome after myocardial infarction (MI) in a RyR2-S2808A mouse model. Our aim was to
determine whether part of this discrepancy is due to the genetic background of the mice, since Marks model was C57Bl/6 and that created
by the Valdivia laboratory was Sv129.
Methods. RyR2-S2808A Sv129 mice were backcrossed for seven
generations with C57Bl/6J mice to obtain the congenic mouse line
with N99% C57Bl/6 genetic background. Congenic RyR2-S2808A mice
and C57Bl/6J wild type (WT) controls were then: 1) evaluated with a
basal echocardiogram one week before MI; 2) subjected to MI by LAD
coronary ligation; 3) followed-up with an echocardiogram one- and
four-weeks post-MI.
Results. The basal echocardiogram of S2808A and WT mice did not
show statistical difference. MIs performed in both groups were signicant, as determined by a decrease in ejection fraction (EF) and fractional
shortening (FS), as well as dilation of the left ventricle (LV). S2808A
mice did not show better outcome than WT mice up to four weeks
post-MI. All parameters measured were comparable between groups,
including EF (30.85 4.14% WT vs. 35.05 3.83% S2808A, p = 0.47),
FS (14.87 2.17% vs. 17.06 2.06% p = 0.47), LV diameter
(5.210.20 mm vs. 5.210.29 mm, p=0.99) and heart weight as percentage of body weight (0.720.03% vs. 0.700.05%, p=0.78).
Conclusions. The genetic background of the mice is unlikely the
source of discrepancy between results obtained by the Marks and Valdivia/Houser groups using different RyR2-S2808A mice. These data support the notion that RyR2-S2808 phosphorylation is not critically
involved in HF progression.
TU-086
CDK6 mediates the effect of attenuation of miR-1 on provoking
cardiomyocyte hypertrophy
Jie ning Zhu, Chun mei Tang, Qiu xiong Lin, Wen si Zhu, Yong heng Fu,
Chun yu Deng, Min Yang, Zhi xin Shan
Guangdong General Hospital, Guangdong Cardiovascular Institute, Guangdong Academy of Medical Sciences, Guangzhou, Guangdong, China
MicroRNA-1 (miR-1) is approved involved in cardiac hypertrophy,
but the underlying molecular mechanisms of miR-1 in cardiac hypertrophy are not well elucidated. The present study aimed to investigate the
potential role of miR-1 in modulating CDKs-Rb pathway during cardiomyocyte hypertrophy. A rat model of hypertrophy was established with
abdominal aortic constriction (AAC), and a cell model of hypertrophy
was also achieved based on PE-promoted neonatal rat ventricular
cardiomyocytes (NRVCs). We demonstrated that miR-1 expression
was markedly decreased in hypertrophic myocardium and hypertrophic cardiomyocytes. Dual luciferase reporter assays revealed that
miR-1 interacted with the 3'UTR of CDK6, and miR-1 was veried to inhibit CDK6 expression at the posttranscriptional level. CDK6 protein expression was observed increased in hypertrophic myocardium and
hypertrophic cardiomyocytes. Morover, miR-1 mimic, in parallel to CDK6
siRNA, could inhibit PE-induced hypertrophy of NRVCs, with decreases in
cell size, newly transcribed RNA, expressions of ANF and -MHC, and the

University of Minnesota, Minneapolis, MN, USA

The Ohio State University, Columbus, OH, USA
3
University of Colorado at Boulder, Boulder, CO, USA
2

Background: Doxorubicin (DOX) is a very effective anticancer medication that is commonly used to treat both hematological malignancies
and solid tumors. Nevertheless, DOX is known to have cardiotoxic effects that may lead to cardiac dysfunction and heart failure. In experimental studies, female animals have been shown to be protected
against DOX-induced cardiotoxicity; however, the evidence of this sexual dimorphism is inconclusive in clinical studies. Therefore, we sought
to investigate whether the genetic background could inuence the sexual dimorphism of DOX-induced cardiotoxicity.
Methods: Male and female Wistar Kyoto (WKY) and Spontaneous
Hypertensive Heart Failure (SHHF) rats were used in this study. DOX
was administered in 8 doses of 2 mg/kg/week; thereafter, the rats
were followed for an additional 12 weeks. Cardiac function was
assessed by trans-thoracic echocardiography, systolic blood pressure
was measured by the tail cuff method, and heart and kidney tissues
were collected for histopathology.
Results: Female sex protected against DOX-induced weight loss and
increase in blood pressure in the WKY rats, whereas it protected against
DOX-induced cardiac dysfunction and the elevation of cardiac troponin
in SHHF rats. In both strains, female sex was protective against DOXinduced nephrotoxicity. There was a strong correlation between DOXinduced renal pathology and DOX-induced cardiac dysfunction.
Conclusions: This study highlights the importance of studying the interaction between sex and genetic background to determine the risk of
DOX-induced cardiotoxicity. In addition, our ndings suggest that DOXinduced nephrotoxicity plays a role in DOX-induced cardiac dysfunction.

TU-088
Rnd3/RhoE is a Pro-Angiogenic Factor
Regulating Responsive Cardiac Angiogenesis
Xiaojing Yue1, Xi Lin1, Tingli Yang1, Xiangsheng Yang1, Xin Yi1, Keith
Youker2, Guillermo Torre-Amione2, Kelsey Andrade1, Jiang Chang1
1

Texas A&M University Health Science Center, Houston, Texas, USA,

Methodist DeBakey Heart & Vascular Center, Houston, Texas, USA

BackgroundThe insufciency of compensatory angiogenesis in response to pathological stimuli contributes to the transition to heart failure. HIF1-VEGF signaling cascade controls angiogenesis in the heart in
response to stress. One of the challenges in reprograming the insufcient angiogenesis is to achieve a sustainable tissue exposure to the
pro-angiogenic factors such as by stabilizing HIF1.
Methods and resultsIn this study, we identied Rnd3, a small Rho
GTPase, as a new pro-angiogenic factor participating in the regulation of
HIF1-VEGF signaling cascade. Rnd3 physically interacts with and stabilizes HIF1, consequently promoting VEGFA expression and endothelial cell tube formation. To demonstrate this pro-angiogenic role of Rnd3
in vivo, we generate Rnd3 knockout mice. Rnd3 haploinsufcient
(Rnd3+/-) mice are viable, yet develop dilated cardiomyopathy with

Abstracts

heart failure after transverse aortic constriction (TAC). The post-TAC

Rnd3+/- hearts show signicantly impaired angiogenesis and decreased
HIF1 and VEGFA expression. The angiogenesis defect and heart failure
phenotype are partially rescued by cobalt chloride treatment, a HIF1
stabilizer, conrming a critical role of Rnd3 in stress-responsive angiogenesis. Furthermore, we generate Rnd3 transgenic mice (MHC-Rnd3)
and demonstrate that Rnd3 overexpression has a cardio-protective effect through reserved cardiac function and preserved responsive angiogenesis after TAC. Finally, we assess the expression level of Rnd3 in the
human heart and detect signicant downregulation of Rnd3 in patients
with end-stage heart failure.
ConclusionsRnd3 acts as a novel pro-angiogenic factor involved in
cardiac responsive angiogenesis through HIF1-VEGFA signaling promotion. Rnd3 downregulation observed in heart failure patients may
explain the insufcient compensatory angiogenesis contributing to the
transition to heart failure. The assessment of Rnd3 expression levels in
patients could be a new reference biomarker for human heart failure.

TU-090
Improved metabolic function and contractility in mdx mice following treatment with morpholino oligomers.
Victoria Johnstone1, Helena Viola1, Abbie Adams3, Steve Wilton3,4, Sue
Fletcher3,4, Livia Hool1,2
1

The University of Western Australia, Crawley, Western Australia, Australia

Victor Chang Cardiac Research Institute, Sydney, New South Wales,
Australia
3
Centre for Neuromuscular and Neurological Disorders, The University of
Western Australia, Crawley, Western Australia, Australia
4
Centre for Comparative Genomics, Murdoch University, Murdoch, Western
Australia, Australia
2

Duchenne Muscular Dystrophy (DMD) is an X-linked muscular disease that involves a fatal cardiac phenotype. DMD-associated cardiomyopathy is underpinned by disrupted cytoskeletal architecture and
mitochondrial dysfunction, and current treatment strategies to date
are limited to minimising symptoms of the disease. Here we report a recovery of metabolic and contractile function in mdx mice (a murine
model of DMD) following treatment with antisense morpholino oligomers to induce skipping of dystrophin exon 23 (M23D). Optimal treatment regimen was rst established by varying dosage and route of
administration using a three week treatment trial in neonates. Activation of the L-type Ca2+ channel (LTCC) facilitates Ca2+ inux required
for contraction, but also causes an increase in mitochondrial membrane
potential (m) in a Ca2+-independent manner. This is dependent on
the cytoskeleton and is disrupted in mdx mice. Recovery of metabolic
function was assessed by monitoring LTCC-dependent increases in m
(JC-1 uorescence) and mitochondrial oxygen consumption (avoprotein autouorescence) in isolated cardiomyocytes. A total weekly dose
of 120mg/kg M23D administered s.c. was optimal in neonatal mdx
cardiomyocytes, and restored the BayK(-)-mediated increase in m
(treated=29.02.0%, n=51; untreated=1.00.4%, n=22) and avoprotein
oxidation
(treated = 16.0 2.0%,
n = 25;
untreated = 2.0 0.5%, n = 21). Using this treatment regimen, 24
week old adult mice with established cardiomyopathy were treated
for 16 weeks. We report a post-treatment restoration of BayK(-)-mediated increases in m (treated = 32.0 3.1%, n = 6; untreated agematched = 1.2 1.2%,
n = 4)
and
avoprotein
oxidation
(treated = 55.4 15.4%, n = 17; untreated age-matched = 4.1 1.6%,
n = 8). In addition, echocardiographic measurements revealed a decrease in end diastolic diameter in systole (treated = 2.5 0.mm,
n = 4; untreated age-matched = 2.8 0.0mm, n = 3) and increase in
fractional shortening (treated = 37.0 1.6%, n = 4; untreated agematched = 31.3 0.5%, n = 3) in adult mice upon completion of 16

S25

weeks treatment. These results indicate that treatment with M23D results in restoration of metabolic function and improvement in contractility in adult mice with established cardiomyopathy.

TU-091
Identication of miR-34 regulatory networks in settings of disease
and antimiR-therapy: Implications for treating cardiac pathology
and other diseases
Jenny Y. Y. Ooi1, Bianca C. Bernardo1, Saloni Singla1, Ruby C.Y. Lin2,3, Julie
R. McMullen1,4
1

Baker IDI Heart & Diabetes Institute, Melbourne, Victoria, Australia,

Asbestos Diseases Research Institute, Sydney, New South Wales, Australia
3
Ramaciotti Centre for Genomics, University of New South Wales, Sydney,
New South Wales, Australia
4
Monash University, Clayton, Victoria, Australia
2

Expression of the miR-34 family (miR-34a, -34b, -34c) is elevated

in settings of heart disease, and inhibition with antimiR-34a/antimiR34 has emerged as a promising therapeutic strategy. Under chronic
cardiac disease settings, targeting the entire miR-34 family is more effective than targeting miR-34a alone. The identication of transcription factor (TF)-miRNA regulatory networks has added complexity
to understanding the therapeutic potential of miRNA-based therapies.
Here, we sought to determine whether antimiR-34 targets secondary
miRNAs via TFs which could contribute to antimiR-34-mediated protection. Using miRNA-Seq we identied differentially regulated
miRNAs in hearts from mice with cardiac pathology due to transverse
aortic constriction (TAC), and these miRNAs were also regulated by
antimiR-34. Two clusters of stress-responsive miRNAs were classied
as pathological and cardioprotective. Using ChIPBase we identied
45 TF binding sites on the promoters of pathological and
cardioprotective miRNAs, and 5 represented direct targets of miR34, with the capacity to regulate other miRNAs. The expression of
two pathological miRNAs (let-7e and miR-31) was independently
experimentally validated in hearts from antimiR-34 treated TAC
mice, and may explain why targeting the entire miR-34 family is
more effective than targeting miR-34a alone. AntimiR-34 regulates
the expression of other miRNAs and this has signicant implications
for drug development.
TU-092
Cardiac Thyrotropin Releasing Hormone (TRH) Inhibition attenuates the post-ischemic damage and improves ventricular function
after myocardial infarction in rats
Mariano Schuman, Ludmila Peres Diaz, Maia Aisicovich, Fernando
Ingallina, Silvina Landa, Silvia Garca
Laboratory of Molecular Cardiology, Institute of Medical Research A. Lanari,
UBA; IDIM-CONICET, Buenos Aires, Argentina
Heart injury induces ventricular remodeling. Particularly acute myocardial infarction causes myocytes damage, reactive hypertrophy and
interstitial brosis in the infarcted area.
We described TRH system hyperactivity in left ventricle (LV)
hypertrophied SHRs hearts. Indeed, TRH inhibition prevents cardiac hypertrophy despite the severe hypertension suggesting its involvement
(Schuman et al, 2011). We observed that LV TRH overexpression in normal rats induces features of the hypertrophic phenotype (Schuman et al
2014).
Microarray studies revealed LV TRH increase after myocardial infarction (Jin H. et al 2004), and added to our reports, we hypothesized that
LV TRH inhibition previous to infarct maneuver could attenuate cardiac
remodeling damage.

S26

Abstracts

Adults Wistar males were infarcted by permanent anterior descending coronary artery ligation simultaneously to 40ug LV SiRNA injection
against TRH or scrambled siRNA (control). At day 6 ventricular function
evaluation was performed (echocardiography) and 24h later animals
were sacriced for heart gene expression quantitation (RT-PCR).
Infarcted rats showed an expected signicant decrease in ejection
fraction and increases in heart rate and end diastolic volume compared
to sham group and according to our hypothesis, the animals in which LV
TRH system was blocked all these changes were not observed pointing
out that LV TRH inhibition prior to injury improves ventricular function
and decreases contractility and heart dilatation.
As expected, we found a LV TRH overexpression in infarcted rats
injected with siRNA-Control accompanied by signicant increases in
BNP, ANP, -MHC and Collagen III and decreases in SERCA2 and actin expressions in harmony to heart tissue damage prole including
the contractility system.
LV TRH inhibition which reduced signicantly TRH gene expression,
blunted BNP, ANP, Collagen III and -MHC increase and normalized the
expression of SERCA2 and -actin.
These novels results demonstrate the participation of TRH in postischemic remodeling and reveal that its inhibition attenuates the damage and improves ventricular function.

TU-093
Functional role of G9a-induced histone methylation in cardiac
hypertrophy
Francesca Rusconi1,2, Pierluigi Carullo2,3, Marco Vacchiano2, Gianluigi
Condorelli2, Roberto Papait2,3
1

Fondazione Umberto Veronesi, Milan, Italy

Humanitas Clinical and Research Center, Rozzano, Milan, Italy
3
Institute of Genetic and Biomedical Research (IRGB) - UOS, Milan, Italy
2

Cardiac hypertrophy is an initially adaptive response of the myocardium to increased work overload and can progress to heart failure (HF).
At the molecular level, its associated with a specic gene expression
program. The role of histone methylation in regulating this program is
poorly understood. Our group has recently shown that an epigenetic
signature dened by methylation and acetylation of histone H3 regulates the gene expression changes accompanying cardiac hypertrophy.
However, the molecular pathways that dene this signature are not elucidated yet. Here, we show that histone methyl-transferase G9a is differentially regulated in cardiomyocytes of mice subjected to
transverse aortic constriction (TAC) a procedure that through pressure overload induces rst compensated hypertrophy then HF and
in stressed human hearts.
G9a is a histone methyl-transferase that specically mono and dimethylates Lys-9 of histone H3 and tri-methylates Lys-27 of histone H3,
leading to transcriptional repression and these histone modications contribute to cellular memory by establishing gene expression programs during development and subsequently stabilizing the differentiated state.
We rst assessed whether G9a had a role in regulating cardiac function at baseline conditions in vivo. To this end, C57BI/6 mice were
treated with a selective inhibitor (BIX-01294) up to four weeks via subcutaneous mini-osmotic pumps. Mice treated with the drug showed a
signicant decrease in cardiac function, as assessed by echocardiographic analysis, compared to control groups (untreated mice and
mice treated with vehicle). Thus, baseline G9a inhibition seemed to
cause progressively heart failure. To conrm that this effect was due
to G9a in cardiomyocytes, we generated conditional cardiac G9a ko
(KO) mice and we analyzed the effects of down-regulated G9a in the
heart of these mice. After 4 weeks of the induction of the myocardial deletion of G9a, by echo analysis, biochemical and histological studies, we

observed a HF phenotype similar to that of mice treated with G9a inhibitor, in KO mice compared to controls.
Data in vitro and in vivo will be presented in support of our hypothesis showing that G9a is important in dening the correct transcription
program of cardiomyocytes and in regulating gene expression reprogramming during cardiac hypertrophy. Our work may lead to the
development of new therapeutic strategies for HF based on the modulation of this epigenetic enzyme.

TU-094
Guanylyl Cyclase-A Signaling Attenuates Deleterious Salt Effect on
Aldosterone-Induced Cardiac Remodeling
Hitoshi Nakagawa, Satoshi Somekawa, Yasuki Nakada, Tomoya Nakano,
Takuya Kumazawa, Kenji Onoue, Hiroyuki Okura, Yoshihiko Saito
Nara Medical University, Nara, Japan
Background: Sodium causes the development of cardiovascular disease such as hypertension, cardiac hypertrophy and heart failure in conjunction with enhanced renin-angiotensin-aldosterone system (RAAS).
Natriuretic peptide (NP), which is an important sodium regulator, prevents pathological cardiac alternations by counteracting RAAS. However, it is not elucidated whether NP inhibits sodium-effect on adverse
cardiac alternations. We investigated whether salt excess exacerbates
cardiac remodeling in mice with impaired NP signaling.
Methods and results: Mice lacking the gene encoding the NP receptor (guanylyl cyclase (GC)-A) and wild type (WT) mice were assigned to
vehicle or subpressor dose of aldosterone (100 ng/kg/min) administration group under low salt (0.001% NaCl), normal salt (0.6% NaCl) and
high salt diet (6.0% NaCl) for 4 weeks. Salt load did not induce cardiac
change in both vehicle and aldosterone groups in WT mice. On the
other hand, cardiac hypertrophy and interstitial brosis were signicantly exacerbated in a salt dependent manner in aldosterone groups
of GC-A KO mice, associated with enhanced gene expression relevant
to hypertrophy, brosis and oxidative stress (BNP, collagen1 and
Nox4, respectively). Of note, salt excess increased the expression of
Sgk1, an important downstream of mineralocorticoid receptor (MR),
in aldosterone groups of GC-A KO mice. These molecular changes
were not observed in WT mice.
Conclusion: The present study demonstrates that salt excess induces
cardiac remodeling in conjunction with aldosterone in GC-A KO, but not
in WT mice. These data indicate that the GC-A signaling attenuates the
deleterious salt effect on aldosterone-induced cardiac remodeling.

TU-095
The role of broblast and endothelial cell NADPH oxidase-2 in the
development of cardiac brosis
Daniel Richards1, Craig Harrison1, Greta Sawyer1, Heloise Mongue-Din1,
Stephanie Telerman2, Fiona Watt2, Ajay Shah1
1

King's College London - BHF Centre of Excellence, London, UK

King's College London - Centre for Stem Cells & Regenerative Medicine,
London, UK
2

Background
NADPH oxidase-2 (NOX2) is elevated in myocardium of heart failure
patients. Global NOX2 knockout (KO) mice have reduced cardiac brosis in models of elevated angiotensin II (Ang II) or chronic pressure
overload. NOX2 is expressed in cardiomyocytes, broblasts, endothelial
cells and inammatory cells, but the NOX2-expressing cell type responsible for these anti-brotic effects is unknown.
Aim: To investigate the role of broblast and endothelial NOX2 in
the development of cardiac brosis.

Abstracts

Methods: We generated inducible broblast-specic or endothelialspecic NOX2 KO mice by crossing Col1a2-Cre or Cdh5-Cre mice with a
novel oxed-NOX2 mouse model. Cre recombinase expression was induced with tamoxifen and the delity of cell-specic targeting was
evaluated by using a STOP-oxed tdTomato reporter strain.
Results: Both models were successfully generated. Flow cytometry
of cardiac cellular digests of STOP-oxed reporter mice indicated that
N96% of broblasts or endothelial cells underwent recombination. In a
model of Ang II infusion (1.1 mg/kg/day), broblast-specic NOX2 KO
mice developed less cardiac brosis than wild-type (WT) littermates
(1.59% vs. 2.58%; pb0.05, n=4-6). However, transverse aortic constriction (TAC) caused a similar extent of cardiac brosis (7.62% vs. 7.21%;
p=0.97) in broblast-specic NOX2 KO and WT controls. Endothelialspecic NOX2 KO subjected to TAC also developed similar brosis to
WT littermates (11.8% vs. 9.61%; p = 0.69). There were no signicant
differences in the extent of cardiac hypertrophy or contractile dysfunction between broblast-specic or endothelial-specic NOX2 KO and
their respective WT littermate controls.
Conclusion: Although broblast NOX2 contributes to the development of Ang II-induced cardiac brosis it has no effect on TAC-induced
brosis. Endothelial NOX2 is also dispensable for TAC-induced brosis.
These results suggest that other NOX2-expressing cell types are required for the development of cardiac brosis in response to chronic
pressure overload.

TU-096
NADPH oxidase-4 mediates cardiac adaptation to volume overload
Moritz Schnelle1,2, Karl Toischer2, Norman Catibog1, Min Zhang1, Katrin
Schrder3, Ralf Brandes3, Gerd Hasenfuss2, Ajay Shah1
1

King's College London BHF Centre, London, UK

Goettingen Heart Centre, Goettingen, Germany
3
Institut fr Kardiovaskulre Physiologie, Goethe-Universitt, Frankfurt am
Main, Germany
2

Background: Chronic pressure and volume overload induce concentric versus eccentric remodelling respectively. Distinct signalling pathways are likely involved in these responses but the underlying
pathways are incompletely dened. NADPH oxidase-4 (Nox4), a reactive oxygen species (ROS)-generating enzyme, reduces detrimental cardiac remodelling during chronic pressure overload but its role in
volume overload-induced remodelling is unknown.
Methods: Aortocaval stula (ACF) was performed to induce volume
overload in male, global Nox4-null mice (KO) and wildtype (WT) littermates. Animals were followed up for 2 weeks.
Results: 2 weeks of ACF in WT mice caused a signicant increase in
cardiac Nox4 mRNA (1.6 fold, pb 0.05) and protein expression (2.0 fold,
pb0.01) compared to sham controls but no change in Nox2 levels. KO
mice developed signicantly less LV hypertrophy (+25% vs +43% increase in LV/tibia length ratio, pb0.01) and less LV dilatation (echocardiographic LVEDD: 4.6 mm vs 5.1 mm, pb 0.01) than WT animals after
ACF. LV ejection fraction was similar in both genotypes following ACF,
as were levels of ANP, BNP and SERCA-2 mRNA. Phospho-Akt levels increased in WT mice after ACF whereas levels decreased in KO mice
(+ 29% vs -21%, p b0.05). The levels of phospho-Erk1/2 decreased to
similar levels after ACF in both genotypes (-37% vs -29% in WT and KO
respectively, p=n.s.).
Conclusion: Nox4 appears to be required for the development of eccentric cardiac remodelling and hypertrophy during chronic volume
overload. Nox4-dependent activation of Akt may be involved since Akt
is implicated in the development of adaptive cardiac dilatation during
volume overload. Ongoing studies are assessing the impact of Nox4 deletion during more prolonged volume overload.

S27

TU-097
Structural and functional changes in the murine heart during
sustained -adrenergic stimulation in vivo
Sarah-Lena Puhl, Kate Weeks, Antonella Ranieri, Metin Avkiran
King's College London, London, UK
Purpose: To determine the structural and functional responses of
the murine heart to sustained -adrenoceptor stimulation in vivo.
Methods: C57/BL6J mice aged 8 weeks were randomly assigned to
receive a subcutaneous infusion of saline or isoprenaline (30 g/g/day)
for 3 days (n = 9/group) or 14-days (n = 8/group). At the end of the
14-day infusion period, the mice in each group were randomly assigned
to receive a single bolus intraperitoneal injection of saline or dobutamine (0.75 g/mg) (n=4/group). Cardiac phenotype was assessed by
high-resolution echocardiographic imaging and standard gravimetric
and histological assays.
Results: -adrenergic stimulation for only 3 days induced cardiac
hypertrophy (signicant increases in left ventricular (LV) wall thicknesses and heart weight, heart weight/body weight ratio and heart
weight/tibia length ratio). Mice subjected to more prolonged adrenergic stimulation for 14 days exhibited comparable differences
in cardiac structure relative to corresponding saline-infused mice, but
at this stage such differences were accompanied also by enhanced LV
function (signicantly greater LV fractional shortening and ejection
fraction) and increased heart rate. Interestingly, relative to mice that
had received saline for an identical period, mice that had received isoprenaline infusion for 14 days exhibited signicantly lower LV fractional
shortening and ejection fraction following acute -adrenergic stimulation with dobutamine, in the presence of a similarly elevated heart rate.
Conclusion: These observations indicate that, during sustained adrenergic stimulation by isoprenaline infusion at 30 g/g/day, structural hypertrophic remodelling occurs predominantly within the initial 3
days and precedes persistent positive inotropic and chronotropic responses. Sustained -adrenergic stimulation for 14 days induces a loss
of contractile reserve, which is revealed only when an acute adrenergic stress is superimposed on the hypertrophied heart. Thus,
acute -adrenoceptor stimulation with dobutamine may be a useful
method to unmask early signs of LV dysfunction in the remodelled
heart, even when basal function appears enhanced.

TU-098
Distinct Roles of Intracellular Calcium Release Channels in Cardiac
and Vascular Remodelling
Gaetano Santulli1, Qi Yuan1, Steven Reiken1, Jingyi Yang1, Alain
Lacampagne1,2, Andrew Marks1
1

Columbia University, New York, NY, USA

Montpellier University, Montpellier, France

Background - Calcium release from intracellular stores controls

countless cellular processes. Ryanodine receptors (RyRs) and inositol
1,4,5-trisphosphate receptors (IP3Rs) are the major calcium release
channels on the endo/sarcoplasmic reticulum (ER/SR). RyRs and IP3Rs
comprise macromolecular signalling complexes that include modulatory proteins which regulate channel activity in response to extracellular
signals eventually resulting in intracellular calcium release.
Methods and results - We investigated the respective functional
roles of different RyR and IP3R isoforms in the pathophysiology of vascular and cardiac dysfunction. To this aim, we generated tissue specic
knockout murine models via a ox/cre recombinant technique, targeting
endothelial cells (EC), vascular smooth muscle cells (VSMC), and ventricular cardiomyocytes. Combining in vivo, ex vivo, and in vitro techniques we demonstrated for the rst time that: 1) IP3R1 is directly

S28

Abstracts

involved in nitric oxide (NO) production in EC via a calcineurin/nuclear

factor of activated T-cells (NFAT) pathway, and its deletion in EC causes
a hypertensive phenotype; 2) IP3R1 in VSMC is a key player in the vasomotor responses both in basal conditions and during neurohormonal
overdrive mediated by both adrenergic and renin-angiotensin systems following myocardial infarction obtained via ligation of the left
anterior descending coronary artery; 3) in ventricular cardiomyocytes,
RyR2, but not IP3R2, has a crucial role in determining mitochondrial
dysfunction in heart failure.
Conclusions - Taken together, our results provide robust evidence
towards a tissue-specic functional predominance within intracellular
calcium release channels: IP3Rs are crucial in modulating vascular
tone whereas RyRs are the main players in the regulation of myocardial
contractility.

TU-099
Inhibition of Rho Kinase (ROCK) Restores Ionic Currents and
Prevents Electrical Remodelling of Heart in Pressure Overload
Induced Hypertrophy Model
Murat Cenk CELEN1, Bilge Eren YAMASAN1, Yusuf OLGAR2, Semir
OZDEMIR1
1

Akdeniz University, ANTALYA, Turkey

Ankara University, ANKARA, Turkey

Background: Various cardiovascular diseases like myocardial infarction, heart failure and cardiac hypertrophy are associated with the
RhoA/Rho kinase (ROCK) signalling pathway. Although electrical remodelling of left ventricle has been studied in pressure overload (PO)
induced cardiac hypertrophy, effect of ROCK inhibition with selective
ROCK inhibitor fasudil on action potential (AP) prolongation and relevant currents have not been studied yet. This study examined the impact of ROCK inhibition on AP duration and repolarizing potassium
currents.
Methods and results: PO model is created by transverse aortic constriction (TAC) of rats. SHAM animals underwent surgery without
banding. All data taken from three groups; SHAM, TAC and fasudiltreated (5 mg/kg and 10 weeks) TAC (Tac+Fas) group. In TAC group,
increased heart weight (HW), HW/body weight ratio and HW/tibia
ratio were observed and fasudil treatment attenuated these ratios.
There was a signicant prolongation in TAC myocytes AP duration
which was similar to control values in Tac+Fas group. Inward rectier
(IK1) and transient outward (Ito) potassium currents were recorded in
whole-cell conguration of patch-clamp by step pulse protocol. Both
currents decreased signicantly in TAC myocytes, despite inhibition of
ROCK reversed these currents to control values.
Expression level of relevant proteins RhoA, ROCK1, ROCK2, Kir2.1
and Kv4.2 were also examined. According to western blot analysis,
Kv4.2 didnt change signicantly while RhoA increased and Kir2.1 decreased in TAC myocardium. ROCK1&2 expressions decreased signicantly after 10 weeks in TAC hearts. Fasudil administration brought
these proteins changed in TAC heart to control levels.
Conclusion: These ndings suggest that fasudil improves AP duration due to restoration of potassium currents and underscore the role
of RhoA/ROCK pathway in development of pathological cardiac hypertrophy. Therefore inhibition of this pathway may be a potential target
for therapeutic purposes in future.

Loyola University Chicgo, Maywood IL, USA

Background: The Troponin-I (TnI) R145W mutation is associated
with the presentation of restrictive cardiomyopathy (RCM), high diastolic
lling pressures, and a severe adverse clinical outcome. The molecular
mechanisms underlying RCM in patients carrying this mutation are uncertain. It has been suggested that increased myolament calcium sensitivity plays an important role in the disease. Myolament calcium
sensitivity and myolament relaxation kinetics determine diastolic stiffness of the heart. These parameters are inuenced by factors that include
both PKA and PKC mediated contractile protein phosphorylation, as well
as sarcomere length (SL) mediated regulation of contractile function via
the process of myolament length dependent activation (LDA).
Methods: Permeabilized multicellular myocardial muscle preparations or single myobrils were isolated from frozen human donor septum, followed by overnight exchange for recombinant troponin
composed of hTnC, hTnT-myc, and WT or mutated/phosphomimetic
hTnI: S23D,S24D (PKA), T144E (PKC), R145W (RCM),
S23D,S24D,T144E (PKA+ PKC), and S23D,S24D,R145W (PKA+RCM).
Force was measured in skinned muscles over a wide range of free
[Ca2 +] at short and long SL to derive myolament Ca2 + sensitivity
and LDA; activation/relaxation kinetics were measured in single myobrils at saturated [Ca2+].
Results: PKA phosphomimetic induced a large reduction in myolament Ca2 + sensitivity and a strong increase in LDA, while PKC
phosphomimetic induced a slight increase in myolament Ca2+ sensitivity, but no change in LDA. RCM induced a large increase in myolament Ca2 + sensitivity and a decrease in LDA. Finally, PKA
phosphomimetic accelerated the kinetics of myobril relaxation while
PKC phosphomimetic was without affect on this parameter. In contrast,
RCM induced a strong slowing of myobril relaxation rate. Both PKC
phosphomimetic (PKA+PKC) and RCM mutation (PKA+RCM) virtually eliminated the impact of PKA phosphomimetic on: myolament Ca2+
sensitivity, LDA, and myobril relaxation kinetics. Finally, the hcTnIR145W mutation caused the threonine phosphorylation target on
hcTnI-144 to be inaccessible to a panel of PKC kinases, thus, rendering
this PKC target effectively phospho-null in RCM.
Conclusions: Phosphorylation of cTnI at PKA target S23/S24 reduces
myolament activation, increased length dependency (LDA), and accelerates relaxation kinetics, events that are expected to lower diastolic
stiffness of the heart. Phosphorylation of PKC target T144 induces opposite properties, expected to enhance diastolic stiffness. The R145W RCM
associated mutation induces a phenotype that is similar to PKC
phosphomimetic, but constitutively. Moreover, the RCM mutation interrupts the normal phenotypical cross-modulation property between
PKA and PKC contractile phosphorylation, such that the relaxing impact
of PKA mediated phosphorylation caused by decreased myolament
Ca2+ sensitivity, enhanced LDA, and accelerated relaxation kinetics is
no longer possible. We propose that these myolament based properties contribute to elevated diastolic stiffness of the heart in RCM patients, especially during episodes of elevated beta-adrenergic
stimulation, such as in exercise.

TU-101
Richard Schell1,2, Florian Leuschner1, Andras Toth2, Hugo A. Katus1,
Johannes Backs2
1

TU-100
Restrictive cardiomyopathy mutation TnI-R145W blocks PKA-PKC
cross-modulation of human myolament length dependent activation and relaxation kinetics
Alexey Dvornikov, Nikolai Smolin, Mengjie Zhang, Jody Martin, Seth
Robia, Pieter de Tombe

University Hospital - Department of Cardiology, Angiology and

Pneumology, Heidelberg, Germany
2
University Hospital - Department Molecular Cardiology and Epigenetics,
Heidelberg, Germany
Rationale: Heart failure is one of the most severe burden of cardiovascular diseases due to its striking prevalence, mortality and morbidity. Besides

Abstracts

the clinical classication of distinct etiologies the underlying molecular

mechanisms still remain unclear. Recent ndings suggest that inammatory
pathways play critical roles remodeling and progression of heart failure.
Myocyte enhancer factor 2 (MEF2) is a crucial inductor of pathologic
cardiac remodeling due to its effects on fetal gene programs. In a lately
in-vitro study, we could show that Prostaglandin E2 (PGE2) leads to a
strong MEF2-activation. Furthermore the investigation provides data
that PGE2 stimulates via EP3-receptor an intracellular signal transmission, which drives a protein kinase D (PKD) dependent
hyperphosphorylation of histone deacetylase 5 (HDAC5) and the
resulting nucleo-plasmatic shuttling of HDAC5 leads to the increased
MEF2 activity.
The aim of the present work is a translation of these ndings in different in-vivo models to gure out its relevance in inammatory
cardiomyopathies.
Results: In an experimental model of cardiac myosin-induced myocarditis, the resulting setting of inammation comes along with increasing levels of the PGE2-forming Cyclooxygenase 2 and Prostaglandin-Esynthase 1. Furthermore we see an increase of catalytic PKD-activity
and consecutively an HDAC5-hyperphosphorylation. Additionally
raised mRNA-levels of MEF2-target genes like Myomaxin and CCL3/
CCL6 represent the induction of cardiac remodeling genes. We validated
the mechanism in other models of inammatory cardiomyopathies and
see conrming results in a model of LPS-induced septic cardiomyopathy
and a model of coxsackie B3-induced viral myocarditis as well.
Conclusion: Inammation and autoimmune response seem to play
crucial roles in the induction of ventricular remodeling and progression
of heart failure. With these in-vivo data, we provide evidence that PGE2
mediates an epigenetic pathway containing EP3-receptor transmitted
phosphorylation of PKD and HDAC5 leading to a nucleo-cytoplasmatic
shuttling of HDAC5 with the consequence of a MEF2 deregulation and
induction of pathologic remodeling. The described pathway provides a
new link between cardiac inammation and the initiation and progression of remodeling and heart failure. Further investigations on the distinct pathway and its relevance in the different etiologies of
remodeling is urgently needed and could provide new therapeutic targets to alleviate the burden of heart failure.

TU-102
Effects of a Selective Class I HDAC 1/2 Inhibitor on Cardiac Remodeling in Mouse TAC
Kersten Small 1, Joseph McCarthy 2, Shu Yu Sun1 , Mark Aronovitz 2 ,
Richard Karas2, Jeffrey Madwed1, Robert Blanton2
1

Merck Research Labs, Kenilworth, NJ, USA

Tufts Medical School, Boston, MA, USA

Pan Class I HDAC inhibitors (HDAC 1/2/3) have shown benets in preclinical heart failure models, however, given the severe cardiac toxicity
phenotype of Hdac3 mutant mice, it remains unclear whether a selective
Class I HDAC 1/2 inhibitor would have enhanced efcacy. Therefore, the
objective of this study was to evaluate the therapeutic potential of a potent and selective small molecule HDAC 1/2 inhibitor (MRL-001; IC50:
HDAC1=2.9nM; HDAC2=27nM; HDAC3=2553nM) to improve cardiac structure and function in a mouse model of chronic moderate thoracic
aortic constriction (TAC). The study was performed blinded in c57/b6
mice (n=15/group), with in-feed doses of MRL-001 (3, 10 and 30
mg/kg/day) with treatment beginning 3-weeks post-TAC for a duration
of 10-weeks. Dose selection was based on the highest tolerated dose in
a 28-day pharmacokinetic /safety study. MRL-001 did not signicantly
alter TAC-mediated increases in anterior and posterior wall thickness or
left ventricular (LV) weight at any dose. Likewise, MRL-001 had no effect
on the TAC-induced reduction in ejection fraction, stroke volume, and LV
lling, or the prolonged LV relaxation as measured by tau. Low doses of

S29

MRL-001, however, signicantly attenuated LV dilatation as measured

by end diastolic dimensions, with highest dose showing no effect. In summary, while MRL-001 did not demonstrate benet on cardiac hypertrophy or function, MRL-001 did demonstrate cardiac structural
improvements at low doses. The highest dose of MRL-001 was not effective and trended to worsen LV function and structure. In conclusion, MRL001 preserved normal LV dimensions after TAC, but at the lower doses.
The lack of effect on cardiac function differed from the literature with
pan Class I inhibitors. It is possible that Class I HDAC 1/2/3 is needed for
enhanced benet, and the cardiac toxicity observed in Hdac3 mutant
mice will not be observed in pharmacological studies. Future studies
will address this hypothesis.

TU-103
Direct and Selective AMPK Activation Fails to Improve Cardiac Structure and Function in Mouse Pressure-Overload
Kersten Small1, Jessica Bradley2, Traci Goodchild2, Craig Zilblich2, Juliann
Ehrhart1, Shu Yu Sun1, Iyassu Sebhat1, Jeffrey Madwed1, David Lefer0
1

Merck Research Labs, Kenilworth, NJ, USA

Louisiana State University, New Orleans, LA, USA

Because chronic treatment with indirect AMPK activators AICAR and

metformin improves cardiac structure and function in multiple preclinical heart failure settings, AMPK activation has been proposed as a
promising therapeutic target for heart failure. AMPK mediated cardiac
benets hypothesized to drive efcacy include increased glucose uptake
and metabolism, improved insulin sensitivity, enhanced endothelial
function, anti-inammatory and anti-brotic effects. Here, we evaluated
the efcacy of a potent, direct, and selective small molecule AMPK activator (MRL-002) in mouse TAC. The study was performed blinded and
included 5 groups, sham-vehicle, sham-10 mg/kg/day, TAC-vehicle,
TAC-1 mg/kg/day and TAC-10 mg/kg/day. Treatment was administered
in feed beginning 4 weeks prior to TAC surgery. Echocardiography and
invasive hemodynamics were performed to assess dose response effects
of AMPK activation on cardiac structure and function over 12 weeks
post-TAC. Exposures for the 10 mg/kg/day dose were consistent with
those required for glucose lowering in lean C57Bl6 mice. Echocardiography revealed expected increases in wall thickness at early time points in
TAC-vehicle mice as well as progressive decreases in fractional shortening and increases in chamber dimensions at later time points. 1
mg/kg/day MRL-002 blunted the increase in interventricular septal
thickness at diastole at 4, 6, and 8 weeks post-TAC. Posterior wall thickness at diastole trended lower in treatment groups at early time points.
Fractional shortening decreased and chamber dimensions increased
similarly in both vehicle and MRL-002 treatment groups. Heart weights
were also similarly increased. Thus, in the long-term, MRL-002 failed
to improve cardiac function or alter progressive remodeling in this
model. In addition and unexpectedly, invasive hemodynamics revealed dose dependent increases in Tau indicating prolonged relaxation following long term treatment. Data from this study fails
to replicate ndings observed with indirect AMPK activators and
refutes the hypothesis that chronic direct activation of AMPK as a
therapeutic approach for heart failure.

WE-001
The transactivation activity of glucocorticoid receptor plays a key
role in protecting heart against stress and that is suppressed under
pressure-overload
Motoaki Sano
Keio University School of Medicine, Tokyo, Japan

S30

Abstracts

[Objective]: We previously reported glucocorticoids protect heart

against ischemia-reperfusion injury (J Clin Invest. 2009, Hypertension
2014) and acute viral myocarditis (J Cardiol. 2013). In the present
study, we investigated the role of cardiomyocyte glucocorticoid receptor (GR) in pressure-overload induced cardiac remodeling.
[Methods and results]: We made cardiac-specic conditional
knockout of GR (GRCKO) mice and mineralocorticoid receptor (MR)
(MRCKO) mice. GRCKO and MRCKO mice had no phenotype in the
steady-state condition. GRCKO mice showed exaggerated cardiac hypertrophy and worse systolic function in comparison with their wildtype (WT) littermates after 4 weeks of transverse aortic constriction
(TAC). MRCKO showed a similar degree of hypertrophy and systolic
function in comparison with their WT littermates after 4 weeks of
TAC. MR and GR, while functionally redundant in some contexts, cardiomyocyte GR played a distinct functional specicities, since neither genetic ablation of MR nor pharmacological blockade of MR by
eplerenone rescued the phenotypes observed for GRCKO mice under
pressure overload. DNA microarray analysis of GRCKO and MRCKO in
the steady-state condition revealed that cardiomyocyte GR has distinct
transcriptional specicities in comparison with MR. Interestingly, GR
transcriptional activities were suppressed under the pressure overloaded.
GR-selective agonist dexamethasone ameliorated TAC-induced hypertrophy and preserved LV systolic function in WT mice.
[Conclusions]: Heart is the most stressful organ in the body. Cardiomyocyte GR transcriptional activities protects heart against pressureoverload. Homeostatic role of glucocorticoids-GR signaling in
cardiomyocytes has been underestimated because of systemic adverse
effects induced by glucocorticoids.

WE-002
Gentianella acuta Improves Cardiac Function in a Model of Coronary
Ligation Induced Heart Failure via a Mechanism of Against Endoplasmic Reticulum Stress-Associated Autophagy
Yu Liu, Aiying Li
Hebei University of Chinese Medicine, Shijiazhuang,Hebei, China
Background: Increased endoplasmic reticulum(ER) stress is known
to be one of the causes of cardiovascular damage. Gentianella acuta
(Michx.) Hulten can treat hepatitis, jaundice, headache and fever in
Mongojia native medicine. However, the cardioprotective effect of
Gentianella acuta has yet to be examined. The aim of the current
study is to investigate the cardioprotective effect of Gentianella acuta
on ER stress-induced heart failure (HF) rats and its possible
mechanisms.
Methods: HF was induced using coronary artery ligation in adult
male Sprague-Dawley rats and Gentianella acuta was used. Thirty minutes after surgery, rats were randomly assigned to 3 groups: HF (n
= 12) alone, HF with high-dose Gentianella acuta, or HF with lowdose Gentianella acuta treatments. Rats in medicine-treated groups
were given 0.06 mL/10 g (once a day) of Gentianella acuta by gavage
based on different doses (1.2 or 0.3- g/Kg). Sham surgery was performed in another group of rats (n =12) without coronary artery ligation. Cardiac function was assessed by echocardiography and cardic
index 4 weeks after HF. After treated with Gentianella acuta for 4
weeks, cellular levels of ER stress marker and autophagy marker were
evaluated by western blot analysis, immunohistochemistry and real
time RT-PCR respectively.
Results: Gentianella acuta effectively inhibited ischemia-induced
heart failure, as evaluated by biometric, echocardiography, and histological examinations. Consistently, western blot analysis and immunohistochemistry showed that the protein level of ER Stress markers and
autophagy marker in cardiac tissue were signicantly lower after treatment with Gentianella acuta than HF group. Meanwhile, Gentianella

acuta signicantly increased p-AKT and p-mTOR expression in cardic

tissue. In addition, Gentianella acuta was also found to inhibit GRP78,
ATF4 and LC3 mRNA expression induced by HF.
Conclusions: Taken toghter, our results suggest that ER stressassociated autophagy is essential for HF, which can be effectively improved by Gentianella acuta.

WE-003
Gentianella acuta prevents isoprenaline-induced myocardial brosis
in rat by reduction of myocardial TGF-1/ CTGF expression
Aiying Li1, Ensheng Ji2, Jingjing Wang2
1
Department of Biochemistry, Hebei University of Chinese Medicine, Shijiazhuang, Hebei, China
2
Department of Physiology, Hebei University of Chinese Medicine, Shijiazhuang, Hebei, China

Gentianella acuta (Michx.) was used as folk medicine to treat hepatitis, jaundice, headache and fever in Mongolia native medicine. It has
been used as a health tea to treat heart diseases for many years in
Hulunbeier districts of inner Mongolia. So we thought that Gentianella
acuta could inhibit myocardial brotic formation. In this study, we investigated the effect and potential mechanisms of the extract of
Gentianella acuta on myocardial brosis. A rat myocardial brosis
model was established by hypodermic injection of isoproterenol (5
mg/kg bw/day) for 7 days, when these rat were simultaneously treated
with extract of Gentianella acuta(1.2 g/kg, 0.6 g/kg, 0.6 g/kg)or saline by
gavage for 21 days. After 21 days, the rats underwent electrocardiograph detection and were sacriced. Myocardial brosis was observed
by Masson staining. NF-B, TGF-1 and CTGF protein expression were
detected by immunohistochemistry and western blotting, TGF-1 and
CTGF mRNA expression were detected Real-Time PCR.Treatment with
Gentianella acuta could signicantly improve myocardial brosis and
decrease the collagen accumulation, hydroxyproline content in myocardial tissue. Gentianella acuta could attenuated the cardiac dysfunction
and decreased the ST-segment-elevation in isoproterenol rats. RealTime PCR results indicated that the mRNA expression of TGF-1 and
CTGF in myocardial tissue was decreased. Importantly, Gentianella
acuta could signicantly decrease the protein expressions of TGF-1,
CTGF and NF-B in myocardial tissue. The results of this research indicated that Gentianella acutal extract improved the cardiac function
and anti-brotic activity by reducted TGF-1 and CTGF expression via
inhibition of NF-B in myocardial tissues.

WE-004
Phosphodiesterase 3A1 Prevents Cardiac Remodeling from Neurohormonal Activation
Masayoshi Oikawa, Shoji Iwaya, Shu-ichi Saitoh, Yasuchika Takeishi
Fukushima Medical University, Department of Cardiology and Hematology,
Fukushima, Japan
Background: -adrenergic receptor (AR) signaling and reninangiotensin-aldosterone system (RAAS) are pivotal mechanisms to induce cardiac remodeling, and recent studies have revealed that there is
direct interaction between AR and RAAS. Phosphodiesterase 3A
(PDE3A) inhibits AR/protein kinase A axis by metabolizing cAMP. Therefore, we hypothesized that overexpressed PDE3A has cardioprotective effects against neurohormonal activation.
Methods and Results: Isoproterenol (ISO, 30 mg/kg/day for 7 days)
or angiotensin II (AngII, 800 ng/min/kg for 10 days) was continuously
infused using osmotic mini-pump in wild-type (WT) mice and

Abstracts

transgenic (TG) mice with cardiac-specic expression of exogenous

PDE3A1. Both ISO and AngII infusion increased heart weight/body
weight ratio in WT mice compared with WT mice given vehicle, but
not increased in TG mice. The 8-OHdG, a marker of oxidative DNA damage, positive area was increased by ISO stimulation in WT hearts compared with vehicle hearts (14.93.7% vs. 7.41.1%, Pb 0.05), but not
in TG hearts (13.91.9% vs. 12.02.8%, ns). Protein expression levels
of Sirt1, which provides anti-oxidative effects, were upregulated in TG
hearts compared to WT hearts in both basal (1.90.2 AU vs. 1.00.1
AU, P b 0.01) and after ISO infusion (2.8 0.2 AU vs. 1.3 0.2 AU,
Pb0.01), suggesting that PDE3A has anti-oxidative effects by upregulating Sirt1-related signaling. AngII induced cardiac brosis in both WT
and TG mice, but the extent of brosis was less in TG mice compared to WT mice (4.2 1.1% vs. 6.9 2.6%, P b0.05). Moreover,
basal expression levels of transforming growth factor- were
lower in TG hearts compared to WT hearts (0.31 0.05 AU vs.
1.00 0.10 AU, P b 0.01), and it remained lower levels after AngII
stimulation in TG hearts compared to WT hearts (0.52 0.09 AU
vs. 1.72 0.29 AU, P b0.01).
Conclusion: We conclude that PDE3A prevents cardiac remodeling
by neurohormonal activation.

WE-005
Angiotension-converting enzyme inhibitor-induced cough prevalence in resistant hypertension patients
Andr Nascimento Publio Pereira, Adilson Machado Gomes Junior,
Camila Barbosa Pereira, Paulo Chenaud Neto, Thiago Matos e Silva,
Andr Oliveira Barbosa, Cristiano Ricardo Bastos de Macedo, Roque
Aras Jnior
Federal University of Bahia, Salvador, Bahia, Brazil
Background: Resistant Arterial Hypertension (RAH) is characterized by persistently high blood pressure values. Angiotensin
Converting Enzyme (ACE) inhibitors in combination with other
antihypertensive drugs are effective for RAH. According to the literature, the adverse effect of cough in patients using ACE inhibitors occurs in 5-20% of patients. However, in practice, the
incidence appears to be higher, making it difcult the therapeutic
adherence.
Objective: To estimate the prevalence of cough induced by ACE inhibitors in patients with RAH.
Methods: Cross-sectional study in a referral hospital in severe hypertensive cardiovascular disease. To assess the adverse effect cough
in the use of ACE inhibitors, patients answered to a questionnaire and
the blood pressure (BP) was measured on the day of the interview.
Statistical analysis: Data were analysed using IBM SPSS Statistics
Program for Mac version 21. Frequency and percentage were used for
qualitative variables and mean standard deviation for quantitative
variables.
Results: 120 patients were analysed and 70% were female (84). The
average age was 62,1 12,4 years. 100% (120) of the patients use or had
used ACE inhibitors. The prevalence of cough was 64,2% (77). 71,7% (86)
of the patients started using an angiotensin II receptor blocker as an ACE
inhibitor substitute. 13,9% (12) of patients reported that the cough continued even after the discontinuation of ACE inhibitor. Patients used an
average of 4,7 1,2 antihypertensive medications. The average systolic
pressure was 151,8 27,6 mmHg and the average diastolic pressure
was 88,6 16,3 mmHg.
Conclusion: We observed a high prevalence of cough associated
with the use of ACE inhibitor in this population. Despite the large number of antihypertensive drugs in use, the BP was not controlled in most
of the patients. It is possible that the non-use of ACE inhibitors may contribute to the low hypertensive control.

S31

WE-006
Inhibition of Class I Histone Deacetylases Blunts Cardiac Hypertrophy via TSC2-dependent mTOR Repression
Cyndi Morales1, Dan Li1, Zully Pedrozo2, Herman I. May1, Nan Jiang1,
Viktoriia Kyrychenko1, Geoffrey Cho1, Julia Kim1, David Rotter1, Beverly
A. Rothermel1, Jay W. Schneider1, Sergio Lavandero2, Thomas G.
Gillette1, Joseph A. Hill1
1

Department of Internal Medicine, UT Southwestern Medical Center, Dallas,

TX, USA
2
Facultad Ciencias Qumicas y Farmacuticas & Facultad Medicina,
Universidad de Chile, Santiago, Chile
Introduction: Histone deacetylases (HDACs) participate in the pathogenesis of pathological cardiac growth, and small molecular inhibitors
of HDACs reduce and regress pathological hypertrophy. The mammalian target of rapamycin complex 1 (mTORC1) is an important regulator
of cell growth. It has been shown that mTORC1 is active during cardiac
hypertrophy, leading to increased protein synthesis. Inhibiting
mTORC1 can repress pathological remodelling. Therefore, we hypothesized that class I HDACs regulate cardiac hypertrophy in an mTORdependent manner.
Results: To test this hypothesis, neonatal rat ventricular myocytes
(NRVMs) were exposed to a variety of growth stimuli, and class I
HDACs were inhibited by either pharmacological means or by knockdown of individual HDAC isoforms. We found that HDAC1, HDAC2 and
HDAC3 act together to facilitate pathological and physiological cardiomyocyte hypertrophy. In addition, inhibition of class I HDACs decreases
mTOR activation by hypertrophic growth stimuli. HDAC inhibition also
decreased mTOR activity in the setting of pressure overload using an
in vivo surgical model of transverse aortic constriction (TAC). Adult
mice with conditional cardiomyocyte-specic knockout of both
HDAC1 and HDAC2 together had improved function following TAC surgery as well as decreased mTOR activity. Tuberin (TSC2) is a component
of the tuberin-hamartin complex, which inhibits mTOR. We found that
inhibition of class I HDACs increased expression of TSC2 in NRVMs, in
mice and in embryonic stem cell-derived cardiomyocytes. Using siRNA
we observed that TSC2 is required for HDAC-dependent inhibition of
mTOR in NRVMs. Furthermore, we showed that overexpression of
TSC2 is sufcient to reduce NRVM hypertrophy.
Conclusion: These ndings point to TSC2-dependent control of
mTOR as a critical component of the mechanism through which HDAC
inhibitors blunt pathological growth. Together, these results enhance
our understanding of the function of HDACs in cardiac pathology and facilitate the ultimate translational application of HDAC inhibitors in the
treatment of heart disease.

WE-007
Myosin Activator improves Actin Assembly and Sarcomere Function
of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes
with a Troponin T Point Mutation
Kathleen Broughton1, Elina Sarmah1, Jieli Li1, Chad Warren1, Ying-Hsi Lin1,
Marcus Henze1, Vero Sanchez-Freire2, R. John Solaro1, Brenda Russell1
1

University of Illinois at Chicago, Chicago,IL, USA

Stanford University, Stanford, CA, USA

Background. We have investigated cardiac myocytes derived from

human induced pluripotent stem cells (iPSC-CMs) from two normal
control and two family members expressing a mutant cardiac troponin
T (cTnT-R173W) linked to dilated cardiomyopathy (DCM). cTnT is a
scaffolding protein of the sarcomeric thin lament. The loss of this
basic charge, which is strategically located to control tension, has consequences leading to progressive DCM. iPSC-CMs serve as a valuable

S32

Abstracts

platform for understanding clinically-relevant mutations in sarcomeric

proteins; however, there are important questions to be addressed
with regard to stress on myocytes and adaptation over time.
Methods and results. We model stress by plating iPSC-CMs on
physiologically stiff substrates (100 kPa). During the rst week of
culture of the iPSC-CMs, we have determined structural and functional characteristics as well as actin assembly dynamics. Shortening,
actin content and actin assembly dynamics were depressed in CMs
from the severely affected mutant at one week of culture, but by
two weeks differences were less apparent. Potential changes due to
sarcomeric troponin and myosin isoform composition were also
assessed. Furthermore, the troponin complex, reconstituted with
wild-type cTnT or recombinant cTnT-R173W, depressed the entry
of cross-bridges into the force generating state, which can be reversed by the myosin activator Omecamtiv Mecarbil. Therapeutic
doses of this drug increased both contractility and the content of Factin in the mutant iPSC-CMs.
Conclusions. Collectively, our data suggest the use of a myosin activation reagent to restore function within patient specic iPSC-CMs may
aid in understanding and treating this familial DCM.

WE-008
Identication of calpastatin as a novel substrate of p38gamma mitogen activated protein kinase.
Aminah Loonat1, Eva Denise Martin1, Sang Hoon Choi1, Francesca Hunt1,
Nicholas T Hertz2, Rebecca Levin2, Kevan Shokat2, Alma L Burlingame2,
Michael S Marber1, James E Clark1
1

King's College London, London, UK

University of California, San Francisco, USA

Despite the high and preferential expression of p38 mitogen activated protein kinase in the myocardium, little is known regarding its
role in the heart. The aim of this study is to elucidate p38 signalling
in the heart, with a particular focus on its role in the progression of pathological hypertrophy following abdominal aortic banding. Comparisons
of cardiac function and structure of wild type (WT) and p38 knock out
(KO) mice in response to abdominal aortic banding found that KO mice
develop less ventricular hypertrophy than their corresponding WT controls, and have preserved cardiac function. Basally, p38 myocardial
staining is primarily localised at the membranes and throughout the cytoplasm. Following aortic constriction nuclear staining of p38 increases
but no nuclear accumulation of the other dominant isoform, p38, occurs.
This suggests differential roles of the two isoforms in the heart.
To elucidate its signalling pathway and identify endogenous substrates
of p38 we have generated an analogue sensitive p38, which is mutated
at a gatekeeper residue, to specically track endogenous substrates in the
myocardium. The mutation allows only the mutant kinase, but not WT kinases, to utilise analogues of ATP that are expanded at the N-6 position and
contain a visible tag on the -phosphate. Transfer of this tag to substrates
allows subsequent isolation and identication by mass spectrometry.
Using this technique we have been able to identify, amongst others,
calpastatin as a novel target of p38. Calpastatin is the natural and endogenous inhibitor of calpain proteases. Calpain proteases are activated by increased calcium signalling during cardiac hypertrophy and inhibition of
calpain shows favourable improvements in cardiac function. We observed
that phosphorylation of calpastatin by p38 reduces the efciency of
calpastatin to inhibit calpain and we propose that this may be a mechanism by which p38 mediates its pro-hypertrophic role in the heart.
WE-009
Increased activity of AMP deaminase by decreased interaction with
PGM1 and depletion of F1,6P: a novel mechanism of diabetic
cardiomyopathy

Yuki Tatekoshi1, Masaya Tanno1, Hidemichi Kouzu1, Atsushi Kuno2,

Satoko Ishikawa1, Toshiyuki Yano1, Wataru Ohwada1, Kei Nakata1,
Keitaro Nishizawa1, Takayuki Miki1, Tetsuji Miura1
1

Department of Cardiovascular, Renal and Metabolic Medicine, Sapporo

Medical University, Sapporo, Japan
2
Department of Pharmacology, Sapporo Medical University, Sapporo, Japan

Background: AMP deaminase (AMPD) critically regulates adenine

nucleotide pool and thereby the amount of ATP production by
catalysing conversion of AMP to IMP. We have recently demonstrated
that afterload-induced diastolic dysfunction in a rat model of type 2 diabetes (T2DM), OLETF, is mediated by excessive activity of AMPD and
consequent ATP depletion. Thus, AMPD is a promising therapeutic target for diabetic cardiomyopathy. Here, we examined the mechanism
by which AMPD activity is increased in T2DM, focusing on its interacting
proteins and its regulation by metabolic alterations in T2DM.
Methods and results: OLETF showed 60% higher AMPD activity in
the left ventricular myocardium than in the non-diabetic control,
LETO. Western blot analyses revealed that protein levels of AMPD3, a
cardiac isoform of AMPD, were comparable in OLETF and LETO, indicating that regulation of AMPD3 activity is modied in OLETF. However, reported regulatory mechanisms of AMPD activity, including PKCmediated phosphorylation of AMPD, its interaction with calmodulin
and tissue inorganic phosphate levels, were similar in OLETF and
LETO. Metabolomic analysis of left ventricular myocardium revealed
that fructose 1,6-diphosphate (F1,6P) level was substantially lower in
OLETF than in LETO (13112 vs. 28956 nmol/g, pb 0.05), indicating
reduced activity of phosphofructokinase 1 (PFK1), a kinase catalysing
conversion from fructose 6 phosphate (F6P) to F1,6P, in OLETF. In
vitro addition of F1,6P (20 mmol/L) to left ventricular tissue lysates reduced AMPD activity by 69% in OLETF, conrming that AMPD activity
depends on the level of F1,6P. We next performed two-dimensional
gel electrophoresis using anti-AMPD3 immunoprecipitates obtained
from left ventricular tissues of OLETF and LETO. Among 15 protein
spots observed, intensities of 2 spots were much lower in OLETF than
in LETO. MALDI-TOF/MS analysis revealed that one of the spots
contained phosphoglucomutase-1 (PGM1), a component of the glycogenolytic sarcoplasmic reticulum complex (GSRC) that regulates local
ATP level in the immediate vicinity of the sarcoplasmic reticulum.
Conclusion: Reduction of F1,6P level by reduced PFK1 activity contributes to T2DM-induced upregulation of myocardial AMPD activity.
The change in PFK1 activity may be attributable to reduction of
AMPD3-PGM1 interaction in GSRC, which potentially modies Ca2+calmodulin-mediated regulation of PFK1.

WE-010
Angiogenesis in patients with angiographically signicant coronary
artery diseases and chronic heart failure: endothelial progenitor
cells, growth factors and cytokines
Karina Khmelnitskaya1,2, Eugenii Shlyakhto1,2
1

First Pavlov State Medical University, Saint-Petersburg, Russia

Almazov Federal Medical Reseach Centre, Saint-Petersburg, Russia

Backround: Angiogenesis is a complex multifactorial process with

involving different cellular , molecular proangiogenic and
antiangiogenic factors and is a zone of intensive researches at the present time. Mature endothelial cells possess limited regenerative capacity.
There is therefore much interest in circulating endothelial progenitor
cells (EPCs). EPCs were rst described in 1997 and have since been
the subject of numerous investigative studies exploring the potential
of these cells in the process of cardiovascular damage, repair and

Abstracts

angiogenesis. Circulating EPCs are capable of differentiating into mature

endothelial cells to assist in angiogenesis and vasculogenesis. Previous
studies have suggested an inverse relationship between levels of circulating EPCs and the presence of coronary artery disease (CAD) or cardiovascular risk factors, whereas other studies have observed increased
numbers of EPCs in the setting of acute ischemia.
Objectives: To investigate whether the number of EPCs in patients
with CHF was associated with severity CAD in patients undergoing coronary angiography, their correlations with the severity of stenosis, cytokines activation, growth factors, other clinic indicators.
Methods: Peripheral blood EPCs assessed both as CD133+ cells and
CD133+ cells coexpressing CD34 and vascular endothelial growth factor (VEGF) receptor-2 cells, plasma tumor necrosis factor- (TNF-),
C-reactive protein, VEGF, granulocyte-colony stimulating factor (GCSF), NT-probrain natriuretic peptide (NT-proBNP) were studied in 82
men with ischemic heart disease and CHF I-IV class (NYHA), undergoing
coronary angiography. Patients with acute coronary syndroms were
excluded.
Results: There was an decrease CD133 +, CD34 +/CD133 +/
VEGFR2+ cells in men with CHF and 3-vessel, 4-vessel CAD compared
with 1-vessel CAD (pb 0.05). Patients with occlusion of coronary artery
had lower CD133+, CD34+/CD133+/VEGFR2+ cells (pb0.05). A signicant decrease blood levels of VEGF were detected with 3-vessel, 4vessel compared 1-vessel CAD (pb0.05). CD34+/CD133+/VEGFR2+
cells negative correlated to age, smoking, NYHA CHF class, left ventricular ejection fraction, number of miocardis infarction, NT-proBNP, and
positive - to VEGF, CD34+, CD133+ cells. VEGF positive correlated circulating endothelial progenitor cells CD34+/CD133+/VEGFR2+ cells,
CD133+ cells.
Conclusion: There were lower number of circulating EPCs was associated with the presence of signicant angiographically CAD and the
number of vessel CAD, and the EPCs number correlated with maximum
angiographic stenosis in patients with CHF. There were VEGF decrease
and cytokine activation in patients with ischemic heart disease with
CHF especially with more severe NYHA class. VEGF level was interdependent with important angiogenesis cells - circulating endothelial progenitor cells in CHF.
WE-011
Effects of phosphodiesterase-5 A (PDE5A) inhibition on the
hypertrophied myocardium of spontaneously hypertensive rats
(SHR).
Daiana Sabrina Escudero1, Romina Gisel Daz1, Maria Soledad Brea1,
Enrique Leo Portiansky2, Nstor Gustavo Prez1
1
Centro de investigaciones cardiovasculares Dr.Horacio E Cingolani, La
Plata, Argentina
2
Laboratorio de Anlisis de Imgenes del Instituto de Patologa, La Plata,
Argentina

In a previous study we showed that an increased protein Kinase G

(PKG) activity after PDE5A inhibition (sildenal, SIL) inhibits the myocardial Na+/H+ exchanger (NHE1). Since NHE1 hyperactivity is linked
to the development of cardiac hypertrophy, our study was aimed to
study the potential antihypertrophic effects of SIL on the hypertrophic
myocardium of SHR. We initially tested the inhibitory capability of SIL
(1M) on NHE1 in isolated cardiomyocytes of SHR by comparing H+ efux (J+
H ) in the absence or presence of SIL at a common pHi of ~6.8, during the pHi recovery from an acidic load (ammonium prepulse in the
absence of bicarbonate where the NHE1 is the only active pHi regulatory
mechanism). SIL signicantly decreased J+
H : (mmol /L/ min)
12.933.80, n= 5vs. 2.090.87, n=4 (P b 0.05), conrming its inhibitory effect on the NHE1. Then 8 months old SHR were chronically treated (3 months) with SIL (100mg/kg/day, orally through drinking water,
n = 4) and compared to age-matched untreated controls (n = 6). SIL

S33

treatment decreased left ventricular weight to body weight ratio (hypertrophy index) from 3.2 0.1 (control) to 2.7 0.1 mg/g (SHR
+ SIL). Accordingly, cardiomyocytes cross-sectional area (CSA) from
treated rats was signicantly reduced (688 39 vs. 496 23m2, P
b0.05). SIL treatment also reduced myocardial interstitial brosis: (in
percentage of total interstitial collagen) 7.010.018 vs. 1.360.003%,
P b0.05), which was in accordance to the lower myocardial stiffness detected in treated hearts by comparing length-tension curves in isolated
papillary muscles (P b0.05, 2-wayANOVA). Finally, we measured kinases upstream NHE1. Not signicant changes in ERK1/2-p90RSK
MAP kinases phosphorylation, or in NHE1 protein expression were
detected between groups. In summary, the results show that
PDE5A acute inhibition by SIL inhibits NHE1 activity in SHR, suggesting that this effect would be responsible for the decreased cardiac
hypertrophy and the lower stiffness observed in hearts from SIL
treated SHR.

WE-012
Cardiomyocyte high Ca2+ operational levels linked with arrhythmogenic vulnerability in a rat model of hypertrophic heart failure with
preserved ejection fraction
James Bell1, Claire Curl1, Antonia Raaijmakers1, Wendy Ip1, Chanchal
Chandramouli1, Tristan Harding1, Kimberley Mellor2,1, Stephen Harrap1,
Lea Delbridge1
1

University of Melbourne, Melbourne, Australia

University of Auckland, Auckland, New Zealand

The pathophysiology of heart failure with preserved ejection

fraction (HFpEF) is characterised by near normal systolic function
coincident with diastolic dysfunction and inadequate ventricular lling at normal pressures. While the extent of hypertrophy is a key diagnostic indicator in HFpEF, the underlying cellular aetiology of this
disease is poorly understood, due partly to a lack of appropriate
models.
The aim of this study was to characterize in vivo cardiac and isolated cardiomyocyte functional status in the Hypertrophic Heart
Rat (HHR), a newly derived model of HFpEF. Echocardiography (GE
Vivid 9) was performed in adult male HHR and NHR (Normal Heart
Rat). Ventricles were used for brosis (picrosirius red staining) and
protein quantication (immunoblotting). Single cardiomyocyte
(fura 2-AM loaded) contractility and [Ca 2 +] i measurements by
edge-detection and microuorimetry (3Hz, 2.0mM Ca2 +, 37o C)
were performed.
Premature death in HHR was preceded by cardiac hypertrophy
(HHR vs NHR: cardiac weight index, 4.60.2 vs 3.20.1mg/g; cardiomyocyte length, 1632 vs 1331m) and in vivo diastolic dysfunction
(E/E, 31.0 3.4 vs 21.7 2.5) with maintained systolic parameters
(ejection fraction, 73.81.5 vs 80.10.9%). Diffuse interstitial brosis
was not prominent in HHR but dispersed brotic foci were observed.
Surprisingly, hypertrophic cardiomyocytes exhibited hypercontractile
status (94% shortening increase) and high Ca2 + operational levels
(91% increase in transient amplitude) linked with arrhythmogenic vulnerability. This was associated with hyperphosphorylation of sarcoplasmic reticulum Ca2+ regulatory proteins.
In the HHR model of HFpEF, a distinctive cardiomyocyte Ca 2 +
dysregulation during progression to overt decompensated heart failure is revealed. This strongly supports the contention that progression to HFpEF has a cellular phenotype which is different to that
observed in failure linked with reduced systolic dysfunction and
ejection fraction (HFrEF). These ndings demonstrate that therapies
directed to increasing cardiomyocyte Ca2 + operational levels as appropriate for HFrEF phenotype may not be effective, and may be detrimental, in the HFpEF context.

S34

Abstracts

WE-013
Alda-1 improves cardiac function in the heart failure mice carrying
human aldehyde dehydrogenase 2 E487K variant
Vanessa Lima1, Ivson Silva1, Cintia Ueta1, Rafael Dariolli2, Leonardo
Jensen2, Jos Eduardo Krieger2, Maria Cludia Irigoyen2, Julio Ferreira1
1

Institute of biomedical science, University of Sao Paulo, Sao Paulo, SP,

Brazil
2
Heart Institute, University of Sao Paulo, Sao Paulo, SP, Brazil
The aldehyde dehydrogenase 2 (ALDH2) located in the mitochondrial matrix is crucial for the maintenance of cellular aldehydic balance. It
plays a important role in metabolizing reactive aldehydes produced
during oxidative stress. Currently, it is estimated that 8% of the world
population have a point mutation in the ALDH2 gene (E487K) which reduces its enzymatic activity by 95%. We assess the impact of the E487K
variant of ALDH2 on cardiac function in myocardial infarction-induced
heart failure. For that, we used heterozygous and homozygous ALDH2
E487K knock-in and WT mice. We observed that sham mice carrying
the ALDH2 variant have a reduced ALDH2 activity and protein levels
compared to WT mice. We have also seen that animals with ALDH2 mutation develop cardiac dysfunction and ventricular remodeling equivalent to WT animals after myocardial infarction. However, animals with
the mutation have a signicant reduction in the basal and maximum oxygen consumption (estimated by respirometry and maximal running
test). Isolated heart mitochondria from mutant mice validated the
in vivo ndings of reduced oxygen consumption. Finally, the sustained
Alda-1 treatment (an ALDH2 allosteric activator) improved cardiac
function of infarcted animals, regardless of genotype. Taken together
our data suggest that mice carrying ALDH2 E487K (which is probably
the most common human enzyme decient worldwide) are responsive
to Alda-1 treatment, even though the have lower ALDH2 protein levels
and activity.

WE-018
Cardiac anti-brotic effects of direct AT2 and Mas receptor stimulation in stroke-prone spontaneously hypertensive rats
Dhaniel Baraldi
Monash University, Melbourne, Victoria, Australia
Background: Angiotensin II type II receptor (AT2R) and Mas receptor (MasR) belong to the protective arm of the RAS, with AT2R or MasR
stimulation known to evoke a number of cardiovascular effects, including acute vasodilatation and chronic anti-brotic effects. Compound 21
(C21) is the prototypical AT2R agonist, while Ang (1-7) has mainly been
used in chronic studies to stimulate MasR, although being relatively
nonselective. Therefore, we examined if selective AT2R (using C21) or
MasR (using AVE0991) stimulation evokes similar anti-brotic phenotypes to that of combination treatment, which may implicate similar
signalling mechanisms.
Methods: To investigate if AT2R and MasR pharmacological costimulation provide additional protection against end-organ damage
in stroke-prone spontaneously hypertensive rats (SP-SHR) than either
treatment alone. Adult male SP-SHR, aged 20-22 weeks, were treated
for 4 weeks with either saline (n = 7), AT2R agonist C21 (0.03
mg/kg/day, n=6), MasR agonist AVE0991 (24 g/kg/h, n=3), or a combination of both (n=4), subcutaneously via osmotic mini-pump. Blood
pressure (tail-cuff) was measured at days 0, 14 and 28 of the protocol.
At the end of treatment, indices of aberrant cardiac remodelling (cardiac
hypertrophy and interstitial brosis) were quantied.
Results: None of the treatments inuenced elevated blood pressure
or cardiac hypertrophy in SP-SHR. However, cardiac interstitial brosis
as collagen volume fraction (assessed by picrosirius red staining) was
strikingly attenuated from control levels (5.1%) to approximately half

those levels by each treatment (2.5%, 2.4% for C21 and AVE0991, respectively, both Pb 0.01 versus untreated) while there was no additive antibrotic effect of combination treatment (2.3%). Similar signicant reductions were noted for vimentin and -smooth muscle actin immunoreactivity suggesting that treatments reduced broblast number and
differentiation to synthetic myobroblasts.
Conclusion: Pharmacological stimulation of AT2R and/or MasR exhibited marked cardiac anti-brotic effects without inuencing blood
pressure. Ongoing studies will address whether similar mechanisms
contribute to altered extracellular matrix.

WE-019
A simplied, Langendorff-free method for concomitant isolation of
viable cardiac myocytes and broblasts from the adult mouse heart
Matthew Ackers-Johnson1,2, Peter Li2, Roger Foo1,2
1

Genome Institute of Singapore, Singapore, Singapore

National University of Singapore, Singapore, Singapore

Objective: Recent advances in mouse genomics, epigenomics and

transgenics offer huge potential for research in murine models of
heart disease. However, the isolation of viable cardiac myocytes from
the adult mouse heart is particularly challenging to most labs worldwide. Every established protocol to date relies on Langendorff apparatus
or equivalent to allow retrograde aortic perfusion and digestion of the
myocardium. This 45-year-old technique presents signicant logistical,
technical and nancial barriers, and requires considerable training investment. We therefore sought to re-invent an alternative.
Method: We have developed a simplied method to introduce
optimised digestion buffers to the intact mouse heart by intraventricular injection. Deep myocardial perfusion via the coronary vasculature
was induced by clamping of the aorta. Myocyte fractions were subsequently separated from non-myocytes by gravity settling, and cells
were analysed by quantitative real-time PCR, immunocytochemistry,
calcium imaging and an xCELLigence RTCA CardioECR system to conrm
healthy, viable properties, in addition to characteristic responses to hypoxia, neurohormonal and electrical stimulation.
Results: The technique elicits viable myocyte yields of up to 80%,
which meets and even exceeds those reported in previous
Langendorff-based protocols. Myocytes could be maintained in
culture for a week and displayed a full range of morphological
and functional contractile characteristics, while cardiac broblasts
could be concurrently cultured from the non-myocyte cellular
fraction.
Conclusion: We propose a robust, convenient protocol for the isolation of adult mouse cardiac myocytes. The procedure is simple, exible,
does not require heparin pre-injection and uses only common surgical
and laboratory equipment. We further demonstrate concurrent isolation and culture of myocyte and broblast populations, from the same
adult mouse heart.

WE-020
Study of a possible paracrine communication between cardiac broblasts and myocytes induced by Galectin-3
Mario Bustamante1,3, Ingrid Oyarn1,3, Georthan Mancilla1,3, Clara
Quiroga1,3, Hugo E. Verdejo1,3, Sergio Lavandero2,3, Pablo Castro1,3
1
Lab. de Sealizacin Cardiovascular, Divisin de Enfermedades
Cardiovasculares, Facultad de Medicina, Ponticia Universidad Catlica
de Chile, Santiago, Chile
2
Lab. de Transduccin de Seales Moleculares, Facultad de Cs. Qumicas y
Farmacuticas, Universidad de Chile, Santiago, Chile
3
Advanced Center for Chronic Diseases (ACCDiS), Universidad de Chile &
Ponticia Universidad Catlica de Chile, Santiago, Chile

Abstracts

Introduction. Galectin-3 is a -galactoside-binding protein that is

being evaluated as a biomarker for the development of heart failure
(HF). By now it is unknown if Gal-3 has a direct role in cardiac disease
progression. On the other side, TGF-1 is a cytokine related largely to
cardiac remodelling and cardiac disease progression. Here, we show
that Gal-3 has no obvious effect over cardiomyocytes biology. Instead,
it activates cell signalling cascades and tgfb1 gene expression in broblasts. Our results suggest that a paracrine communication between broblasts and cardiomyocytes by means of TGF-1 is established in
response to Gal-3, explaining to some extent the deleterious actions of
Gal-3 over cardiac tissue.
Methodology. Primary cultures of cardiac myocytes and broblasts
were stimulated with Gal3 10 ug/ml. Cell death was evaluated by
Flow cytometry through PI incorporation and MTT assay. The activation
of signaling pathways was evaluated by western blot, while mRNA expression was analysed by RT-qPCR.
Results. The results obtained showed that Gal-3 has no effect over
cardiomyocytes, at least at the times and concentrations used here.
When broblasts were stimulated with Gal-3 the phosphorylation of
ERK1/2 and AKT as well as the expression of tgfb1 was increased. The
supernatant obtained from Gal-3-stimulated broblasts provokes a hypertrophic effect onto cardiomyocytes and the increase of anp mRNA
expression. TGF-1 released into the culture media of broblasts could
mediate this effect.
Conclusions. Gal-3 is a biomarker for the development of HF, showing a straightforward relationship between plasma levels of Gal-3 and
the impairment of cardiac function. Our results show for the rst time
the activation of pro-survival signaling pathways and TGF-1 expression in broblasts in response to Gal-3.
Supported by: FONDECYT #3160287 (MB), #11140470 (CQ),
#1150359 (HEV), #1141198 (PC) and FONDAP #15130011 (SL, PC)

WE-021
Identication of emerging micro-rna markers for heart failure
development
Geortan Mancilla1,2, Ingrid Oyarzn1,2, Rocio Artigas2, Ignacio
Wichmann2, Alejandro Corvalan2, Clara Quiroga1,2, Hugo Verdejo1,2,
Pablo Castro1,2
1

Lab. de Sealizacin Cardiovascular, Divisin de Enfermedades

Cardiovasculares, Facultad de Medicina, PUC, Santiago, Chile
2
Advanced Center for Chronic Diseases (ACCDiS), Universidad de Chile &
Ponticia Universidad Catlica de Chile, Santiago, Chile., Santiago, Chile
Purpose: Heart failure is the nal stage of several cardiovascular diseases. Despite the health burden associated with this pandemic, biomarkers aimed to assess the individual risk of developing HF are still
lacking. Advances in bioinformatics have accelerated the pre-analytic
phase of biomarker research using data mining strategies; we aim to
identify potential microRNA (miR) biomarkers in plasma of nonischemic HF patients combining in silico and in vivo approaches.
Methods: We systematically reviewed the literature for miR proling
and HF. Four studies fullled quality criteria for analysis. Raw data was
obtained from public databases. The largest dataset was normalized and
analyzed by unsupervised hierarchical clustering to identify differentially
expressed miRs using a fold change of 2 as cut-off with a false discovery
rate b 1%. Results from the discovery dataset were contrasted with previously reported miRs using robust rank aggregation. We validate our predicted miRs in plasma samples from 12 HF patients and 5 healthy controls
by RT-qPCR. miR-39 was used to standardize for extraction procedures.
Results: We identied nine differentially expressed miRs (let-7b,
miR-100, miR-103, miR-199a and miR-23a), including three previously
unreported (miR-125b, miR-140 and miR-15b). Relative expression of
miR-23a was markedly up-regulated in plasma of HF patients
(p = 0.05). On the other hand, miR-140 was signicantly down-

S35

regulated HF patients (p=0.03). Interestingly, miR-140 participates in

the regulation of several genes of the Wnt and Akt/mTOR pathways,
critical in the transition from compensate hypertrophy to overt HF
(p=1.21e-08 for interaction).
Conclusion: Bioinformatics analysis allows to identify previously
unreported miRs involved in HF development. This is a novel approach
using public access data for identifying new potential biomarkers such
as miR-140; its biological role in HF development or progression remains to be elucidated.
Support by: FONDECYT #11140470 (CQ), #1150359 (HEV),
#1141198 (PC) and FONDAP #15130011 (AC, PC).

WE-022
Role of carbonic anhydrase ix and hypoxia-inducible factor 1 in infarcted rat heart.
Mariela Nolly, Andrs Pinilla, Juliana Fantinelli, Patricio Morgan
Centro de Investigaciones Cardiovasculares, CONICET., La Plata, Argentina
Background. Acute myocardial infarction (MI) remains a leading
cause of morbidity and mortality worldwide. MI refers to an oxygen
blood perfusion reduction, hypoxia, severely altering cardiac function
and myocardial energy metabolism. Studies made on hypoxic tissues
of solid tumors revealed an increase expression of Hypoxia-Inducible
Factor 1 (HIF-1). This transcription factor translocates to the nucleus,
binds to DNA elements and stimulates the transcription of several
genes including Carbonic Anhydrase IX (CAIX). During MI the increase
expression of HIF-1 reduces infarct size, improving cardiac function.
We have previously shown that heart CAIX plays a critical role in regulating myocardial intracellular pH, interacting with bicarbonate transporters (BT). However, the role of CAIX in MI is unknown.
Objetive. Our goal is to evaluate CAIX expression in ischemic myocardium and its relation to HIF-1 and BT.
Methods. To analyze the role of HIF-1, CAIX and BT in MI we used
adult male Wistar rats. MI was produced by permanent ligation of the
left anterior descending coronary artery in vivo and analyzed by histology. Heart samples were obtained from infarct, peri-infarct and remote heart regions. Expression of HIF-1, CAIX and BT was analyzed by
western blot. Also, interaction of CAIX-BT was assessed by coimmunoprecipitation and colocalization.
Results and conclusions. Infarcted Wistar rats showed a signicant
increased expression of HIF-1 and CAIX in the peri-infarct regions, compare to the remote heart areas. Peri-infarct regions show a marked
physical interaction between CAIX and sodium bicarbonate cotransporter (NBC1). These results suggests that HIF-1 and its downstream target, such us CAIX, interacting with BT may improve cellular
pH surroundings and survival mechanisms possibly attenuating progression of cardiac dysfunction after MI.

WE-023
The cMyBP-C E258K HCM-causing mutation does not affect mRNA
splicing
Willem De Lange, Nicole Bednarz, Richard Moss, Carter Ralphe
University of Wisconsin, Madison, Wisconsin, USA
Hypertrophic cardiomyopathy (HCM) is the most commonly
inherited cardiovascular disease, affecting approximately 0.2% of the
general population. Mutations in MYBPC3, encoding cardiac myosin
binding protein-C (cMyBP-C), are common causes of HCM. Many
MYBPC3 mutations cause aberrant mRNA splicing, leading to cMyBP-C
truncation and cause disease through a mechanism of
haploinsufciency. The E258K mutation in MYBPC3, a prevalent cause
of HCM, has been postulated to alter splicing due to its location in the

S36

Abstracts

exon 6 splice donor site. Our previous data, however, indicated that it
may act in a dominant negative manner by altering interactions with
myosin-S2 and actin.
Here we investigate whether the E258K mutation alters RNA splicing and act through a mechanism of cMyBP-C haploinsufciency, or as
a true dominant negative missense mutation by assessing mRNA and
protein levels in an E258K knock-in mouse model.
Applying an array of RT-PCR primers designed to detect all potential
miss-spliced transcripts arising from this mutation no aberrantly spliced
Mybpc3 transcripts were found in mice heterozygous for E258K. Additionally, Myocardium expression of cMyBP-C protein in either heterozygous or homozygous E258K mice was similar to that of wild type control
littermates and lacked evidence of truncated cMyBP-C. Interestingly, the
E258K mutation results in reduced phosphorylation levels of cMyBP-C
at S273 and S302, without affecting phosphorylation S282.
In this murine model, the E258K mutation does not affect mRNA
splicing and does not appear to act through a mechanism of cMyBP-C
haploinsufciency. We previously showed that E258K cMyBP-C reduces
its afnity for myosin S2 while increasing its afnity for actin, resulting
in reduced twitch force amplitude and accelerated contractile kinetics.
Taken together, these results suggest that this mutation acts in a dominant negative fashion.

developing novel ways to modulate the actomyosin contractile apparatus is of growing interest and importance. The nucleotide analog 2deoxyadenosine triphosphate (dATP) has recently garnered interest as
potentially having therapeutic benet for treatment of systolic and/or
diastolic heart failure. dATP has been previously reported to enhance
cardiac contractility, increase +dP/dt, and improve diastolic relaxation
parameters in transgenic mice with elevated levels of dATP in the heart
(Korte, 2011). To better understand potential therapeutic benets of
dATP on actomyosin, we characterize the mechanism of action for
dATP using bovine cardiac myosin subfragments S1 and HMM in a
variety of steady-state, transient, and single-molecule experiments.
We report a 40% increase in unloaded in vitro motility sliding velocities, as well as increased ATPase activity, ADP- and phosphaterelease rates, and actin-binding afnities with dATP compared to
ATP. The combination of transient kinetic rates and equilibrium constants of the actomyosin ATPase cycle, as well as basal myosin parameters, implicate ADP release as the primary contributor to the
differences observed between the two nucleotides. We propose a
model by which enhancing both cardiac contraction and relaxation
kinetics can improve cardiac function and potentially serve as a therapeutic for genetic heart disease.

WE-024
Neuregulin-1 Modulates Doxorubicin Cardiotoxicity In Mouse
Marina Bonanno, Abigail Perez Abraham, Agustn Rizzo, Hernn Garca
Rivello, Cecilia M. Hertig

WE-026
Frailty, not age, predicts age-dependent cardiac contractile dysfunction under basal and ischemic conditions in Langendorff-perfused
hearts from C57BL/6J mice
Hirad Feridooni1, Arash Boroumandi2, Nazari Polidovitch3, Robert Rose1,
Robert Tsushima2, Susan Howlett1

INGEBI, Buenos Aires, Argentina

Neuregulin-1 (NRG1) signaling through tyrosine kinase receptors
erbB2 and erbB4 is required for cardiac morphogenesis, and plays an essential role in maintaining the myocardial architecture during adulthood. Targeted immunotherapies blocking the survival of erbB2 +
cancer cells revealed that an impaired NRG1 signal under anthracycline
chemotherapy may lead to dilated cardiomyopathy in a subpopulation
of treated patients. The ventricular-specic deletion of ErbB4 (erbB4KO) manifested dilated cardiomyopathy, aggravated by the administration of anthracyclines (doxorubicin) (KOD). The exacerbated toxicity in
KOD induced genes of the ubiquitin-proteasome system and autophagy.
Myobril proteins were largely ubiquitinated with the commonality of a
subgroup of proteins in the erbB4-KO and the doxorubicin mice WTD.
We aimed to investigate the activities underlying cardiomyocyte damage and moreover, to evaluate the therapeutic effect of recombinant
NRG1 peptides. We rst examined biomarkers of apoptosis and autophagy (e.g. active caspase3, LC3II/I), then characterized the
ubiquitination prole of myobrils in 2D gels towards the monitoring
of the rNRG1 effect through the reversion of the molecular modications observed in cardiotoxic conditions. We have identied new consistent biomarkers of pathology and suggest that rNRG1 protects
from cardiotoxic injury.

WE-025
2-deoxy-ATP enhances multiple kinetic parameters to improve cardiac function
Ivan Tomasic, Marcus Henze, Ferdinand Evangelista, Anu Anto, Hector
Rodriguez, Sadie Bartholomew Ingle
MyoKardia, Inc., South San Francisco, CA, USA
Hypertrophic cardiomyopathy (HCM) is a form of genetic heart disease often caused by point mutations in sarcomeric proteins. As the underlying mechanisms of genetic HCM continue to be unraveled,

Dalhousie University, Halifax, Canada

York University, Toronto, Canada
3
University of Toronto, Toronto, Canada
2

Frail patients with cardiovascular disease (CVD) experience

worse outcomes and higher mortality than non-frail patients, but
the links between frailty and myocardial function are unclear. Here
we investigated the impact of age and frailty on cardiac hemodynamic function under control conditions and after ischemia/reperfusion. Frailty was measured in male C57BL/6J mice (755-882 days; n
= 18) using a novel frailty index (FI) developed in our laboratory
based on the clinical assessment of health decits. Hypertrophy
was assessed by measuring heart weight to tibia length (HW:TL) ratios. The HW:TL ratio increased with frailty (r = 0.42, P = 0.01) but
not with chronological age (r = 0.24, P = 0.15). Langendorffperfused hearts were used to measure left ventricular developed
pressure (LVDP), rate of pressure development (+ dP/dt), rate of
pressure decay (-dP/dt), and incidence of arrhythmias under
normoxic conditions. Under these conditions, LVDP (r = 0.64;
P = 0.004), + dP/dt, (r = 0.61; P = 0.01), and -dP/dt (r = 0.58;
P = 0.01) declined dramatically as FI scores increased. However,
chronological age did not affect LVDP, + dP/dt, or -dP/dt (r = 0.11,
0.10, and 0.11 respectively; P = 0.66, 0.70, and 0.65 respectively).
Furthermore, frailty increased the incidence of arrhythmias
(r = 0.55; P = 0.02) while age did not (r = 0.11; P = 0.66). Hearts
were then exposed to 30 min of ischemia followed by 40 min of reperfusion. Interestingly, contracture, a marker of ischemic damage,
increased with frailty (r = 0.52; P = 0.03) but not with increasing
age (r = 0.03; P = 0.92). By contrast, recovery of contractile function
was poor after reperfusion in all aged hearts regardless of FI score or
age. These results suggest that age-dependent hypertrophy, cardiac
contractile dysfunction, and arrhythmias are more closely linked to
frailty than chronological age. Thus, frailty disrupts cardiac structure,
function, and may increase susceptibility to ischemic damage in the
aging heart.

Abstracts

WE-028
Cardiac-protection of acetylcholine on ischemia/reperfusion injury
via regulation of TNF-/TNFR signal pathway
Dong-Ling Li, Jin-Jun Liu, Xiao-Jiang Yu, Wei-Jin Zang
Department of Pharmacology, Health Science Center, Xian Jiaotong University, Xian city, Shaanxi Province, China
Background: Recent studies reported ischemic heart disease is accompanied by substantial withdrawal of vagal activity, and overproduction of tumor necrosis factor-alpha (TNF-) worsen cardiac injury. However, it is not fully clear that the replacement of ACh for myocardial ischemia/reperfusion (I/R) modulated the production of TNF- and
TNF- receptor1/2 (TNFR1/2) signal pathway.
Methods: Langendorff- perfused rat hearts and H9c2 cells were subjected to global ischemia and reperfusion, or hypoxia/reoxygenation, respectively. Real-time PCR, western blot, TUNEL and Si RNA were used.
Results: 1) ACh abolished hypoxia-induced up-regulation of TNF-
mRNA and protein, caspase-3 activation, and reactive oxygen species
(ROS) in cardiomyocytes. ACh treatment prevented the hypoxiainduced increase in p38 MAPK and JNK phosphorylation, and increased
ERK phosphorylation in H9c2 cells. Co-treatment with atropine, a nonselective muscarinic acetylcholine receptor antagonist, or
methoctramine, a selective type-2 muscarinic acetylcholine (M2) receptor antagonist, abrogated the above effects of ACh.
2) Following Langendorff- perfused rat myocardial I/R injury, the
cardiac dysfunction and myocardial infarction signicantly increased
and the expression of TNFR1, apoptosis signal regulating kinase 1
(ASK1) and activated caspase-8 were increased in left ventricle. Instead
of TNFR1, TNFR2, Akt and ERK were not affect by I/R. Treated with ACh
not only improved the cardiac function, decreased infarction area and
apoptosis by TUNEL and Bcl-2/Bax, but also down-regulated the expression of TNF- and TNFR1, and reduced the activity of ASK1 and caspase8, nally inhibiting the cardiomyocyte apoptosis. Meantime, ACh upregulated TNFR2 expression, Akt and ERK phosphorylation, which involved in survival pathway to protect myocardium against I/R injury.
3) Si RNA TNFR1 in H9c2 cells reduced HR-induced phosphorylation
of ASK1 and caspase-3 activation. In addition, Si RNA TNFR2 eliminated
ACh-increased phosphorylation of Akt and ERK after HR in H9c2 cells.
Conclusion: ACh protected myocardium against I/R injury via inhibition TNF- production and regulation of TNFR1/2 pathway.

WE-029
Acute hyperglycemia abolishes cardioprotection by remote ischemic
perconditioning
Tams Baranyai1, Csilla Terzia Nagy1, Gbor Koncsos1, Zsa Ondi1,
Andrs Makkos1, Zoltn V. Varga1, Pter Ferdinandy1,2, Zoltn Giricz1,2
1

Department of Pharmacology and Pharmacotherapy, Semmelweis University, Budapest, Hungary

2
Pharmahungary Group, Szeged, Hungary

Background: Remote ischemic perconditioning (RIPerC) has a

promising therapeutic insight to improve the prognosis of acute myocardial infarction. Chronic comorbidities such as diabetes are known
to interfere with conditioning interventions by modulating
cardioprotective signaling pathways, such as e.g., mTOR pathway and
autophagy. However, the effect of acute hyperglycemia on RIPerC has
not been studied so far. Therefore, here we investigated the effect of
acute hyperglycemia on cardioprotection by RIPerC.
Methods: Wistar rats were divided into normoglycemic (NG) and
acute hyperglycemic (AHG) groups. Acute hyperglycemia was induced
by glucose infusion to maintain a serum glucose concentration of
1520 mM throughout the experimental protocol. NG rats received
mannitol infusion of an equal osmolarity. Both groups were subdivided

S37

into an ischemic (Isch) and a RIPerC group. Each group underwent reversible occlusion of the left anterior descending coronary artery
(LAD) for 40 min in the presence or absence of acute hyperglycemia.
After the 10-min LAD occlusion, RIPerC was induced by 3 cycles of 5min unilateral femoral artery and vein occlusion and 5-min reperfusion.
After 120 min of reperfusion, infarct size was measured by triphenyltetrazolium chloride staining. To study underlying signaling mechanisms,
hearts were harvested for immunoblotting after 35 min in both the
NG and AHG groups.
Results: Infarct size was signicantly reduced by RIPerC in NG, but
not in the AHG group (NG + Isch: 46.27 5.31 % vs. NG + RIPerC:
24.65 7.45 %, p b 0.05; AHG + Isch: 54.19 4.07 % vs. 52.76
3.80 %). Acute hyperglycemia per se did not inuence infarct size,
but signicantly increased the incidence and duration of arrhythmias. Acute hyperglycemia activated mechanistic target of
rapamycine (mTOR) pathway, as it signicantly increased the phosphorylation of mTOR and S6 proteins and the phosphorylation of
AKT. In spite of a decreased LC3II/LC3I ratio, other markers of autophagy, such as ATG7, ULK1 phopsphorylation, Beclin 1 and
SQSTM1/p62, were not modulated by acute hyperglycemia. Furthermore, acute hyperglycemia signicantly elevated nitrative stress in
the heart (0.87 0.01 vs. 0.50 0.04 g 3-nitrotyrosine/mg protein,
p b 0.05).
Conclusions: This is the rst demonstration that acute
hypreglycemia deteriorates cardioprotection by RIPerC. The mechanism of this phenomenon may involve an acute hyperglycemiainduced increase in nitrative stress and activation of the mTOR pathway.

WE-030
LAPTM4b protects hearts from ischemia/reperfusion injury by
promoting autophagy ux
Shan-Shan Gu, Jin-Long Liu, Ji-Liang Tan, Yan-Jun Zheng, Xu-Xia Li,
Qiang Li, Huang-Tian Yang
Institute of Health Sciences, Shanghai Institutes for Biological Sciences
(SIBS), Chinese Academy of Sciences & Shanghai Jiao Tong University
School of Medicine, shanghai, China
Myocardial injury following ischemia/reperfusion (I/R) is a common
clinical scenario in patients suffering from ischemic heart disease. During myocardial I/R over-activated autophagy and accumulated
autophagosome contribute to cardiomyocytes death. Thus, understanding how to accelerate autophagosome clearance and promote autophagy ux is important for the development of new cardioprotective
approaches to alleviate I/R injury. Lysosome is responsible for the eventual degradation of autophagosome, however, how to promote lysosome ux via the regulation of lysosome function remains poorly
understood. In the present study, we found a lysosomal membrane protein lysosomal-associated transmembrane protein 4b (LAPTM4b) was
down regulated during myocardial I/R. Overexpression of LAPTM4b in
neonatal rat cardiomyocytes preserved cell viability from hypoxia/reoxygenation (H/R) injury. Moreover, overexpression of LAPTM4b activated lysosomal function and promoted autophagy ux
characterized by a decrease in the autophagosome of H/R
myocytes,while knockdown of LAPTM4b blocked autophagy ux
and aggravated cell death. We then constructed LAPTM4b knockout
(LAPTM4b-/-) mice by using Crispr/Cas9 system. The size of myocardial infarction/area at risk after I/R was signicantly larger in
LAPTM4b-/- mice than that in wide-type mice. Our results rstly report the involvement of a lysosomal protein LAPTM4b in myocardial
I/R injury through the regulation of autophagy ux and acceleration
of autophagosome clearance.
Key words: LAPTM4b; autophagy ux; ischemia/reperfusion injury;

S38

Abstracts

WE-031
The Role of Calcium-sensing Receptors and Spermine in Hypoxiainduced Pulmonary Vascular Remodeling and the Mechanism
Can Wei, Xue Peng, Guangwei Li, Changqing Xu
Harbin Medical University, Harbin, China
Background: Pulmonary vascular remodeling(PVR) is an importent
pathological feature of hypoxia-induced pulmonary hypertension
(HPH), which exact mechanism is unknown. Calcium-sensing receptor
(CaSR) is an G-protein coupling receptor, and spermine is a polyamine.
Methods: We estabolished rat hypoxia models in vivo and in vitro
by nitrogen or cobalt chloride (CoCl2), and observed CaSR expression,
polyamine metabolism, PVR related parameters and signal pathways
by RT-PCR, Western blotting, immunouorescence, immunohistochemistry, confocal laser scanning microscopy, ow cytometric assay etc.
Results: Under hypoxic conditions, the expressions of CaSR, SSAT(a
key enzyme of polyamine degradation), PCNA, OPN (osteopontin) and
p-ERK, the intracellular concentration of calcium, the survival rate of
cells and cell proliferation index (PI) were markedly increased, while
the expressions of ODC(a key enzyme of polyamine biosynthesis),
SM-actin (SMA) and calponin were signicantly reduced. The agonists of CaSR (GdCl3,Neomycin) enhanced but antagonist of CaSR
(NPS2390) weakened the hypoxic effect. PD98059 (a MEK1 inhibitor)
or LY294002 (a PI3K inhibitors) reversed the upregulation of PCNA expression and the increase of cell proliferation index induced by
hypoxiain PASMCs. Exogenous spermine at low concentrations signicantly inhibited hypoxia induced PASMC proliferation, leading to cell
cycle arrest at the G1/G0 phase, decreased cyclin D1 expression, increased p27 expression, and suppressed the phosphorylation of ERK1/
2, PI3K and AKT.
Conclusions: CaSR activation and polyamine disbalance are involved in the proliferation, phenotypic modulation of PASMCs and pulmonary vascular remodeling induced by hypoxia through MEK1/ERK1.2
and PI3K/AKT pathway. Exogenous spermineat inhibits the proliferation
of PASMCs. Our study thus offer new insight into the prevention and
treatment of hypoxia-induced pulmonary hypertension (HPH).

WE-032
Exogenous H2S Contributes to Recovery of Ischemic PostConditioning-Induced Cardioprotection in the Aging Rat and
Cardiomyocytes and the Related Mechanism
Hongzhu Li, Weiming Sun, Lina Li, Changqing Xu
Harbin Medical University, Harbin, China
Background: Ischemic post-conditioning (PC) plays an important
role in cardioprotection from ischemia/reperfusion (I/R) injury in
young heart but not in aging. The physiological and pathological roles
of hydrogen sulde (H2S) in the regulation of cardiovascular functions
have been recognized. Whether H2S is involved in the recovery of PCinduced cardioprotection in aging cardiomyocytes is unclear.
Methods: The aging rats (24-months-old) and the aging
cardiomyocytes induced by D- galactose suffer from I/R (H/R) and PC.
Western blotting, real time-PCR, TUNEL staining, confocal laser scanning microscopy, ow cytometric assay were used to detect apoptosis,
oxidative stress and related signal pathways.
Results: Both I/R (H/R) and PC decreased cystathionine--lyase
(CSE) expression and the production rate of H2S in aging heart. Supplementation of NaHS protected against I/R (H/R)-induced apoptosis, production increase of reactive oxygen species (ROS), the expression of
cleaved caspase-3 and cleaved caspase-9, the release of cytochrome c
(Cyt c), and mPTP opening. The addition of NaHS also counteracted
the reduction of cell viability caused by I/R (H/R) and increased the

phosphorylation of ERK1/2, PI3K, Akt, GSK-3 and mitochondrial membrane potential. Additionally, NaHS increased Bcl-2 expression, promoted PKC- translocation to the cell membrane, and activated
mitochondrial ATP-sensitive K channels (mitoKATP). PC alone did not
provide cardioprotection in I/R (H/R)-treated aging cardiomyocytes,
which was signicantly restored by the supplementation of NaHS.
Conclusion: The exogenous H2S restores PC-induced
cardioprotection in aging rat and cardiomyocytes via inhibition of oxidative stress and the inhibition of mPTP opening by the activation of
the ERK1/2-GSK-3, PI3K-Akt-GSK-3 and PKC--mitoKATP pathways.
These ndings provide a novel potential target for the treatment of
aging ischemic cardiomyopathy.

WE-033
Cardiomyocyte-specic Runx1 deciency protects the heart from
ischemia-reperfusion injury in vivo.
Ashley Cochrane1, Weihong He1, Charlotte McCarroll1, Peter Bowman1,
Stuart Nicklin1, Ewan Cameron2, Christopher Loughrey1
1

Glasgow Cardiovascular Research Centre, Institute of Cardiovascular and

Medical Sciences, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, UK
2
School of Veterinary Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow, Garscube Campus, Bearsden Road, Glasgow,
UK
Coronary artery blockage leading to prolonged myocardial ischemia
and cardiomyocyte cell death (myocardial infarction; MI) is a leading
cause of death worldwide. Current treatment options include reperfusion of the myocardium to salvage reversible damage and limit further
irreversible cell death. Paradoxically, the efciency of this treatment is
limited by ischemia-reperfusion injury. Identication of novel targets
that have the potential to limit cardiac dysfunction caused by
ischemia-reperfusion injury are urgently required. The transcription
factor, Runx1, is activated in human and mouse cardiomyocytes postMI. The functional role of Runx1 during ischemia-reperfusion injury remains unknown. Furthermore, whether Runx1 is also increased in an intermediate sized species, which could be utilised for future translational
studies, has not been characterised. Here we show the importance of
Runx1 during ischaemia-reperfusion injury using an in vivo mouse
model of Runx1 deciency and in separate experiments demonstrate
that Runx1 is increased in rabbit myocardium post-MI. Echocardiography of a cardiomyocyte-specic Runx1-decient mouse with ischemia
reperfusion injury demonstrated preserved left ventricular function as
measured by fractional shortening compared to control mice (45% versus 29% at 5 wk post-ischemia reperfusion; Pb0.05). Western blot analysis revealed increased expression of Runx1 protein levels in the border
zone and left ventricular region of post-MI rabbit hearts to 175% and
250% of sham levels respectively (Pb0.05). These results demonstrate
that Runx1 is a novel therapeutic target post-MI; in particular deciency
of Runx1 within cardiomyocytes is cardio-protective against ischemiareperfusion injury. Increased expression of Runx1 within the rabbit reveals that this larger species could be used in future translational
studies.

WE-034
Simultaneous Ultrasound Diagnosis and Treatment of Thrombosis
using Activated Platelet Targeted Theranostic Microbubbles
Xiaowei Wang1,2, Yannik Gkanatsas1, Jathushan Palasubramaniam1, Jan
David Hohmann1, Christoph Hagemeyer2, Karlheinz Peter1,2
1

Baker IDI Heart and Diabetes Institute, Melbourne, Australia

Monash University, Melbourne, Australia

Abstracts

Molecular ultrasound imaging is an attractive non-invasive technology widely available for rapid clinical diagnosis. We hypothesized that
thrombolytic drugs loaded microbubbles (MBs), which are selectively
targeted to activated platelets, will allow high-resolution, real-time imaging of thrombosis, and at the same time offer potent thrombolytic efcacy without bleeding complications, and enable the immediate
monitoring of success or failure of thrombolysis.
Our therapeutic agents/imaging particles, targeted theranostic
microbubbles (TT-MB), consist of a fusion construct that combines the
brinolytic drug urokinase, echo-enhancing microbubbles for visualization by ultrasonography, and an activated-platelet-specic single-chain
antibody for targeting specically to thrombi. In the ferric-chloride induced carotid artery thrombosis mouse model, treatment with TT-MB
signicantly reduced thrombus size after 45 min, while no signicant
difference was observed in the MB that were targeted but without urokinase (37.09 5.6 vs. 97.16 4.3, mean % change SEM, normalized
to baseline thrombus size, p b0.001). The same degree of efcient
thrombolysis was only achievable using a high dose of urokinase (NS).
We also show that the targeting and thus clot-enrichment effect of TTMBs results in a highly potent brinolysis that could only be matched
using high doses of non-targeted urokinase. However, the latter is associated with a highly prolonged bleeding time (79.25 6.5 vs. 1079.25
260.7, seconds SEM, pb0.001). In contrast, TT-MB does not prolong
bleeding time (NS).
In conclusion, activated platelet targeted microbubbles conjugated
with recombinant urokinase represent a novel and unique theranostic
approach to simultaneously diagnose and treat thrombosis as well as
to immediately monitor success or failure of thrombolysis. This unique
technology holds promise for major progress towards rapid diagnosis
and bleeding-free, potent therapy of the vast number of patients suffering from thrombotic diseases.

WE-035
Acetylcholine to Improve Calcium Dyshomeostasis in Cardiovascular
Disease: Attenuated ER-PM contacts
Ming Zhao, Long-Zhu Liu, Yi Lu, Xi He, Hang-Huan Jia, Xiao-jiang Yu,
Man Xu, Dong-Ling Li, Wei-jin Zang
Department of Pharmacology, Xian Jiaotong University Health Science
Center, Xian, China
Background: The endoplasmic reticulum (ER) is an important
organelle for the protein homeostasis and calcium (Ca2 +) storage
in cells. It forms discrete junctions with the plasma membrane
(PM) and membranes of organelles (such as mitochondria) that
play critical roles in Ca2 + signaling during cellular bioenergetics,
apoptosis and autophagy. We have conrmed that acetylcholine
(ACh), the neurotransmitter of vagal nerve, could inhibit ER stress
and protected cells in inammatory injury, as well as inhibit the
formation of ER-mitochondria junctions to attenuate [Ca2 +]mito
overload in hypoxia/reoxygenation HUVEC. However, limited researches focus on the formation or dissociation of ER-PM complex
in cardiovascular disease.
Objectives: In this work, we studied the structure and function of supramolecular complex involved in regulating Ca2+ homeostasis in cardiovascular disease.
Methods: The nanometers apart of ER-PM and ultrastructure of cell
were measured by transmission electron microscope. Protein-protein
interactions were measured by immunoprecipitation. Ca2+ concentration was measured by confocal microscope. The siRNA was employed
to silence specic proteins.
Results: 1. Our results rst demonstrated that the peripheral ER
translocation into PM-junction sites, while ER dilation and [Ca2+]ER depletion was induced by TNF-alpha. There was new NCX1-TRPC3-IP3R1
complex formed in the PM-junction sites. 2. The abdominal aortic

S39

coarctation promotes STIM1 junctional accumulation in rat heart, and

then formed the STIM1-Orai1 complex. 3. Above two ER-PM complex
involve in the [Ca2+]cyt overload and apoptosis. 4. Interestingly, the activated M3AChR by ACh could uncouple the NCX1-TRPC3-IP3R1 complex, then inhibit [Ca2 +]cyt overload and apoptosis. In fact, the
protective effect of ACh was depended on the M3/AMPK pathway.
Conclusion: ER mediated Ca2+ transport by connection of PM or mitochondria. ACh ameliorated Ca2+ dyshomeostasis by inhibition of ERPM and ER-mitochondria connection simultaneously. It is suggested
that the inhibition of ER-PM junction may be play an important role in
cardiovascular protection.
Keywords: acetylcholine; endoplasmic reticulum; ER-PM contacts;
[Ca2+]cyt overload; cardiovascular disease
Supported by: grant from National Natural Science Foundation of
China (Major International Joint Research Project, No. 81120108002;
General Project, No. 81473203 and 81402924), Specialized Research
Fund for the Doctoral Program of Higher Education (No.
20130201130008).
*Corresponding author:
Prof. Wei-jin Zang;
Department of Pharmacology, Xian Jiaotong University Health Science Center
P.O.Box 77 #, No.76 Yanta West Road, Xian, Shannxi 710061, (PR
China)
Tel: +86-29-82655150; Email: [email protected]

WE-037
Extracellular RNA induces ischemia/reperfusion injury by Tumor
Necrosis Factor (TNF-) Shedding: The role of TNF-Receptor 1
Hector Cabrera-Fuentes1,2, Sandrine Lecour3, Marisol Ruiz-Meana4,
David Garcia-Dorado4, Klaus Schlter5, Derek Hausenloy2, Klaus
Preissner1
1

Institute of Biochemistry, Medical School, Justus-Liebig-University, Giessen,

Germany
2
Cardiovascular & Metabolic Diseases Program, Duke-NUS Graduate Medical School, Singapore, Singapore
3
Hatter Institute for Cardiovascular Research, University of Cape Town,
Cape Town, South Africa
4
Hospital Universitari Vall dHebron, Laboratorio de Cardiologa Experimental, Barcelona, Spain
5
Institute of Physiology, Medical School, Justus-Liebig-University, Giessen,
Germany
Background - During acute myocardial infarction, cardiomyocyte
death has a great impact on the quality of life and survival of patients.
Despite reopening/reperfusion of stenosed vessels, major organ damage
remains. The initial mechanistic triggers of this myocardial ischemia/
reperfusion (I/R) injury remain greatly unexplained. Hypothesis - We
hypothesized that extracellular-RNA (eRNA), derived from damaged
tissue, and tumor-necrosis-factor-a (TNF-), may dictate cardiac I/R
injury.
Methods and results - Following myocardial I/R in mice or I/R induced in the isolated Langendorff rat heart, increased eRNA levels
were found together with cardiac injury markers such as troponin-I,
creatine-kinase and LDH. Likewise, eRNA was released from
cardiomyocytes under hypoxia and subsequently induced TNF- liberation by triggering TNF--converting-enzyme (TACE) to provoke cardiomyocyte death. Conversely, TNF- promoted eRNA release
especially under hypoxia, feeding a vicious cell damaging cycle during
I/R. Administration of RNase1 or TAPI (a TACE-inhibitor) prevented
cell death and myocardial infarction. Likewise, RNase1 signicantly reduced I/R-mediated energy exhaustion, opening of mitochondrialpermeability-transition-pores as well as oxidative damage in

S40

Abstracts

cardiomyocytes. Furthermore, as compared to isolated wild-type

cardiomyocytes, in TNF-receptor-1 and TNF- knockout cells, upon exposure to hypoxia cell viability decreased in a similar manner, but was
not further reduced in the presence of eRNA. In contrast, in TNFreceptor-2 knockout cells eRNA signicantly induced cell death, indicating that the lack of TNF- and TNF-receptor-1 prevented eRNA-induced
cell death. These ndings were corroborated by the observation that
TAPI-treatment of the isolated rat heart during an interval of 30min
prior to the ischemic phase signicantly decreased LDH release in comparison to the untreated I/R group.
Conclusions - RNase1 and TAPI provide novel therapeutic regimen
to interfere with the adverse eRNA-TNF- interplay and signicantly reduce or prevent the pathological outcome of ischemic heart disease.
This as yet unrecognized fundamental pathomechanism is likely to operate in other organs and tissues as well.

WE-038
Endogenous annexin-A1 is cardioprotective against myocardial infarction in mice in vivo
Cheng Xue Qin1,2, Siobhan B Finlayson1,3, Sarah Rosli1, Colleen J
Thomas3, Annas AI-Sharea1, Andrew Murphy1, Helen Kiriazis1, Yuan H
Yang4, Eric F Morand4, Xiao-Jun Du1, Xiaoming Gao1, Rebecca H
Ritchie1,2
1

Baker IDI Heart and Diabetes Institute, Melbourne, Australia

Department of Pharmacology, University of Melbourne, Melbourne,
Australia
3
La Trobe University, Bundoora, Australia
4
Centre for Inammatory Diseases, Monash University, Clayton, Australia
2

Background: Annexin-A1 (ANX-A1) is an endogenous antiinammatory protein that preserves left ventricular (LV) viability and
function after an ischemic insult in vitro. However, its cardioprotective
actions in vivo are largely unknown. The aim of this study was to test
the hypothesis that ANX-A1 decient (ANX-A1-/-) mice have an exaggerated detrimental response to myocardial infarction (MI) in vivo compare to their wild type counterparts.
Methods: Adult male ANX-A1+/+ and ANX-A1-/- mice were subjected to left anterior descending (LAD) coronary artery occlusion (1h)
followed by reperfusion (24h or 48h), permanent LAD occlusion (8
days) or sham operation.
Results: Compared to ANX-A1+/+ mice, ANX-A1-/- mice exhibited increased infarct size (24h; 34.81.7 vs. 49.35.4% pb0.05, n=8-9) and
increased LV macrophage content (48h; 54671 vs. 87386 macrophages/mm2; pb0.05, n=5-6). Eight days post-MI, there was a signicant 2-fold up-regulation of hypertrophic ANP expression in ANX-A1+/+
mice compared to sham animals (pb 0.05), which tended to be further
increased in ANX-A1-/- mice (p = 0.08). This corresponded with increased heart weight in ANX-A1+/+ compare to ANX-A1-/- mice
(5.6 0.2 vs. 7.3 0.4mg/g; p b0.001) and LV weight (4.2 1.2 vs.
4.9 0.2mg/g; p b0.05) relative to body weight. In addition, proinammatory TNF- and pro-brotic CTGF gene expression were increased 2-fold in ANX-A1+/+ mice, compared to a 7-fold elevation in
ANX-A1-/- mice (pb 0.05 vs. ANX-A1+/+), and this was associated with
increased LV collagen deposition after MI (192 vs. 41 7%, p b0.01,
n=5-7). Moreover, ANX-A1-/- mice exhibited greater expansion of the
hematopoietic stem cell population and altered pattern of mobilization
relative to ANX-A1+/+ mice after MI. Further, circulating neutrophil and
platelet (but not monocyte) numbers were signicantly increased in
ANX-A1-/- mice after MI compared to ANX-A1+/+, possibly as result of increased monocyte/macrophage inltration into the injured ANX-A1-/myocardium after MI.
Conclusion: In summary, ANX-A1-deciency increased cardiac necrosis, inammation, hypertrophy and brosis following MI. These ndings suggest endogenous ANX-A1 limits LV damage in vivo and supports

further development of novel ANX-A1 based therapies to improve cardiac outcomes after MI.

WE-039
HAX-1 regulates contractile recovery after ischemia/reperfusion
injury by preventing SERCA2a degradation
Philip Bidwell, Guan-Sheng Liu, Chi Keung Lam, Jack Rubinstein,
Evangelia Kranias
University of Cincinnati, Cincinnati, OH, USA
Cardiac SR calcium handling is critical for control of contractility,
bioenergenetics, and cell death. We have previously shown that a mitochondrial protein, HAX-1, is an interacting partner of phospholamban (PLN) and can modulate SERCA2a activity. HAX-1
overexpression increases the inhibitory effects of PLN on the Caafnity of SERCA2a, resulting in depressed Ca handling and contractility. To examine the functional role of endogenous HAX-1 in the heart,
we generated an inducible cardiac specic HAX-1 knockout model
(HAXcKO). Full ablation of HAX-1 in adult hearts signicantly enhanced SERCA2a activity, cardiomyocyte contractile parameters and
Ca-kinetics without altering levels of Ca handling proteins (SERCA2a,
PLN, RyR). The increased activity was half of that observed with PLN
ablation or isoproterenol stimulation, suggesting that 50% of the physiological inhibition of PLN is mediated by HAX-1. Additionally, no alterations in apoptotic and ER stress markers (caspase 3/12, GRP94,
and IRE-1) were associated with ablation of the anti-apoptotic HAX1 protein. However, HAX-1 decient hearts exhibited signicantly reduced functional recovery upon ex vivo ischemia/reperfusion injury
(I/R; 40 min no ow ischemia/60 min reperfusion). The rates of contraction/relaxation and left ventricular developed pressure recovered
to only 25% of pre-I/R levels in HAXcKO hearts, compared to the
50% recovery in WTs. This diminished recovery was partially attributed to 40% reduction in SERCA2a protein in HAXcKO hearts, compared
to a 20% decrease in WT. Accordingly, HAX-1 overexpression
prevented loss of SERCA2a protein after I/R and enhanced contractile
recovery. The alterations in SERCA2a degradation did not reect
changes in calpain 1 and 2 protein levels, while calpain activity was
equally increased in the HAX-models due to loss of the endogenous
calpain inhibitor, calpastatin. Thus, HAX-1 depresses SR Ca-cycling
but enhances functional recovery after ischemia/reperfusion, in part
by preventing the degradation of SERCA2a protein.

WE-040
Adenosine A1 receptor biased agonism in cardiac ischemiareperfusion injury
Jo-Anne Baltos, Chung Chuo, Andrew Kompa, Manuela Jorg, Henry
Krum, Arthur Christopoulos, Peter Scammells, Paul White, Lauren May
Monash University, Melbourne, Victoria, Australia
Background. Stimulation of the adenosine A1 G protein-coupled receptor (A1AR) is a powerful protective mechanism in cardiac ischemiareperfusion injury (IRI). Despite this, therapeutic targeting of the A1AR
has been largely unsuccessful due to on-target adverse effects, including
pronounced bradycardia, atrioventricular block and hypotension. Biased agonism has the potential to overcome these limitations by enabling the separation of therapeutic from adverse effects.
Aims. To compare the in vitro, ex vivo and in vivo signaling prole of
the A1AR biased agonist VCP746 to A1AR prototypical agonists in cardiac
IRI.
Methods. In the isolated heart model, perfused rat hearts were
subjected to ischemia (30 min) and reperfusion (60 min). In the
acute myocardial infarction model, the left anterior descending

Abstracts

coronary artery was temporarily occluded for 30 min, followed by

120 min reperfusion. In both models, compounds were added at reperfusion and infarct size, heart rate and blood pressure (in vivo
only) assessed. Signalling proles were determined in isolated rat
neonatal ventricular cardiomyocytes.
Results. VCP746 and prototypical agonists stimulated an A1ARdependent reduction in infarct size and an improvement in cardiac
function post-IRI. However, in contrast to prototypical agonists,
VCP746 had no signicant haemodynamic effects. In isolated rat
cardiomyocytes, A1AR agonists stimulated ERK1/2 phosphorylation,
inhibited cAMP accumulation, promoted cardiomyocyte cell survival
and decreased glycolysis and oxidative metabolism after a period of
simulated ischemia. Prototypical agonists stimulated a potent reduction
in cardiomyocyte beat rate frequency via G protein-coupled inwardlyrectifying potassium (GIRK) channels. In contrast, VCP746 stimulated
only a weak decrease in cardiomyocyte beat rate, suggesting signal divergence at the level of GIRK channel activation.
Discussion. Collectively, these studies demonstrate that VCP746 can
promote cardioprotection in the absence of bradycardia, a prole suggestive of ligand bias. Insights into the signaling prole of VCP746 in
cardiomyocytes suggest this signal divergence may involve GIRK channel activation.
WE-041
Deletion of the NADPH Oxidase Organizing Protein NoxO1 promotes
angiogenesis
Katrin Schrder, Sabine Harenkamp, Jeremy Epah, Christoph
Schrmann, Juri Vogel, Beliza Rashid, Flavia Rezende, Ralf P. Brandes

S41

Clinic of Cardiovascular Surgery, Inselspital, Bern University Hospital,

University of Bern, Berne, Switzerland
Background: Donation after circulatory death (DCD) could improve
cardiac graft availability, which is currently insufcient to meet transplant demand. However, in DCD heart transplantation, organs undergo
an inevitable period of warm ischemia and most cardioprotective approaches can only be applied at reperfusion (procurement) for ethical
reasons. Therefore, we investigated whether strategies applied at the
onset of reperfusion may improve heart recovery after warm ischemia.
Methods: Isolated hearts of male Wistar rats were perfused in
working-mode for 20 min (baseline), subjected to 27 min global ischemia (37C), and 60 min reperfusion (n=43). Mild hypothermia (MH;
30C, 10 min), mechanical postconditioning (MPC; 2x30 sec), hypoxia
(HY; no O2, 2 min) and low pH (pH 6.8-7.4, 3 min) were applied at
the onset of reperfusion and compared with controls (i.e. no strategy
applied). Data (mean SD) were compared using t-tests; p-values
were corrected for multiple comparisons.
Results: Post-ischemic recovery was higher in MPC, MH and HY
treated hearts compared to controls. No difference was measured for
low pH (see Table below).
Conclusions: MPC, MH and HY, but not pH, seem to improve hemodynamic recovery vs controls. Reduced necrosis (MH), increased oxidative metabolism (MPC) and decreased mitochondrial damage (HY) may
contribute to improved functional recovery. Cardioprotective strategies
applied at graft procurement, could improve DCD graft recovery and
limit further injury; however, optimal reperfusion strategies remain to
be identied.

Goethe University, Frankfurt am Main, Germany

Reactive oxygen species contribute to angiogenesis and vascular repair. NADPH oxidases are the main source of ROS in the vasculature.
NoxO1 is a cytosolic protein facilitating assembly on the constitutively
active NADPH oxidase of epithelial cells. Being constitutively active,
we speculate that NoxO1 contributes to basal ROS formation in the vascular system and modulates angiogenic responses. This hypothesis was
tested in NoxO1 knockout mice and cells obtained from these animals.
Blood ow recovery after femoral artery occlusion was better in
NoxO1-/- as compared to WT animals. Similar, ex vivo spheroid outgrowth assays revealed increased tube formation capacity in lung endothelial cells obtained from NoxO1-/- mice as compared to WT animals.
In a spheroid confrontation assay, in which color labeled cells from
WT and NoxO1-/- animals are directly studied within the same spheroid, the number of NoxO1-/- cells at the tips was higher than that of
wildtype cells. These results suggest that deletion of NoxO1 favors the
expression of a tip cell like phenotype.
The NOTCH pathway is one of the main switches for an endothelial
cell from a tip cell into a stalk cell phenotype and activation of the
NOTCH pathway results in expression of a stalk cell phenotype. Physiologically, NOTCH mediated signalling requires proteases, among them
the alpha-secretase ADAM17, to eventually result in the formation of
the active NOTCH intracellular signalling domain. Importantly,
ADAM17 activity was indeed reduced in NoxO1-/- cells when compared
to wildtype as measured by the degradation of an articial substrate.
We conclude that NoxO1 controls alpha-secretase activity. Deletion
of NoxO1 therefore promotes a tip cell phenotype which results in increased angiogenesis.
WE-042
Cardioprotective reperfusion strategies improve cardiac recovery
after global, warm ischemia in an isolated working rat heart
model of donation after circulatory death
Emilie Farine, Petra Niederberger, Rahel Wyss, Natalia Mndez
Carmona, Thierry Carrel, Hendrik Tevaearai Stahel, Sarah Longnus

Control
MH
MPC
HY
Low
pH

LV
Work
[%]

Cardiac
Output
[%]

dPdt
max [%]

O2 cons. Coronary LDH

Flow
release
[mL/min] [Umin-1g
wet-1]

Cyt c
release
[ngmin-1g
wet-1]

447
627
658
61ir
4513

58
2018
2719
8+16
1211

5710
7412
747
85+20
6014

4115
5513
6114
5Q12
4410

4520
31 23
4417
2810
38+18

133
142
173
163
153

396276
112128
303357
265318
449628

All parameters are reported as 60 min reperfusion values expressed as percentage recovery of baseline, except for coronary ow, cytochrome c (Cyt c) and lactate dehydrogenase
(LDH) release, expressed as the absolute value at 10 min reperfusion.
pb0.05 vs control; left ventricular (LV) work (developed pressure*heart rate) 1 dPdt
max (maximum contraction rate) / O2 cons (O2 consumption)

WE-043
High circulating fatty acids prior to warm ischemia decrease cardiac
recovery in an isolated rat heart model of donation after circulatory
death
Petra Niederberger, Emilie Farine, Maria Arnold, Rahel Wyss, Natalia
Mndez Carmona, Thierry Carrel, Hendrik Tevaearai Stahel, Sarah
Longnus
Clinic of Cardiovascular Surgery, Inselspital, Bern University Hospital,
University of Bern, CH-3010 Bern, Switzerland
Background: Insufcient cardiac graft availability could potentially
be improved with donation after circulatory death (DCD). Preclinical
studies suggest that high pre-ischemic levels of circulating fatty acids,
as may expected with DCD, affect post-ischemic cardiac recovery. Therefore, we investigated whether acute cardiac exposure to high levels of
fatty acids prior to global warm ischemia alters subsequent recovery.
Methods: Isolated hearts of male Wistar rats underwent 20 min
baseline working-mode perfusion with glucose (11 mM) and either
high fat (1.2 mM palmitate; HF) or no fat (NF), followed by 27 min global ischemia (37C), and 60 min glucose only reperfusion (n=16). Additional hearts underwent 10 min reperfusion with radiolabelled glucose

S42

Abstracts

for measurement of glucose oxidation (GOX) and glycolysis (GLY;

n =2-4). Release of lactate, cytochrome c and tissue glycogen content
were also monitored. Data (meanSD) were compared using t-tests;
p-values were corrected for multiple comparisons.
Results: After 60 min reperfusion, percent recovery of rate-pressure
product (peak systolic pressure*heart rate) was two-fold lower in HF vs
NF hearts (31 17% vs 69 17% baseline; p b0.01). Trends toward
lower GLY and GOX rates, with a greater imbalance between GLY and
GOX was measured in HF vs NF hearts during early reperfusion. Furthermore, lactate (102 vs 62 mol*g tissue-1; pb 0.05) and cytochrome
c release (189 vs 52 ng*min-1*g wet-1; pb 0.01) were greater in HF
vs NF hearts at 10 min reperfusion. Glycogen consumption during ischemia was not different between groups.
Conclusion: Acute pre-ischemic exposure of hearts to high fat significantly decreases hemodynamic and metabolic recovery upon reperfusion compared to no fat. Thus, pre-ischemic circulating fatty acid levels
should be taken into consideration in pre-clinical models and clinical situations involving cardiac ischemia-reperfusion. In the context of DCD,
pre-ischemic interventions are limited, but optimizing energy substrate
metabolism at the time of procurement may facilitate use of these hearts.

WE-044
T185- Study and characterization of p38MAPKs key residue involved
in Ischaemic Heart Disease
Dibesh Thapa, Denise Eva Martin, Gian De Nicola, Michael Marber
Kings College London, London, UK
p38 has been studied over the years and over hundred studies have
shown it to be implicated in Ischaemic heart disease. p38 belongs to a
family of MAPK and gets activated via classical 3 tiers of MAPKKK cascade. However during ischaemic condition, p38 gets activated via atypical activation mechanism involving a scaffolding protein Transforming
growth factor--activated protein kinase 1 binding protein 1 (TAB1),
where TAB1 binds to p38 in a bipartite manner to induce structural
changes within p38 that leads to its autoactivation. Both in-vivo and
in-vitro model have shown this specic pathway of p38 activation to
be the root of harmful outcomes seen during and after Myocardial Infarction. This specic pathway of p38 activation makes it a very attractive therapeutic target, as adverse effect from small molecules has
been the major Achilles heel in drug discovery of p38 inhibitors. We recently published the crystal structure of p38 with TAB1 peptide in
NSMB, and in this structure we made an observation that led us to
hypothesise that Thr185 residue of p38 could play a pivotal role in the
autoactivation process. Following our investigation, we present evidence to support our hypothesis that T185 plays a critical role in the
structural changes during TAB1 induced auto-activation of p38 and
without it the process is signicantly compromised. Furthermore, with
our on-going investigations weve collected some preliminary results
to indicate that this residue may have additional functional role to
play upon TAB1 induced autoactivation of p38 which could shed light
on p38s mechanism of action under ischaemic stimuli, however further
experiments are required.

WE-045
The inhibition of proteasomes prevents Mitofusin 2 and Miro 1 degradation in cardiomyocytes during ischemia-reperfusion
Ivonne Olmedo1, Gonzalo Pino1, Cecilia Anrquez1, Zully Pedrozo1,2,
Paulina Donoso1, Gina Snchez1
1
Instituto de Ciencias Biomdicas, Facultad de Medicina, Universidad de
Chile, Santiago, Chile
2
Advanced Center for Chronic Diseases, Facultad de Medicina, Universidad
de Chile, Santiago, Chile

During cardiac ischemia reperfusion (I/R) diverse mitochondrial

proteins are degraded altering mitochondrial dynamics and inducing
mitochondrial fragmentation (ssion). Extensive mitochondrial ssion
impairs mitochondrial function and causes cardiomyocyte death. One
strategy to reduce heart damage during I/R is the use of proteasome inhibitors, however the mechanism by which these inhibitors induce protection during I/R is still unknown. Mitofusin 2 (Mfn2) and Miro 1 are
proteins implicated in transport and dynamics of the mitochondria.
The consequences of I/R on Mfn 2 and Miro1 content in cardiomyocytes
have not been studied. The aim of this work was to evaluate the content
of these proteins and whether inhibition of the proteasome is able protect the mitochondria from I/R injury. Cultured neonatal rat
cardiomyocytes were subjected to simulated I/R (sI/R) in the absence
or the presence of the proteasome inhibitor MG132. Cell death was evaluated by lactate dehydrogenase release (LDH) and the relative content
of mitochondria was determined by qPCR. Mitochondrial fusion and ssion were evaluated by confocal microscopy using mitotracker green
and the protein levels of Mfn2 and Miro1 were determined by
inmunowesternblot (WB).
In the absence of proteasome inhibitor, sI/R decreased the relative
content of mitochondria, increased mitochondrial ssion and produced
cardiomyocytes death. Also, sI/R decreased the protein content of Mfn2
and Miro1. The inhibition of proteasomes by MG132 preserved the content of Mfn2 and Miro1. Mitochondrial ssion was also prevented
resulting in an increased number of cells that survived sI/R. Taken together, these data suggest that inhibition of the proteasome preserves
the mitochondria explaining at least in part the protective effect of proteasome inhibition after I/R.
Acknowledgement to FONDECYT Postdoctorado 3140449,
FONDECYT 1150887, 1130407, FONDAP 15130011

WE-046
The new St Thomas' Hospital polarized cardioplegia shows noninferiority and improved efcacy of myocardial protection in pigs
undergoing cardiopulmonary bypass compared to St. Thomas 2
cardioplegia
Felix Nagel1, David Santer1, Anne Kramer1, Attila Kiss1, Wolfgang Dietl1,
Karola Trescher1, Klaus Aumayr3, Seth Hallstrm2, Hazem Fallouh4,
David J Chambers4, Bruno K Podesser1
1
Ludwig Boltzmann Cluster for Cardiovascular Research, Department for
Biomedical Research, Medical University of Vienna, Vienna, Austria,
2
Institute of Physiological Chemistry, Center for Physiological Medicine,
Medical University of Graz, Graz, Austria
3
Clinical Institute for Pathology, AKH Wien, Medical University of Vienna,
Vienna, Austria
4
Cardiac Surgical Research, The Rayne Institute (Kings College London),
Guys and St Thomas NHS Foundation Trust, St Thomas Hospital, London,
UK

Objectives: Increasingly, patients undergoing cardiac surgery are

more elderly, sicker and hence require improved protection. We compared cardioprotective efcacy of a new St Thomas Hospital Polarizing
cardioplegia (STH-Pol: esmolol, adenosine, magnesium) to conventional
St Thomas Hospital cardioplegia (STH2: potassium, magnesium) in a
pig model of cardiopulmonary bypass (CPB). Our hypothesis was the
non-inferiority of depolarized versus polarized arrest.
Methods: Pigs (47 4kg) were anesthetized and monitored for
baseline hemodynamic function. After sternotomy, CPB and aortic
cross-clamping, hearts were arrested via antegrade warm (37C) STHPol (n=7) or STH2 (n=6) for 60min ischemia followed by 60min onpump reperfusion. After weaning from CPB, hearts were monitored for
further 120min off-pump reperfusion before sacrice and tissue sampling (for high-energy phosphates and electron microscopy). Recovery
was measured as % of baseline (meanSEM).

Abstracts

Results: Baseline hemodynamics were comparable. After 180min

reperfusion, recovery of mean arterial pressure and heart rate were similar; however, in STH-Pol hearts had improved recovery of left ventricular systolic pressure (133 8 vs. 97 5 %, p b .01) and external heart
work (14516 vs. 8810%, pb .05) than STH2 hearts. Coronary ow/
heart weight was also higher during early (430 59 vs. 211 59%,
p b.05) and late reperfusion (269 43 vs. 90 16 %, p b.01) in STHPol. Total creatine kinase release was lower in STH-Pol hearts during reperfusion (2016262 vs. 1232199 U/L, pb.05). Creatine phosphate
levels in ST-POL hearts were higher (133 31 vs. 63 2 nmol/mg,
pb.05). There was no difference in ultrastructure between groups.
Conclusion: Polarized cardiac arrest improves myocardial protection and reduces ischemic damage in a model of CPB in pig hearts. We
therefore think this new concept of polarized cardioplegia should have
clinical relevance.

WE-047
Quantitative assay of microvascular hyper-permeability following
cardiac ischemia-reperfusion
Li-Ping Han1,2, Xiao-Ming Gao2, Xiao-Lei Mao2, Yi-Dan Su2, Xiao-Jun Du2
1

Wenzhou Medical College, Wenzhou, Zhejiang, China

Baker IDI Heart and Diabetes Institute, Melbourne, Victoria, Australia

Background: Microvascular hyper-permeability is a common event

following ischemia-reperfusion (IR) and impacts the efcacy of therapeutic interventions. Currently there has been lack of quantitative
assay of microvascular hyperpermeability. We attempted to establish
a quantitative method to determine microvascular permeability at
both organ level and cellular level.
Methods: Male C57Bl/6 mice were subjected to left coronary artery
occlusion (0.25, 0.5, 1 and 4h) followed by reperfusion for 24 h (IR).
Evans blue (EB, i.v.) was given 3h prior to termination. Heart tissues
were homogenized and EB was extracted in trichloroacetic acid solution
and quantied using spectrophotometer. Experiments were conducted
to determine an optimal dose of EB (3.3, 10, 20 and 33 mg/kg, respectively). For in vitro permeability assay, mouse cardiac endothelial cells
(H5V) were cultured in transwells for 4 days to conuency and maturation. Hypoxia-reoxygenation was induced. The permeability across endothelial cell monolayer was measured by adding FITC-dextran
(mw = 40KDa, 1M) as uorescent tracer into the upper chamber of
transwells. Concentration of FITC-dextran in the lower chamber was
determined.
Results: Compared to other doses, EB at 20 mg/kg yielded a high
level of EB content in the infarct zone while EB content in non-infarct
zone was minimal. So this optimal dose was used. And the results
showed that IR induced a 15-fold increase in EB content in the infarct
zone. The extent of microvascular leakage was dependent on the duration of ischemia of 0.25, 0.5, 1 and 4h, Pb0.001 by ANOVA, n=6 each).
FITC-dextran leaking experimental results showed that the permeability of monolayer H5V cells increased over 50% at both hypoxia 16h
and reoxygenation at 2h and 6h.
Conclusion: The microvascular permeability induced by myocardial
IR can be quantied in vivo by using EB extraction method described
and ex vivo by using FITC-dextran as indicator.
Key words: microvascular damage, ischemia-reperfusion, Evans blue

WE-048
Effects of hydrogen sulphide (H2S) on oxidative stress in acute myocardial ischemia injury in isolated hearts in rats
Zhang Jianxin, Liu Fang, Li Lanfang, Zhang Qinzeng, Xie Lijun
Hebei Academy of Medical Sciences, 050021, 97 Huaian road,Shijiazhuang,
Hebei, China

S43

Objective: To observe the effects of H2S on oxidative stress in myocardial ischemia injury in isolated heart in rats.
Methods: The myocardial ischemia injury model was established by
the ligation of coronary artery. Forty male SD rats, weighing 27020g,
were randomly divided into ve groups: sham, model, and low, middle,
high dose groups of NaHS. The left anterior descending coronary artery
was ligated in rats of the model group, but the rats in the sham group
were only threaded without ligation. The normal perfusate was replaced with NaHS perfusate (5mol/L,10mol/L,20mol/L) accordingly
in low dose,middle dose and high dose group of NaHS at 2h after ischemia. The content of MDA, the activities of LDH, SOD and GSH-PX were
respectively measured by spectrophotometry. The ultrastructural alterations of myocardium were observed by electric microscope.
Results: Compared with those of the sham group, the activity of LDH
in perfusate was signicantly increased in the model group. Compared
with those of the model group, the activity of LDH in perfusate was signicantly decreased in low, middle and high dose groups of NaHS. The
content of MDA in cardiac tissue was signicantly increased, and the activities of SOD and GSH-PX in cardiac tissue were signicantly decreased
in model group compared with those of sham group .The content of
MDA was signicantly decreased and the activities of SOD and GSH-PX
in cardiac tissue were signicantly increased in the low, middle and
high dose groups of NaHS compared with those of model group. The ultrastructure of the myocardial cells exhibited the myocardial cells were
characterized by mitochondrial swelling, disappearance or deformation
of mitochondrial cristae, disruption of nuclear membrane, and nuclear
condensation in the model group. Compared with those of the model
group, The myocardial ischemia injury was signicantly decreased in
NaHS treatment groups.
Conclusion: It could be concluded that H2S has certain protective effect against acute myocardial ischemic injury and the mechanism may
be related to anti oxidation.

WE-049
Effects of Simvastatin on the Expression of P47phox in Renal Ischemia Reperfusion Injury
Xiao-hong Xia, Jiao Jing, Li-jing Niu, Yan-ling Wang, Zhi-hui Zhi-hui
Miao
Hebei Academy of Medical Sciences, Shijiazhuang, China
Objective: To investigate the effects of Simvastatin (SIM) on the expression of P47phox in renal ischemia-reperfusion injury (RI/RI).
Methods: Sixty male Sprague-Dawley rats were divided into ve
groups randomly: (1) Sham group; (2) ischemia-reperfusion group (I/
R); (3) low-dose SIM group (Sim-L, 5mg/kg/d); (4) middle-dose SIM
group (Sim-M, 20mg/kg/d); (5) high-dose SIM group (Sim-H ,
40mg/kg/d). Sim-L, M and H group rats were given oral SIM 5, 20 and
40 mg/kg/d treatment respectively for 2 weeks. The model of RI/RI
was induced by bilateral clamping the renal artery and vein for 45 minutes followed by reperfusion. After 6 and 24 hours of reperfusion, the
blood samples were taken for detecting contents of serum creatinine
(Scr), urea nitrogen (BUN). After blood was taken, both side of kidney
were excised for observing renal histological examination, content of
Nitric Oxide (NO), activity of superoxide dismutase (SOD), the content
of malondialdehyde (MDA) and the protein expression of P47phox
were measured respectively.
Results: After RI/RI, the renal tubule epithelial cells showed signs of
damage in I/R group rats. the contents of Scr, BUN and MDA were significantly increased in I/R group than that of sham group (P0.01); Compared with the I/R group rats, contents of Scr, BUN and MDA were
signicantly lower in Sim-L , M and H groups (P 0.05). Contents of
NO and activity of SOD were signicantly increased (P b0.01) in SimM and Sim-H groups; The expression of positive immunoreactive

S44

Abstracts

particles and protein of P47 phox were increased in I/R group rats than
that of in Sham group rats. Compared with I/R group rats, both of positive immunoreactive particles and protein expression of P47phox were
decreased in Sim-M and Sim-H group rats, but not in Sim-L group rats.
Conclusions: These results suggest that SIM could reduce renal tissue injury and down-regulated the expression of P47phox of renal tissue in RI/RI rats. It is indicated that the protective effects of SIM to the
RI/RI may be related to block the NAD (P) H oxidate pathway and
anti-free radical damage.

WE-061
A pathogenic MYBPC3 25-bp polymorphic variant causes hypertrophic cardiomyopathy in South Asian descendants
Sakthivel Sadayappan
Loyola University Chicago, Maywood, IL 60153, USA
South Asians account for 25% of the worlds population, but they
hold a disproportionate 60% of the worlds cardiovascular disease burden. Hypertrophic cardiomyopathy (HCM) is predominantly caused
by mutations in sarcomeric genes, including MYBPC3, the most common
HCM-associated gene. Previously, we identied a MYBPC3 25-bp polymorphic variant (MYBPC3Int32), which is inherited in 4% of South
Asian descendants. MYBPC3Int32, an intronic 25-bp deletion in
MYBPC3 at the 3 region, is characterized by incomplete penetrance
and expressivity. While those carrying this variant are at high risk for
developing HCM and heart failure, its functional and molecular effects
remain unknown. Using cultured adult rat cardiomyocytes in vitro, we
showed that MYBPC3Int32 was unable to incorporate into the sarcomere, which resulted in contractile dysfunction. In the current study, a
genetically engineered mouse model expressing a moderate amount
of MYBPC3Int32 was established with HCM phenotype, including diastolic dysfunction. Furthermore, to determine the prevalence of this variant among South Asians in the United States, we screened 1162
subjects and determined a variant frequency of 6.80% and an allele frequency of 3.57%, a higher prevalence than was initially expected in this
cohort study. Four homozygous subjects were identied. Following
prevalence studies, clinical studies, including echocardiogram and electrocardiogram analyses, were performed on 15 positive subjects, compared to 15 non-carriers, to determine the presence of any sign of
HCM. Our data again conrmed incomplete penetrance. Overall, therefore, we determined that MYBPC3Int32 alone is sufcient to promote
the development HCM, implicating the translational importance of
these studies in the context of the development of heart disease
among South Asian populations.

WE-062
Lack of essential myosin light chain phosphorylation impairs
cardiac ability to adapt to augmented physical demand.
Selina Hein1, Lisa Scheid2, Matias Mosqueira2, Mandy Kossack1,
Benjamin Meder1, Rainer Fink2, David Hassel1
1

Heidelberg University Hospital, Heidelberg, Germany

Heidelberg University, Heidelberg, Germany

Cardiac ability to adapt its function to the bodys demand is pivotal for normal heart function. Modulatory proteins adjunctive to actin
and myosin largely accounts for this ability. Among others, the regulatory (RLC) and the essential myosin light chain (ELC) are part of
myosin molecules and contribute to modulation of cardiac contraction. Mutations in RLCs and ELCs cause cardiomyopathy in humans.
While the role of RLCs in cardiac physiology and pathophysiology is
well established, the precise function of ELCs in the heart and its

contribution to human cardiomyopathy remains unclear. Similar to

RLCs, ELCs are subject to phosphorylation. However, the exact role
of ELC phosphorylation for normal heart function and in disease is unknown. To model human haploinsufciency, we used the adult heterozygous zebrash mutant lazy susan (lazm647) carrying a nonsense
mutation in ELC, resulting in the removal of the highly conserved
phosphorylation site S195. By echocardiography we found that these
zebrash display signs of systolic dysfunction. When subjected to
forced swimming, heart function severely deteriorated, causing heart
failure and sudden death. We used native heart tissue to show that
ELC becomes phosphorylated after physical stress. Additionally,
in vitro motility analysis of zebrash actin sliding on ventricular myosin derived from wildtype (wt) and laz mutant zebrash after rest or
physical stress reveals that C-terminal phosphorylation critically modulates cross-bridge activity, cycling kinetics and lament velocity, specically after stress. Our model enabled us to analyze acto-myosin
interaction in native composition of wt and mutant protein. Further, calcium-dependent force measurements and calcium transient recordings demonstrates impaired calcium handling in laz
mutant cardiomyocytes, again specically after physical stress.
Our study employed for the rst time adult heterozygous zebrash
to provide novel mechanistic insights into ELC-mediated adaptation of cardiac function after physical stress and might contribute
to a better understanding of pathomechanisms in ELC-linked
cardiomyopathy.

WE-063
CYP2C19 and PON1 genetic variants as potential predictors for the
risk of bleeding in antiplatelet-treated patients
Yu Zhang1, Mengzhen Zhang2, Zhoucuo Qi2, Qiuxiong Lin2, Bin Zhang3,
Jiyan Chen3, Shilong Zhong2,3
1
School of Pharmaceutical Sciences, Guangzhou Medical University,
Guangzhou, Guangdong, China
2
Medical Research Center, Guangdong General Hospital, Guangzhou,
Guangdong, China
3
Guangdong Cardiovascular Institute, Guangdong Academy of Medical
Sciences, Guangzhou, Guangdong, China

Bleeding has emerged as an important outcome in antiplatelet

treatment after percutaneous coronary intervention (PCI), but the
study on the relationship between genetic variations with interpatient variability of bleeding is lack, compared to major adverse
cardiac events (MACE). The present study was aimed to evaluate
the contribution of 13 genetic variants to the risk of MACE and
the occurrence of bleeding events in Han Chinese patients after
PCI. Five hundred and twenty Han Chinese patients undergoing
PCI and received dual-antiplatelet therapy were sequentially recruited and followed up to 1 year. Thirteen variants in ABCB1,
CYP2C19, PON1, P2RY12, P2RY1 and ITGB3 were genotyped. The
effect of genetic variants on MACE in 1 year and bleeding in 6
months was assessed. CYP2C19*2 allele was signicantly associated with a higher risk of the efcacy endpoint of MACE (HR per allele, 2.00; 95%CI: 1.02-3.92), while a low risk of safety endpoint of
bleeding events (OR, 0.42; 95%CI, 0.25-0.70). Univariate analysis
indicated PON1 g.*2435 A allele and p.Gln192Arg G allele were associated with a lower risk of bleeding (OR, 0.54; 95%CI, 0.30-0.99,
and OR, 0.47; 95%CI, 0.25-0.86), while p.Leu55Met T allele was associated with a higher risk of bleeding (OR, 2.83; 95%CI, 1.127.15). PON1 g.*2435G NA and p.Leu55Met were still signicantly
associated with the risk of bleeding in 6 months when adjusted
for other variables. This study conrmed CYP2C19*2 and identied for the rst time PON1 genetic variants as potential predictors for the risk of bleeding events in clopidogrel-treated
patients after PCI.

Abstracts

WE-064
ADP-stimulated contraction: a predictor of thin-lament activation
in cardiac disease
Vasco Sequeira1, Aref Naja1, Paul J.M. Wijnker1, Cris dos Remedios2,
Michelle Michels3, Diederik W.D. Kuster1, Jolanda van der Velden1
1

VU University Medical Center, Amsterdam, The Netherlands

Muscle Research Unit, Bosch Institute, University of Sydney; Anderson
Stuart Building (F13), Sydney, Australia
3
Cardiology, Erasmus Medical Center, Rotterda, The Netherlands
2

Background: Diastolic dysfunction is general to all idiopathic dilated

(IDCM) and hypertrophic cardiomyopathy (HCM) patients. Relaxation
decits may result from increased actin-myosin formation during diastole due to altered tropomyosin position, which block myosin-binding
to actin in the absence of Ca2 +. We investigated if ADP-stimulated
force development (without Ca2 +) can be used to reveal changes in
actin-myosin blockade in human cardiomyopathy cardiomyocytes.
Methods: Force measurements were performed in single membranepermeabilized cardiomyocytes at sarcomere length of 2.2 m in the absence of Ca2+, but in the presence of mM levels of ADP. Exogenous protein
kinase A (PKA)-treatment was performed to determine whether myolaments are sensitive to kinase treatment. Cardiac samples from HCM patients, harboring thick- (MYH7mut, MYBPC3mut) and thin-lament
(TNNT2mut, TNNI3mut) mutations, and IDCM, were compared with sarcomere mutation-negative HCM (HCMsmn) and non-failing donors.
Results: Myolament ADP-sensitivity was higher in IDCM and HCM
compared with donors, while it was lower for MYBPC3. Increased ADPsensitivity in IDCM, HCMsmn and MYH7mut was caused by low phosphorylation of myolament proteins, as it was normalized to donors
by PKA treatment. Troponin exchange experiments in a TNNT2mut sample corrected the abnormal actin-myosin blockade. In MYBPC3trunc samples, ADP-sensitivity highly correlated with cardiac myosin-binding
protein-C (cMyBP-C) protein level. Incubation of cardiomyocytes with
cMyBP-C antibody against the actin-binding N-terminal region reduced
ADP-sensitivity, indicative of cMyBP-Cs role in actin-myosin regulation.
Conclusions In conclusion, ADP-stimulated contraction can be used
as a tool to study how protein phosphorylation and mutant proteins
alter accessibility of myosin-binding on actin. Our data provides a mechanism of how phosphorylation alterations and/or expression of mutant
proteins increase actin-myosin interactions, that precede Ca2+ rise, and
could contribute to the impaired myocardial relaxation observed in
human cardiomyopathy.

WE-065
Cross-bridge dynamics is determined by two velocity dependent
kinetics; implications on the adaptive and synchronous cardiac
function
Daria Amiad Pavlov1, Michal Horowitz2, Amir Landesberg1
1

Faculty of Biomedical Engineering, Technion IIT, Haia, Israel

Faculty of medicine, the Hebrew University, Jerusalem, Israel

Introduction: The cardiac muscle has a remarkable ability to adjust

function to changes in demands, as described by the Frank-Starling Law,
the Fenn effect, and the high contractile efciency. The loading conditions
determine the force per cross-bridge, cross-bridge recruitment and the
sarcomeric energy consumption. The length, stress, and velocity of shortening were suggested as possible modulators of cross-bridge dynamics.
Methods: The study tested the effects of the initial length or isometric
stress (n=9), and the sarcomere shortening velocity (n=9) on crossbridge dynamics in the intact rat trabeculae, under constant activation.
Sarcomere length was measured by laser diffraction and ramp shortenings at various velocities were imposed with a fast servomotor.

S45

Results: Both stress decline and redevelopment responses revealed

two distinct kinetics: a fast and a slower phase. The fast (3 msec) and
slow phases depicted linear dependencies of the rate of stress changes
on the instantaneous stress levels. The rate coefcients of the two phases
were independent of the initial length or stress level. However, they were
tightly dependent on the shortening velocity (VSL). An increase in the VSL
expedited the rates of both phases in a linear mode; the rate coefcients
for the rst and second phases were 28628 + 392 VSL [s-1] and
35.74.8 + 9.51.4 VSL [s-1], respectively. The fast kinetics is more
than 5 time faster than the slow kinetics, at all velocities. The fast kinetics
determines the force per cross-bridge and the second is ascribed to crossbridge cycling and determines the number of strong cross-bridge.
Conclusion: Cross-bridge dynamics is modulated by the velocity
and not by the length or stress. These features shed light on theories
of muscle contraction, and are essential for the regulation and synchronization of all the sarcomeres and myocytes in the myocardium and for
adapting function to match the demands.

WE-066
The functional association between the Sodium/Bicarbonate
Cotransporter and the Soluble Adenylate Cyclase (sAC) modulates
basal cardiac contractility
Mara Sofa Espejo, Mara Carolina Ciancio, Alejandro Orlowski, Ernesto
Alejandro Aiello, Vernica Celeste De Giusti
Centro de Investigaciones Cardiovasculares, La plata, Buenos Aires, Argentina
In addition to the adenylate cyclase (AC) embedded in the plasma
membrane, another source of cyclic AMP (cAMP) was identied in the
heart, the soluble AC (sAC). However, the cardiac physiological function
of sAC is unknown. On the other hand, the cardiac Na+/HCO-3
cotransporter (NBC) promotes the cellular co-inux of HCO-3 and Na+.
Since sAC activity is mainly regulated by HCO-3, our purpose was to investigate the potential impact on cAMP-dependent cardiac contractility
of the relationship between the activity of NBC and sAC. Rat ventricular
myocytes were loaded with Fura-2 or Fluo-3 in order to measure Ca2+
transient amplitude (CaT) by epiuorescence or Ca2+ sparks frequency
(CaSF) by confocal microscopy, respectively. Sarcomere shortening as
contractility index was measured simultaneously with epiuorescence.
The NBC blocker S0859 (10 M) induced a negative inotropic effect
(NIE) in the presence of HCO-3 (Control: 19.1 3.2% vs. S0859:
14.6 2.6%; n = 9, P b0.05) which was associated with a decrease of
18.52.6% in CaT. S0859 did not induce a NIE in the absence of HCO-3.
The selective inhibitor of sAC, KH7 (1M) decreased contractility (Control: 15.7 0.7% vs. KH7: 11.3 0.9%, n = 5, P b 0.05) and CaT
(15.74.9%) only in HCO-3. Moreover, S0859 did not add more NIE in
the presence of KH7 (KH7+S0859: 11.10.9%, n=5). Since cAMP activates the kinase PKA, which in turn increases Ca2 + release through
sarcoplasmic reticulum RyR channels, CaSF was measured as an index
of RyR open probability. The increase in CaSF observed when eld stimulation frequency was increased from 0.5 to 3 Hz (Control variation
ratio: 1.23 0.1) was reversed in the presence of S0859 (0.62 0.2,
n=5, Pb0.05) only when HCO-3 was present in the extracellular medium. In summary, the results demonstrated that the complex NBC-sAC
plays a relevant role in Ca2+ handling and basal cardiac contractility.

WE-067
Proteomic analysis of excitation-contraction coupling abnormalities in a rat model of heart failure with preserved ejection fraction
Daniel Soetkamp, Romain Gallet, Ronald Holewinski, Vidya
Venkatraman, Xin Yue, Rui Zhang, Eduardo Marbn, Joshua I. Goldhaber,
Jennifer E. Van Eyk

S46

Abstracts

Cedars-Sinai Medical Center, Los Angeles, CA, USA

Background: Heart failure with preserved ejection fraction (HFPEF)
is a chronic heart disease with high morbidity and mortality. HFPEF is
characterized by diastolic dysfunction, which leads to elevated cardiac
lling pressures. Currently, there is no effective treatment, perhaps because the underlying cellular mechanisms of HFPEF remain to be elucidated. In this study we focus on investigating of HFPEF-associated
maladaptive calcium responsive changes induced with in a rat model
of salt-sensitive hypertension.
Methods: Dahl salt-sensitive rats received either a high salt (hypertension-induced HFPEF) or a low salt diet (control) for 6 weeks. Following treatment, rat hearts were either harvested for 1) hemodynamic
characterization followed by cell isolation for analysis of intracellular
calcium (Ca2 +) excitation-contraction (EC) coupling or 2) liquid
chromatography-tandem mass spectrometry analysis quantifying protein amounts.
Results: Using the patch clamp technique in isolated myocytes obtained from control and HFPEF hearts, we found defective EC coupling
with reduced EC coupling gain, slowed Ca2+ uptake, and slowed relaxation consistent with myolament phosphorylation. Consistent with
these ndings, proteomic analysis revealed HFPEF-induced changes
(pb 0.05) in the protein quantity of Ca2+ binding and/or Ca2+ handling
proteins (e.g., for HFPEF vs control, the RyR2-inhibiting protein chloride
intracellular channel protein 2 increased by 108%; Sodium/potassiumtransporting ATPase subunit beta-1 decreased by 20%), as well as associated kinases and their activators (e.g., Transforming protein RhoA increased by 69%, Serine/threonine-protein kinase PAK 2 increased by
325%; cAMP-dependent protein kinase (PKA) type I-alpha regulatory
subunit decreased by 15%).
Conclusion: Maladaptive changes in key Ca2 +-signaling proteins
lead to disruption of EC coupling and slowed cardiac relaxation. Pharmacologic targeting of these proteins may be of benet for treating
HFPEF.

WE-068
Insulin Treatment Did Not Prevent Cardiac and Baroreex Dysfunctions in a Model of Type 1 Diabetes
Sarah Cristina Ferreira Freitas1, Iris Callado Sanches3, Jacqueline Freire
Machi2, Paulo Magno Martins Dourado2, Maria Claudia Irigoyen2, Ktia
De Angelis1
1

Universidade Nove de Julho, So Paulo, Brazil

Heart Institute Hospital, So Paulo, Brazil
3
So Judas Tadeu University, So Paulo, Brazil
2

Background: The mechanisms underlying the increased risk in Type

1 Diabetes mellitus (T1DM) patients even on insulin treatment are not
well understood.
Objective: Evaluate the effects of insulin replacement therapy on cardiac, autonomic and oxidative stress (OS) parameters in a model of T1DM.
Methods: Male Wistar rats (230-260g) were divided into 3 groups
(n=7/group): control (C), diabetic (D, streptozotocin 50mg/kg) and diabetic treated daily with insulin subcutaneously (DTI). At 30 days, cardiac function was assessed by echocardiogram. Baroreex sensitivity and
cardiac autonomic tonus were evaluated. OS analysis was performed in
heart tissue.
Results: The diabetic groups showed hyperglycemia (N 350mg/dL)
at the beginning of the protocol. Insulin therapy normalized the glycemia (DTI:12610, C:1287 vs. D:43921mg/dL). There was a reduction in the left ventricle mass (LVM) in D group and these changes were
not observed in DTI group (LVM- C:1.04 0.04, D:0.82 0.03 and
DTI:1.04 0.03g). It was observed impairment in systolic function
(shortening fraction) of diabetic group that was reversed with insulin

treatment. Regarding diastolic function, the isovolumetric relaxation

time (IVRT) was increased and E/A wave ratio (EA) was decreased in
D group, which was not reversed in DTI group (IVRT-C:1.29 0.11,
D:1.68 0.11
and
DTI:1.60 0.08ms/bpm;
EA-C:2.45 0.38,
D:1.530.10 and DTI:1.69 0.17). The baroreex sensitivity was impaired in D group in relation to C in both bradycardic and tachycardic responses (C:-1.36 0.11, D:-1.06 0.05, DTI:-1.32 0.07 and
C:3.180.17, D:2.590.18, DTI:2.580.15bpm/mmHg, respectively).
The tachycardic responses dysfunction was not normalized by the insulin treatment. The insulin treatment normalized mean arterial pressure,
heart rate, intrinsic heart rate, as well as the vagal and sympathetic
tonus which were impaired in D group. These benets of insulin treatment were reected on the analysis of OS, where the diabetic group
had higher oxidized glutathione (D:0.0242 0.0008 vs.
C:0.0169 0.0012, DTI:0.0183 0.0009nmol/g tissue) and increased
lipid peroxidation (D:2.54 0.21 vs. C: 1.95 0.10, DTI:
1.590.18moles/mg protein).
Conclusion: Despite the insulin treatment normalized blood glucose, cardiac morphometry and systolic function, cardiac autonomic
control and oxidative stress, it was not able to attenuate diastolic dysfunction and the tachycardic response of baroreex, suggesting a remaining cardiovascular risk even after insulin replacement in this
model of experimental T1DM.

WE-069
Nitric oxide and CaMKII: critical steps in the inotropic response to
IGF-1
Juan Ignacio Burgos, Alejandra Yeves, Irene Ennis, Martn Vila Petroff
Centro de Investigaciones Cardiovasculares de LP, La Plata, Argentina
Cardiac adaptation to aerobic exercise training includes improved
cardiomyocyte contractility, by a non-yet claried mechanism in which
nitric oxide (NO) and CaMKII have been implicated. At the cellular
level, IGF-1 is the main mediator of the adaptive response to exercise.
Our purpose was to explore the effect of IGF-1 on mice cardiomyocyte
contractility and the underlying signaling pathway.
IGF-1 (10nmol/L) increased cardiomyocyte shortening
(128.124.62%, n=8 vs basal; p0.05), effect abrogated by inhibition
of NO production with the non-selective nitric oxide synthase inhibitor
L-NAME (2.5 mmol/L; 103.23.02%, n=5) or nitroguanidine (NG, 240
nmol/L), specic inhibitor for the neuronal isoform (nNOS, 97.41.21%,
n=5) and by CaMKII inhibition with KN93 (101.502.04%, n=6). In
agreement, a signicant increase in NO production in response to IGF1 (133.752.17%, n=16) was detected by epiuorescence with DAFFM. Again, this was prevented by L-NAME (110.36 3.20%, n = 11)
and NG (114.44 1.83%, n= 9), conrming the involvement of nNOS
but not altered by KN93 (135.221.36%, n=9) suggesting that CaMKII
activation was downstream NO production. We explored the pathway
involved in nNOS activation by measuring AKT phosphorylation. As expected, IGF-1 increased P-AKT (185.9010.18%, n=3; p0.05). Since
NO-dependent CaMKII activation has been proposed, we next determined CaMKII activity (P-CaMKII) and the phosphorylation of its downstream target Thr17-phospholamban, detecting a signicant increase in
both in the presence of IGF-1 (227.19 29.43% and 143.34 5.44%,
n=3 respectively) but not when NO production was prevented by NG
(126.61 5.48 and 65.76 15.04, n = 3 respectively). Interestingly,
similar results showing nNOS and CaMKII activation were obtained in
the hypertrophied myocardium of mice subjected to swimming
training.
In conclusion, our results support a critical role of CaMKII in the positive inotropic effect of IGF-1. Our ndings suggest that IGF-1 through
the IGF-1R triggers the phosphorylation of AKT which in turn activates
nNOS and increases NO production which would be responsible for
CaMKII activation.

Abstracts

WE-070
oxiCaMKII-dependent RyR2 phosphorylation mediates contractile
dysfunction associated with sepsis.
Marisa Seplveda1, Luis Gonano1, Manuel Viotti1, Micaela Lpez
Alarcn2, Isalira Ramos2, Adriana Bastos Carvalho2, Emiliano Medei2,
Martn Vila Petroff1
1

Centro de Investigaciones Cardiovasculares Dr. Horacio E. Cingolani, La

Plata, Argentina
2
Universidade Federal do Rio de Janeiro Centro de Ciencias da Sade
Instituto de Biosica Carlos Chagas Filho, Rio de Janeiro, Brazil
In sepsis, there is a recognized association between cardiac dysfunction and mortality. Contractile dysfunction associated with sepsis has
been attributed to a decrease in the amplitude of the intracellular
Ca2+ transient and recent studies have proposed that altered ryanodine
receptor (RyR2) function is responsible for sarcoplasmic reticulum (SR)
Ca2+ loss and reduced Ca2+ transients.
We examined the subcellular mechanisms involved in SR Ca2+ loss
and contractile dysfunction associated with sepsis.
Using a colon ascendens stent peritonitis mouse model of sepsis
(CASP) and Sham controls, we observed that after 24hs CASP mice
had signicantly elevated proinammatory cytokine levels, reduced
ejection fraction and fractional shortening (EF%54,76 0,67;
FS%27,530,5) compared to sham (EF%73,570,2; FS%46,750,38).
At the cardiac myocyte level, CASP cells showed reduced cell shortening,
Ca2 + transient amplitude and SR Ca2 + content compared to Sham
cardiomyocytes. CASP hearts showed a signicant increase in
oxidation-dependent calcium and calmodulin-dependent protein kinase II (CaMKII) activity (CASP 0.92 0.1 AU, Sham 0.56 0.05 AU)
which could be prevented by pretreating animals with the antioxidant
Tempol (1mM for 7 days in drinking water).
Pharmacological inhibition of CaMKII with 2.5M KN93 prevented
the decrease in cell shortening, Ca2+ transient amplitude and SR Ca2+
content in CASP myocytes. Contractile function was also preserved in
CASP myocytes isolated from transgenic mice expressing a CaMKII inhibitory peptide (AC3-I) and in CASP myocytes isolated from mutant
mice that have the RyR2 CaMKII-dependent phosphorylation site (Ser
2814) mutated to alanine (2814A). Furthermore, CASP 2814A mice
showed preserved EF and FS (EF%59,543,42; FS%35,885,58) compared to sham 2814A mice EF%65,896,95; FS%33,332,38) .
Results indicate that oxidation and subsequent activation of CaMKII
has a causal role in the contractile dysfunction associated with sepsis.
CaMKII, through phosphorylation of RyR2 would lead to Ca2 + leak
from the SR, reducing SR Ca2+ content, Ca2+ transient and contractility.

WE-071
Silencing of the epidermal growth factor receptor (EGFR) blunts the
slow force response to myocardial stretch
Mara Soledad Brea, Romina Gisel Daz, Patricio Eduardo Morgan,
Claudia Irma Caldiz, Nstor Gustavo Prez
Centro de Investigaciones Cardiovasculares Dr. Horacio E. Cingolani, La
Plata, Buenos Aires, Argentina
Myocardial stretch induces a biphasic force increase: A rst phase
due to the Frank-Starling mechanism, followed by a slower one called
slow force response (SFR). The SFR is due to a complex autocrine mechanism that appears to involve Angiotensin II (AII)-triggered EGFR
transactivation and the consequent generation/release of reactive oxygen species (ROS) leading to Na+/H+ exchanger (NHE1) activation. In
order to conclusively prove the role of the EGFR in the SFR, we developed a lentivirus carrying a siRNA against EGFR (siEGFR), and injected
it into the rat cardiac left ventricular wall (n =8). A scramble (siSCR)

S47

sequence was used as control (n=9). After 4 weeks, EGFR protein expression showed a 4815% reduction in siEGFR-injected hearts compared to siSCR (1006%, pb 0.05). Isolated rat papillary muscles from
both groups were then stretched from 92 to 98% of Lmax. The SFR was
131 2% of initial rapid phase in siSCR (p b0.05 vs. rapid phase) and
was blunted in siEGFR-expressing muscles (102 1%, p b0.05 vs.
siSCR). Basal myocardial oxidative stress estimated by T-BARS was not
affected by the reduction in EGFR expression: (in nmol/gr tissue)
1.29 0.09 siEGFR vs. 1.38 0.06 siSCR. However, AII or EGFmediated ROS production (assessed by lucigenin method in cardiac tissue slices) was signicantly reduced in siEGFR-injected hearts: AII
(1nM) from 226 27 siSCR to 113 9 siEGFR (p b0.05); EGF (0,1ug/
ml) from 17519 to 1027 (pb0.05) respectively. Finally, we studied
the EGFR silencing effect over the reported AII-dependent NHE1 activity
by measuring pHi (BCECF, papillary muscles) in bicarbonate-free medium. 1nM AII signicantly increased pHi by 0.180.06 units in the siSCR
group (pb0.05), effect that was completely blunted in the siEGFR one (0.120.03). Taken together, we can conclude that EGFR activation after
stretch is crucial for the development of the SFR, effect that would result
from preventing ROS-mediated NHE1 activation.

WE-072
Thioredoxin 1 (TRX1) overexpression cancels the slow force
response (SFR) development
Maite R Zavala1, Romina G Diaz1, Martin Donato2, Ricardo J Gelpi2, Mara
C Villa-Abrille1, Nstor G Prez1
1

Centro de Investigaciones Cardiovasculares Dr. Horacio E. Cingolani,

Universidad Nacional de La Plata, La Plata, Argentina
2
Instituto de Fisiopatologa Cardiovascular, Universidad de Buenos Aires,
Buenos Aires, Argentina
The stretch of cardiac muscle increases developed force in two
phases. The rst phase occurs immediately after stretch and is the expression of the Frank-Starling mechanism, while the second one or
slow force response (SFR) occurs gradually and is due to an increase
in the calcium transient amplitude. An important step in the chain of
events leading to the SFR generation is the increased production of reactive oxygen species (ROS) leading to redox sensitive ERK1/2, p90RSK
and NHE1 phosphorylation/activation. Conversely, suppression of ROS
production blunts the SFR. The purpose of this study was to verify
whether overexpression of the ubiquitously expressed antioxidant molecule TRX1 affects the SFR development and NHE1 phosphorylation.
We did not detect any change in basal phopho-ERK1/2, phophop90RSK and NHE1 expression in mice with TRX1 overexpression compared to wild-type (pERK1/2:105.4 9.9%, n = 4; p-p90RSK:
11115%, n=3; NHE1:10013%, n=4, ns) Isolated mouse papillary
muscles (wild-type, WT, or with TRX1 overexpression) were stretched
from 92 to 98 % of Lmax. The SFR was 1371% of the initial rapid phase
in wild-type mice (n = 8, P b0.05 vs. rapid phase) while it was
completely canceled in TRX1 overexpressing animals (1003%, n=7,
Pb0.05 vs. control SFR). The increase in NHE1 phosphorylation induced
by stretch was signicantly higher in WT mice (1566% of control, n
=3, P b 0.05) compared toTRX1 overexpressing mice (126.18.4% of
control, n = 3, ns). These results, although preliminary, suggest that
mitigation of ROS formation after stretch by increasing the myocardial
antioxidant defense precludes the NHE1 phosphorylation induced by
stretch and consequently the SFR development.

WE-074
Effect of aging on heart function and calcium handling: impact of
NOX inhibition
ALVARO VALDES, GUILLERMO BARRIOS, NIKOL PONCE, DANIEL
GONZALEZ

S48

Abstracts

UNIVERSIDAD DE TALCA, TALCA, CHILE

Cardiac aging is characterized by alterations in contractility and calcium handling. It has been suggested that oxidative stress may be involved in this process. The superoxide generating system NADPH
oxidase (NOX) is expressed in the heart (NOX2 and 4). We and others
have reported that in cardiac failure, the NOX-derived superoxide is increased, with a negative impact on calcium and contractility. We tested
the hypothesis that calcium transients and contractility in aged rat
cardiomyocytes are disturbed by NOX.
Hearts and cardiomyocytes were obtained from adult (5 months-old)
and aged (20 months-old) Sprague-Dawley rats, and were treated with
apocynin (50 mol/L), a NOX inhibitor. Cells were eld-stimulated from
0.5 to 4 Hz and [Ca2+]i was monitored with fura-2.Contractility was evaluated as dP/dtmax in isolated hearts, challenged with isoproterenol.
Cardiac response to isoproterenol was depressed in aged hearts
compared to adults (pb0.005), but was restored by apocynin treatment.
[Ca2+]i transients amplitude was increased in aged cardiomyocytes
(pb0.005) and was further increased by apocynin treatment. Time-50
to peak [Ca2+]i was increased in aged myocytes (pb0.05), suggesting
impairment in RyR2, and was improved by apocynin treatment. Time50 to maximal relaxation was increased in aged myocytes (p b0.05)
and reduced towards normal by NOX inhibition.
Using thapsigargin to block SERCA2 function, we submitted
myocytes to tetanic stimulation to evaluate the myolaments Ca2+ sensitivity. By comparing the amplitude of the tetanic contraction achieved,
with the level of [Ca2+]i evoked, we found that myolaments Ca2+ sensitivity was reduced in aged myoctes (pb 0.05).
At the protein level, SERCA expression was reduced in aged hearts
while phospholamban was not different between both groups.
In conclusion, contractility in response to isoproterenol was depressed in aged hearts, and in aged myocytes [Ca2+]i level was higher,
as a result of diminished myolaments Ca2+ sensitivity. NOX inhibition
increased [Ca2+]i transients amplitude and improved Ca2+ kinetics, and
improved contractility. These results suggest a NOX dependent effect in
aged myocytes at the level of RyR2 and SERCA2 and myolaments.

WE-075
Mechanisms of sex-difference in serotonergic and 1-adrenergic vasoconstriction in the internal mammary artery of patients going
through coronary artery bypass graft
Victor Lamin1, Amenah Jaghoori1, Michael Worthington2, James
Edwards2, Fabiano Viana2, Robert Stuklis2, David Wilson1, John
Beltrame1
1

School of Medicine, The University of Adelaide, Adelaide, South Australia,

Australia
2
Cardiothoracic Surgical Unit, The Royal Adelaide Hospital, Adelaide, South
Australia, Australia
Background: Females have poorer outcomes following coronary bypass surgery (CABG) than males and sex-differences in the internal
mammary artery (IMA) vasoconstrictor properties have been proposed
to contribute to this differential outcome. The objective of this study
was to determine the role of: (1) endothelial integrity, (2) nitric oxide
(NO) and (3) prostaglandins (PG) in mediating sex- differences in
IMA vasoconstriction to serotonin (5HT) and 1-adrenergic agonist
phenylephrine (PE).
Methods: Contractile responses of male (n = 60) and female
(n=50) IMA to 5HT or PE were generated in the presence or absence
of an intact endothelium. Nitric oxide synthase (NOS) and cyclooxygenase (COX) inhibitors were used to evaluate the role of NO and PG in mediating the sex-dependent vasoconstriction in the presence of 5HT or
PE. Electron paramagnetic resonance (EPR) was used to quantify NO release in response to the endothelium-dependent vasodilator (A23187).

Results: Female IMAs had increased sensitivity to 5HT and PE than

males. (1) Endothelial denudation abolished this sex-difference for
both 5HT and PE, implicating the involvement of an endothelial factor.
(2) NO did not contribute to the sex-difference for either agonist since
EPR-assessed NO production did not differ or NOS inhibition have no
impact. (3) However, COX inhibition abolished female IMA hypersensitivity to 5HT and PE.
Conclusions: These data indicate that the female IMA hypersensitivity to the 5HT and PE are mediated via an endothelium-dependent COX
pathway. Ongoing studies are investigating the potential autocoids involved. Therapies targeting this pathway may negate the sexdifference and improve outcomes amongst women undergoing CABG.

WE-076
The Effects of Sildenal, phosphodiesterase 5 inhibitor, on the
Expression of and Myosin Heavy Chains in Hypoxia induced
Right Ventricular Hypertrophy in Mice
Said Khatib1, Mukhallad Al-Jinabi2, Nayaf Gharaibeh2, Anwar Alkhayat2
1

Faculty of Medicine, King Fahad Medical City, Riyadh, Saudi Arabia,

Faculty of Medicine. Jordan University of Science and Technology, Irbid,
Jordan
2

Introduction: Myosin heavy chains are known to be the main contractile protein in muscles. It is present in the cardiac muscle in two
forms alpha (), and beta (). The contractile properties of the cardiac
muscle are determined by the types of myosin heavy chain (MHC) present in the muscle . The expression of these MHCs can be altered by many
physiological and pathological conditions. In this study we investigated
the effect of sildenal on MCHs isoforms in hypoxia induced right ventricular hypertrophy in mice.
Method and results: Right ventricular hypertrophy was induced by
exposing the animals to low oxygen tension (11%) in normobaric chamber for 20 days. 32 mice were were distributed randomly into: 10 as
control (C). 10 were exposed to hypoxia for 20 days without sildenal
treatment (I) and 12 were given sildenal orally at dose of 30 mg.Kg1.day-1 while they were exposed to hypoxia for 20 days (II). MCHs isoforms were detected using two ELISA kits containing antibodies against
and MHCs. Compared to control group C, mice exposed to hypoxia
(group I) showed a signicant increase in right ventricle weight to
body weight mg/g ratio, (0.890.13 in group C and 1.3 0.3 in group
I, PN 0.001) but signicant changes in ratio of group II, 0.91+ 0.15). Expression of MHC isoform was signicantly decreased in mice group I
(PN0.001), while mice exposed to hypoxia and treated with sildenal
showed signicant shift of MHC towards isoform (P N 0.000).
Hypertrophied right ventricle expresses more myosin heavy chain
and this is benecial to the heart since hearts with more MHC have
more ATPase activity and powerful and fast contraction. Conclusion: sildenal reduced hypoxia induced right ventricular hypertrophy and
caused a shift in MCH towards form whichs makes the heart contraction more economical (i.e using less ATPase).

WE-077
Bisphenol S depresses myocardial function through an estrogen
receptor- -dependent cascade
Melissa Ferguson, W. Glen Pyle
Centre for Cardiovascular Investigations, Department of Biomedical Sciences,
University of Guelph, Guelph, Ontario, Canada
Bisphenol A (BPA) is an estrogenic endocrine disrupting chemical
that has been linked to a variety disorders including diabetes, cancer,
and cardiovascular disease. The link between BPA exposure and widespread health concerns led to its reduced use in consumer products

Abstracts

such as food packages and baby bottles. Bisphenol S (BPS) is a common

substitute for BPA even though it has similar estrogenic potential. Like
BPA, BPS leaches from products and accumulates in people who use
BPS containing products. Despite the similar potential to cause health
problems, investigations into the cardiovascular effects of BPS exposure
are limited. We treated Langendorff perfused mouse hearts with BPS
(10-9M), BPA (10-9M), or DPN ( -estrogen receptor agonist, 10-9M) for
15 min and measured the impact on myocardial contractility. In females
BPS depressed left ventricular developed pressure by 15% through a reduction in systolic pressure. The effects of BPS were greater than the depressant effects of BPA (10%), but they were similar to those seen with estrogen receptor activation. The -estrogen receptor antagonist PHTPP
blocked the effects of BPS. Although the myocardial impact of -estrogen
receptor activation was similar in males and females (~15%), the effects
of BPA and BPS were attenuated in males. BPS had no impact on cardiac
myolament activation in either males or females suggesting that its
mechanism of action is through another molecular pathway. This is
the rst study showing that BPS rapidly and signicantly depresses
heart function through -estrogen receptors. These data call into question the safety of BPS in consumer products.

WE-078
Sarcoplasmic reticulum (SR) calcium transport in atrial myocytes
isolated from healthy human hearts
Jair Trap Goulart1, Orlando Petrucci2, Karlos Alexandre de Souza
Vilarinho2, Felipe Augusto da Silva Souza2, Pedro Paulo Martins de
Oliveira2, Lindemberg Mota Silveira-Filho2, Jos Wilson Magalhes
Bassani1,3, Rosana Almada Bassani3
1
Department of Biomedical Engineering, School of Electrical and Computer
Engineering, University of Campinas, Campinas, SP, Brazil
2
Department of Surgery, Faculty of Medical Sciences, University of
Campinas, Campinas, SP, Brazil
3
Center for Biomedical Engineering, University of Campinas, Campinas, SP,
Brazil

Background: The current information on Ca2+ handling in human

myocardium comes from studies using tissue from explanted hearts or
patients undergoing cardiac surgery. As Ca2+ handling may be altered
in these diseased hearts, it is possible that information from these experiments carry an inherent bias in the case of control preparations. Because data from healthy human hearts are scarce, our aim was to
investigate SR-cytosol Ca2 + transport during a twitch in atrial
cardiomyocytes isolated from healthy human.
Methods: A segment of the left atrium of donor hearts, resected during orthotopic transplantation, was used for cardiomyocyte isolation
with collagenase. Cytosolic free Ca2+ concentration ([Ca2+]i) was measured with indo-1 AM, in cells paced at 0.5 Hz at room temperature. The
SR content (SR[Ca2+]), the SR fractional release during a twitch (FR),
and the integrated Ca2 + uxes carried by SR Ca2 +-ATPase (SERCA)
and Na+/Ca2+ exchanger (NCX) were estimated from the [Ca2+]i decline phase of different types of Ca2+ transients (Gen Physiol Biophys
31:401-8, 2012). The protocols were approved by the institutional Committee for Ethics in Research (CAAE/FCM/UNICAMP, number
32931014.0.0000.5404).
Results: In 10 cells (3 hearts), SR[Ca2+] was 104.48.8 moles/liter
of non-mitochondrial cell water, and FR was 0.410.08. The uxes mediated by SERCA and NCX (and their respective percent contribution to
cytosolic Ca2 + removal) were 43.06 9.62 mol/L (86%) and
3.800.82 mol/L (9%), respectively, while the remaining 5% were carried by slower transporters.
Conclusions: According to our knowledge, we here describe for the
rst time relevant quantitative data on Ca2+ handling in atrial myocytes
from non-diseased human hearts, which may contribute to the establishment of reference values for comparison with those from cells

S49

isolated from diseased patients, and be helpful in the choice of the

best animal models.
Acknowledgments: R&D-CEB staff, CAPES, CNPq, Brazilian Ministry
of Health, FINEP, Dr. Lars S. Maier.

WE-079
Endothelial ATP-binding Cassette G1 in Mouse Endothelium
Protects against Hemodynamic-induced Atherosclerosis
Jinlong He1, Jiaxing Wang2, Xu Zhang1, Wei Pang2, Ding Ai1, Yi Zhu1,2
1

Tianjin Medical University, Tianjin, China

Peking University Health Science Center, Beijing, China

Abstract
AimsActivated vascular endothelium inammation under persistent hyperlipidemia is the initial step of atherogenesis. ATP-binding cassette G1 (ABCG1) is a crucial factor maintaining sterol and lipid
homeostasis by transporting cholesterol efux to high-density lipoprotein. In this study, we investigated the protective effects of ABCG1 in endothelial inammation activation during early-stage atherogenesis in
mice and the underlying mechanisms.
Methods and resultsEndothelial cell-specic ABCG1 transgenic
(EC-ABCG1-Tg) mice were generated and cross-bred with lowdensity lipoprotein receptordecient (Ldlr-/-) mice. After a 4-week
Western-type diet, compared with Ldlr-/- mouse aortas, EC-ABCG1Tg/Ldlr-/- aortas showed decreased early-stage lesions, as evidenced
by decreased lesion area, lipid content, collagen deposition and macrophage inltration. In addition, the expression of EC activation markers
and inammatory factors was decreased in EC-ABCG1-Tg/Ldlr-/- aortas.
Adenoviral overexpression of ABCG1 blunted cholesterol- and TNFactivated ECs in vitro. Furthermore, the lesion area in the EC-ABCG1Tg/Ldlr-/- mouse aortic arch but not thoracic aorta was signicantly reduced, which suggests a protective role of ABCG1 under atheroprone
ow. In vitro, adenoviral overexpression of ABCG1 attenuated EC activation caused by oscillatory shear stress. In exploring the mechanisms
of ABCG1 attenuating endothelial inammatory activation, we found
that ABCG1 inhibited oscillatory ow-activated nuclear factor kappa
B and NLRP3 inammasome in ECs.
Conclusions ABCG1 may play a protective role in early-stage atherosclerosis by reducing endothelial activation induced by oscillatory
shear stress via suppressing the inammatory response.

WE-080
High-throughput screens to discover inhibitors of leaky ryanodine
receptor calcium channels
Robyn Rebbeck, Maram Essawy, Florentin Nitu, David Thomas, Donald
Bers, Razvan Cornea
1

University of Minnesota, Minneapolis, Mn, USA

University of California, Davis, California, USA

Using uorescence lifetime (FLT) detection of uorescence resonance energy transfer (FRET), we have developed and validated highthroughput screening (HTS) methods to discover compounds that modulate the ryanodine receptor (RyR) Ca2+ (Ca) channel for therapeutic
applications. Regulation of cellular Ca homeostasis is critical for skeletal
and cardiac muscle function, and RyR is a central player. In
cardiomyocytes, high Ca leak via the cardiac isoform of RyR (RyR2),
and reduced SR Ca uptake, conspire to reduce the SR Ca content and elevate diastolic [Ca]i, both of which are hallmarks of heart failure (HF).
RyR2s that open inappropriately during diastole contribute to both systolic and diastolic dysfunction and arrhythmias in HF. Therefore, inhibitors of the RyR2 leaky state could become highly effective drugs. Our
HTS methods specically detect binding of key RyR modulatory proteins

S50

Abstracts

(CaM and FKBP12.6) that have been implicated in controlling the pathological RyR2 leak. Thus, under oxidizing conditions that mimic a pathological state, a drug that restores normal CaM and/or FKBP binding may
correct the leaky RyR2 state. Integration of uorescently labeled
FKBP12.6 and CaM and FRET enables translation of these tools into ultrasensitive HTS assays to assess the RyR leaky conformation. We have
carried out a pilot screen of the 727-compound NIH Clinical Collections,
which yielded several compounds that changed FRET by N3SD (a typical
threshold used to select hits in primary HTS). Ongoing studies will show
how the HTS structural readout correlates with effects on RyR function.

WE-081
Functional crosstalk of RyR2 and InsP3R2 mediated SR-Ca2+ release
in atrial cardiomyocytes
Marcel Wullschleger, Marcel Egger
Physiology, UniBE, Bern, Switzerland
Inositol 1,4,5-trisphosphate (InsP3)-induced intracellular Ca2+ release (IP3ICR) has been implicated in modulatory functions of
excitation-contraction coupling (ECC) in cardiac myocytes. Recently
augmented inositol 1,4,5-trisphosphate receptor (InsP3R2) expression
and function has been linked to a variety of cardiac pathologies including cardiac arrhythmogenicity although its role in ECC in atrial and ventricular myocytes is not conclusively characterized. We aimed to
elucidate local crosstalk mechanisms between InsP3R2 and cardiac
ryanodine receptors (RyR2s) in an InsP32 TG mouse model that exhibits
increased cardiac specic InsP3R2 activity.
Using rapid two-dimensional Ca2+ spark analysis (x,y confocal images, 150 Hz), we report in this study that in cardiac cells, local Ca2+ release by InsP3R (Ca2 + puffs) directly activates RyRs to trigger
elementary Ca2+ release events (Ca2+ sparks) with a 266 ms delay of
onset and vice versa, but with a delay of 47 ms. Endothelin-1 (ET-1),
which activates phospholipase C (PLC) and subsequent InsP3 production, triggered an increase in Ca2 + spark frequency from 2.3 to 9.2
Ca2+ sparks 1000 m-2 s-1. Inhibition of the intracellular InsP3 pathway
in the presence of phenylephrine by application of the PLC inhibitor U73122 abolished the Ca2 + puff occurrence and lead to a decrease of
Ca2 + spark frequency from 5.1 to 1.8 Ca2 + sparks 1000 m-2 s-1.
IP3ICR is under local control of Ca2+ release by RyRs open probability.
In our study, this was mimicked by UV-ash photolysis of caged Ca2+,
promoting Ca2+ puffs in the presence of intracellular InsP3.
These results strongly support the concept that IP3ICR can effectively modulate RyRs openings and Ca2+ spark probability in order to shape
global Ca2+ transients and contractility in cardiac myocytes. We conclude that IP3ICR and highly efcient InsP3 dependent SR-Ca2+ ux is
the main mechanism of functional crosstalk between InsP3Rs and
RyRs leading to increased ECC sensitivity. By using this TG mouse
model which exhibits cardiac specic functional overexpression of
InsP3Rs in a similar fashion to human cardiac disorders, this work provides novel perspectives for local control of Ca2+ signaling mechanisms
in cardiac myocytes under physiological and pathophysiological
conditions.

WE-082
Inuence of ACE inhibitors on frailty and cardiac function in middleaged female C57BL/6 mice
Kaitlyn Keller, Susan Howlett
Dalhousie University, Halifax, Nova Scotia, Canada
Objective: ACE inhibitors improve exercise capacity in functionally
impaired older adults without cardiovascular disease and improve
physical performance in aged rodents. This suggests these drugs might

attenuate frailty. We determined whether chronic treatment with ACE

inhibitors attenuates frailty and whether this is accompanied by changes in cardiac function in middle-aged mice.
Methods: One-year old female C57BL/6 mice were treated with either an ACE inhibitor (enalapril; 40 mg/kg/day; n = 5) or placebo
(n=5) for 3 months. Frailty was quantied with a frailty index (FI)
as accumulation of clinically apparent health decits. Blood pressure
(BP) was measured with a tail-cuff; in vivo cardiac function was measured using echocardiography. Contractile function and calcium homeostasis (fura-2) were measured simultaneously in eld-stimulated
cardiomyocytes (2 Hz).
Results: Results showed that FI scores were higher in placebo mice
when compared to enalapril-treated mice after 3 months of treatment (0.21 0.03 vs. 0.14 0.01, pb0.05). BP was not signicantly different between the drug and placebo groups. Echocardiography showed
no changes in in vivo heart structure or systolic and diastolic contractile
function with enalapril treatment. Heart rate was unaffected by drug.
Cardiomyocytes obtained from enalapril-treated mice showed increased cell shortening (1.59 0.22 vs 3.01 0.47 % resting cell length,
p b0.001), increased velocity to peak contraction (0.068 0.005 vs
0.133 0.016 m/ms, pb 0.001) and increased velocity to relaxation
(0.044 0.005 vs 0.100 0.016 m/ms). No signicant changes occurred in underlying calcium transients.
Conclusion: These results suggest that ACE inhibitor treatment
may enhance cellular contractile function independent of effects on
calcium homeostasis. Furthermore, ACE inhibitors attenuate frailty
in middle-aged animals, even in the absence of cardiovascular
disease.

WE-083
Chronic testosterone withdrawal modies cardiac contraction and
calcium homeostasis in ventricular myocytes isolated from
gonadectomised C57BL/6 male mice
Omar Ayaz, Robert Rose, Susan Howlett
Dalhousie University, Halifax, Canada
Objective: The inuence of testosterone on cardiac function is not
well understood. This study determined the impact of chronic testosterone withdrawal on cardiac contractile function and calcium
homeostasis.
Methods: Male C57BL/6 mice had either a bilateral gonadectomy
(GDX) or a sham operation at 4 weeks of age. Ventricular myocytes
were isolated (age 7-11 mos) by enzymatic digestion and cells were
used for eld-stimulation, current clamp, and voltage clamp studies (2
Hz; 37C). Western blot experiments used protein from ventricular homogenates. Contractions and calcium transients (fura-2) were measured simultaneously.
Results: Peak calcium transients and contractions were similar in
myocytes from GDX and sham-operated controls, although calcium
transients (442.3 vs 542.7 ms, Pb 0.05) and contractions (281.5
vs 393.1 ms, Pb 0.05) were prolonged by GDX. Action potential duration also was prolonged in GDX myocytes compared to sham controls
(56 3.0 vs 74 4.6 ms, Pb 0.05) although resting membrane potentials were not different. When the duration of depolarization was controlled with voltage clamp, GDX suppressed peak contractions and
calcium transients, with no difference in E-C coupling gain. Calcium currents from GDX myocytes had a smaller peak (5.90.4 vs 4.50.4 pA/
pF, Pb0.05), prolonged decay (130.8 vs 171.6 ms, Pb0.05), with no
difference in current density compared to sham. Sarcoplasmic calcium
content (10 mM caffeine) was attenuated by GDX, while fractional release was unaffected. Western blots of key calcium handling proteins
(Cav1.2, NCX, SERCA, RYR) showed no change in expression in sham
vs GDX hearts. By contrast, calcium sparks in uo-4 loaded myocytes
were smaller (0.381 vs 0.373 F/F0, P b0.05), less frequent (7.90.9 vs

Abstracts

5.8 0.9 /100m/sec, P b0.05), and decayed more slowly in GDX

myocytes (200.6 vs 221.6 ms, Pb 0.05) when compared to sham.
Conclusion: Low testosterone levels disrupt calcium homeostasis
and prolong cardiac relaxation. This may promote diastolic dysfunction
in older men with very low testosterone levels.

WE-084
Force-frequency relationship in rat ventricular myocytes; elucidating the intracellular mechanisms.
Vernica De Giusti, Ignacio Aiello, Mara Sofa Espejo, Mara Carolina
Ciancio, Ernesto Alejandro Aiello
Centro de Investigaciones Cardiovasculares, Facultad de Ciencias Mdicas,
UNLP-CONICET, La Plata, Argentina
The forcefrequency relationship (FFR) is an important intrinsic regulatory mechanism of cardiac contractility. While an increase in contractile force after elevation of the stimulation frequency (positive
FFR) is elicited in ventricular myocytes of most mammalian species, a
decrease (negative FFR) or no effect (at FFR) in contractile force in response to an elevation of the stimulation frequency is also present in
some species or pathological situations, including rat and in human
heart failure. It is known that reactive oxygen species (ROS) can act as
intracellular signaling molecules activating diverse kinases as CaMKII
and p38 MAPK. In addition, it was demonstrated that p38 MAPK activation induces a negative inotropic effect in ventricular myocytes mediated by a decrease in myolament response to Ca2+. The involvement of
ROS and p38 MAPK activation during the FFR, however, has not been
studied yet. Therefore, our aim was to evaluate the FFR in rat ventricular
myocytes and elucidate the intracellular molecules implicated in such
process. Cell shortening was recorded with an edge detector in isolated
cardiac ventricular myocytes of Wistar rats. The stimulation frequency
was set to 0.5, 1 or 2 Hz. In parallel experiments, Ca2+ transient and
pHi were also recorded by epiuorescence. Data are shown as percentage change at 2 Hz vs 0.5 Hz. * indicates pb0.05 vs Control. Increasing
frequency from 0.5 to 2 Hz decreased Control cell shortening (15.414,02 %; n=20). This negative FFR was changed to positive FFR
when the myocytes were pre-incubated with the ROS scavenger MPG
2 mM (27,874,60 %; n=11*), the NADPH oxidase blocker, Apocynin
300 M (16,483,20 %; n=10*) or inhibiting mitochondrial ROS production with 5-hydroxidecanoate (5-HD) 500 M (30,31 5,81 %;
n = 6*). Similar results were obtained when the cells were preincubated with the CaMKII blocker, KN93 2.5 M or the p38 MAPK inhibitor, SB-203580 10M (23,01 6,28 %; n = 7*, 37,13 7,62 %;
n=7*; respectively). Ca2+ transients or pHi did not signicantly change
in Control or after ROS production inhibition. In conclusion, our results
indicate that the activation of the intracellular pathway involving ROSCaMKII-p38 MAPK is responsible for the negative FFR of rat
cardiomyocytes, likely by desensitizing the response of contractile myolaments to Ca2+.

WE-085
RyR2 haploinsufciency in a rabbit model is compensated by netuning channel activity
Francisco J. Alvarado, Jonathan Hernandez, Y. Eugene Chen, Hector H.
Valdivia
University of Michigan, Ann Arbor, MI, USA
Several reports suggest that RyR2 expression is decreased in Heart
Failure and Hypertrophic Cardiomyopathy, but the contribution of
RyR2 downregulation to the pathology of the disease is unknown.
Using CRISPR/Cas9 technology, we generated a RyR2 knockout rabbit
model to determine the cardiac effects of RyR2 deciency. Mating of

S51

heterozygous knock-out rabbits does not yield homozygotes, highlighting RyR2 relevance for development (105 kits, pb0.001). Heterozygous
hearts show haploinsufciency, with 33.86.1% of RyR2 expression in
the left ventricle (LV) and 54.219.5% in the atria (n=5 per genotype,
pb 0.05). Remarkably, heterozygous animals subjected to echocardiography (n=7 per genotype) and electrocardiography (n=5-9 per genotype) are not different to wild-type littermates. Additionally,
haematoxylin/eosin and Massons trichrome staining of LV and atrial biopsies showed no difference in the general microscopic structure between genotypes (n= 3 per genotype). To determine the mechanism
that prevents an abnormal phenotype in heterozygous knock-out
hearts, we looked at the expression of excitation-contraction proteins.
The expression of SERCA2a, NCX, Cav1.2 and phospholamban (n=3-5
per genotype) in heterozygous rabbits was comparable to that observed
in wild-type animals. Using [3H]ryanodine binding assays, we tested the
sensitivity of RyR2 to increasing [Ca], between 10 nM and 100 M. Wild
type and heterozygous LV samples showed the same Ca-dependent activation (EC50 1.15 0.09 and 1.03 0.05 M, respectively; n= 5 per
genotype). However, the maximum [3H]ryanodine binding normalized
to RyR2 density was 1.91-fold higher in heterozygous samples, suggesting that remaining RyR2 channels are more active. Finally, the phosphorylation of RyR2-S2808 and RyR2-S2814 was not different
between genotypes, but RyR2-S2031 was signicantly more dephosphorylated in the heterozygous LV (n = 3 per genotype, p b0.05).
These data suggest that a large RyR2 protein reserve sustains normal
cardiac function, at least under basal (non-stimulated) conditions.
Moreover, RyR2 activity can be ne-tuned through phosphorylation to
compensate for protein deciency.

WE-086
Thyroid Stimulating Hormone can directly modulates the cardiac
electrical activity
Julieta Fernandez Ruocco1, Hiart Alonso2, Gallego Monica2, Layse
Malagueta Vieira3, Ainhoa Rodriguez De Yurre1,2, Oscar Casis2, Emiliano
Medei1
1

Carlos Chagas Filho Biophysical Institute, Federal University of Rio de Janeiro

(UFRJ), Rio de Janeiro/ Rio de Janeiro, Brazil
2
Departament of Physiology, Pais Vasco University (UPV/EHU), Pais Vasco,
Spain
3
Department of Biophysics and Radiobiology, Federal University of Pernambuco, Recife, Brazil
Background: The electrocardiogram of hypothyroid patients shows
a series of abnormalities of cardiac repolarization due to a reduction of
some repolarizing K + currents and an increase of the L-type calcium
current. Experimental and clinical works called into question the unique
role of T3 and T4 in these mechanisms and correlated serum TSH levels
with the repolarization abnormalities in patients with subclinical and
overt hypothyroidism. The aim of the present study was to investigate
the direct effects of TSH upon cardiac electrical properties.
Methods: We studied the direct acute (30 min) and long term
(24 h) effects of the activation of TSH receptor by the TSH
(30mUI/ml) on adult rat cardiac preparations. We rst studied
acute effects in electrocardiograms records of Langendorff perfused hearts and also, using patch clamp technique, we recorded
Ito current in ventricular myocyte. The long term effects were studied in action potentials of epicardial strips and the transient outward K + current, Ito, and the L-type Ca2 + current, ICa-L, in
ventricular myocytes are recorded too. Finally, Kv and Cav channels subunits mRNA expression was determined by qRTPCR.
Results: TSH has no acute effects on cardiac electrical activity. However, prolonged exposition to TSH increased the action potential duration through a reduction of the amplitude of Ito current in adult
ventricular myocytes due to a reduction in Kv4.2, Kv4.3 and KChIP2

S52

Abstracts

mRNA expression. Interestingly, TSH had no effect on either ICa-L current or Cav1.2 mRNA expression.
Conclusion: These results support the idea that some of the electrical abnormalities seen in hypothyroid hearts, such as increase in ICa-L, are due to
the reduction of T3 levels, and introduce the possibility that others, such as
TSH elevation, could also be involved in this cardiac electrical disturbances.

WE-087
miR-19b deciency impairs cardiac repolarization in zebrash
Alexander Benz, Dominik Auth, Claudia Seyler, Edgar Zitron, Hugo A.
Katus, David Hassel
Department of Medicine III, Cardiology, Heidelberg University Hospital,
Heidelberg, Germany
The most fatal complication of heart failure (HF) is sudden cardiac
death which results mostly from impaired electrical activity of the
heart and arrhythmias. During HF electrical remodeling includes the
prolongation of the ventricular action potential duration (APD) that
may be interpreted as an acquired long-QT syndrome. Molecular
mechanisms contributing to the action potential (AP) perturbation
are still inadequately understood. microRNAs are small noncoding
RNAs that post-transcriptionally ne-tune gene expression by translational repression or transcript destabilization. By now, several
microRNAs are known to be dysregulated during HF, suggesting a potential involvement in the development and progression of the disease. Here, we identied miR-19 to be an important regulator of
heart function. Zebrash lacking miR-19 developed severe bradycardia and reduced cardiac contractility. While the mammalian genome
encodes for two isoforms of miR-19, zebrash express four members
(19a-d). We found that the reduction of miR-19b specically
deploying morpholino mediated knockdown and CRISPR/Cas9 induced knockout is sufcient to cause bradycardia and reduced cardiac
contractility. Moreover, miR-19b deciency results in increased sensitivity to an AV-Block, which is a characteristic feature of long QTSyndrome in zebrash. Recordings of ventricular APs from paced
hearts demonstrated that APD is signicantly prolonged and repolarization is impaired in miR-19b decient zebrash. Strikingly, by reduction of miR-19b we were able to normalize the arrhythmogenic
phenotype of a short QT-mutant zebrash. Mechanistically, miR19b regulates the expression and thereby modulates the function of
several cardiac ion channels crucially involved in shaping the AP. In
conclusion, we identied miR-19b as a novel and essential modulator
of the electrical activity of the heart and establish miR-19b as a potential candidate gene causative for human long QT.

WE-088
Early intravenous low/high doses of Metoprolol in myocardial
infarction dogs on the effects of cardiac sympathetic activities
and electrophysiological properties
Danning Wang, Dening Liao
Department of Cardiology,Changzheng Hospital,Second Military Medical
University, Shanghai, China
Objective: Observed effects of early intravenous low/high doses of
Metoprolol in myocardial infarction dogs on cardiac sympathetic activities and electrophysiological properties
Methods: 32 dogs were randomly divided into three groups. After
establishing the MI model, the low-dose group was given metoprolol
0.6mg / kg iv, the high-dose group was given 1.6mg / kg, while the control group was injected with normal saline. Catecholamine levels in the
coronary sinus blood, ERP and the incidence of VA were all measured.

Results:
1. NE and E were all increased compared with the values before ligation; Changes in the control group was the biggest; The low-dose and
high-dose group performs no signicant differences (p N0.05);
2. ERP after MI was signicantly shorter in all groups compared with
the rst measurement; The low and high dose group shortened approximately, there were no statistically differences; All exhibited uneven
shortness of ERP among different regions, infarcted area was signicantly shortened (pb 0.05);
3. In control group there was 4 dogs induced PVT/VF, the low-dose
group had 5, the high-dose group had 4. There was no signicant difference among all groups (p N 0.05);
Conclusion: Low and high dose of metoprolol performed similarly
in reducing the catecholamine concentrations in dogs with anterior
myocardial infarction, the same effects also observed in the reduction of regional ERP, but there were no differences in induced
arrhythmias.

WE-089
Inhibition of small Conductance Ca 2 +-activated K+-Channels
converts and prevents Reinduction of atrial Fibrillation in Pigs
where Vernakalant fails
Jonas Goldin Diness 1,2, Lasse Skibsbye2, Jesper Hastrup Svendsen3,
Tobias Speerschneider1,2, Nils Edvardsson4, Ulrik Svane Soerensen1,
Thomas Jespersen2, Morten Grunnet1,2, Bo Hjorth Bentzen1,2
1

Acesion Pharma, Copenhagen, Denmark

The Danish National Research Foundation Centre for Cardiac Arrhythmia,
University of Copenhagen, Copenhagen, Denmark
3
The Danish National Research Foundation Centre for Cardiac Arrhythmia,
The Heart Centre, Rigshospitalet, Copenhagen, Denmark
4
Sahlgrenska Academy at Sahlgrenska University Hospital, Gothenburg,
Sweden
2

Introduction: Evidence has emerged that small conductance Ca2+activated K+-channels (SK-channels) constitute a promising new atrialselective target for treatment of atrial brillation (AF). Current antiarrhythmic therapy suffers from ventricular adverse effects and becomes
less effective as the disease progresses. We therefore tested the antiarrhythmic properties of a new SK channel inhibitor in a porcine model
of AF in a setting of remodelled atria that completely abolished efcacy
of the marketed antiarrhythmic drug vernakalant.
Methods: Eight pigs were subjected to atrial tachypacing (AT-P)
until they developed sustained AF that could not be converted by
vernakalant (4 mg/kg infusion over 10 minutes). In these pigs the efcacy of a new SK channel inhibitor, AP14145, was investigated.
The effects of AP14145 and vernakalant (constant rate infusion producing a clinically relevant plasma concentration of ~ 4000 ng/ml) on
the effective refractory periods (ERP) in the atria and ventricles and
the effects on acute burst pacing-induced AF were examined in openchest experiments in anaesthetized pigs subjected to 7 days AT-P as
well as sham operated control pigs.
In both sets of experiments AP14145 was given as bolus injections of
5 mg/kg, 8 mg/kg, and 8 mg/kg with 30 minutes intervals.
Results: The time for the development of vernakalant-resistant AF
was 17.6 5.2 days of A-TP. In 8/8 pigs, AP14145 converted
vernakalant-resistant AF to sinus rhythm. 4 pigs converted after the
low dose, 3 pigs after the middle dose and 1 pig after the maximal
dose. Reinduction attempts (3xburst pacing) failed in all pigs after conversion with AP14145.
In open-chest experiments, vernakalant and AP14145 signicantly
prolonged atrial ERP by 68 31ms and 107 10ms, respectively in
the AT-P pigs and by 49 32 ms and 100 19ms in the control pigs
and signicantly reduced AF-duration without affecting the ventricular
ERP or blood pressure in pigs subjected to 7 days AT-P and control pigs.

Abstracts

Conclusion: SK current inhibition was effective even after some remodeling when vernakalant was no longer effective. This implies that
SK inhibition may have advantages over current treatments and is therefore a promising concept for further development for treatment of AF.

WE-090
Comparing R2CHADS2 and CHADS2VASC Scores in Stroke Patients
With Non-Valvular Atrial Fibrillation and renal failure.
Mohinder Reddy Vindhyal, Shravani Vindhyal, Travis Haneke, Paul
Ndunda, Freidy Eid, Kenneth J Kallail
KU School Of Medicine - Wichita, Wichita, Kansas, USA
Introduction: Atrial brillation (AF) is the most common rhythm
disorder in hospitalized patients. CHA2DS2-VASc and R2CHADS2 are
the stroke risk assessment tool scores for patients with atrial brillation
(2). Even though renal failure is independently associated with stroke
(1), it has not been included in the CHADS2-VASc risk stratication system, which is used for anticoagulation recommendation in non-valvular
AF patients as endorsed by ACC/AHA. Our study retrospectively compared R2CHADS2 to CHA2DS2-VASc scores in stroke patients with a
past medical history of non-valvular AF to assess differences in
predicting stroke in patients with renal failure.
Methods: 171 patients admitted over two years from one hospital
with a diagnosis of atrial brillation and strokes were reviewed. Data
variables included: age, medical record number, sex, race, renal function
and any previously documented CHA2DS2-VASc scores. If the CHA2DS2VASc and R2CHADS2 scores were not documented, they were calculated
based on information within the medical record. GFR was calculated
using the Chronic Kidney Disease Epidemiology Collaboration formula
Results: The median CHA2DS2-VASc score was 6 (range 2-9) and the
median R2CHA2DS2 score was 4 (range 2-8). The average GFR was 69.77
(range 6-108). A weak, but signicant, correlation was found between
renal function and CHA2DS2-VASc score (r = -0.263; p = 0.0005). A
stronger and signicant correlation was revealed between the
R2CHADS2 and GFR (r = -0.70; p b 0.00001). CHA2DS2-VASc and
R2CHADS2 scores also were signicantly correlated (r = 0.627; p b
0.00001).
Discussion: The risk of stroke in patients with impaired renal function is high. Although CHA2DS2VASc and R2CHADS2 are signicantly
correlated to each other, using R2CHADS2 would be benecial to assess
stroke risk in patients with decreased renal function and non-valvular
atrial brillation.
References
1. Stroke Prevention in Atrial Fibrillation study. N Engl J Med
1990;322:8638.
2. Piccini.et.al.circulation-2013 Jan 15: 127(2):224-32.doi:10 1161/
CIRCULATIONAHA. 112.107128.

WE-091
Characterization of Catecholaminergic Polymorphic Ventricular
Tachycardia Using Patient-Specic Human Induced Pluripotent
Stem Cells and a Transgenic Mouse Model Harboring the Mutation
H2464D in the Cardiac Ryanodine Receptor.
Jonathan J. Hernndez1,2, Yanting Zhao1, Carmen Valdivia1, Todd
Herron1, Jianhua Zhang2, Kathleen R. Maginot3, Timothy J. Kamp2,3,
Jos Jalife1, Hctor H. Valdivia1
1

Center for Arrhythmia Research, University of Michigan, Ann Arbor, Michigan, USA
2
University of Wisconsin-Madison, Madison, Wisconsin, USA
3
University of Wisconsin School of Medicine and Public Health, Madison,
Wisconsin, USA

S53

CPVT is a disease characterized by adrenergic-induced ventricular

tachycardia caused by mutations in the cardiac Ryanodine Receptor
(RyR2). We aimed to perform a characterization of the mechanisms of
CPVT using recombinant RyR2 channels, human induced pluripotent
stem cell-derived cardiomyocytes (hIPS-CMs) and a transgenic mouse
model, all harboring the mutation RyR2-H2464D (HD), to determine
mechanisms of arrhythmia and assess patient-specic therapeutic
drugs.
Methods: hiPSCs generated from human broblasts carrying the HD
mutation (HD1 and HD2) and from his mother as a healthy control (HC)
and a non-relative control (HNRC). HD mice were generated by homologous recombination. Ca2 +-dependence of [3H]ryanodine binding
served as an index of the activity of rRyR2. hiPS-CM single cells loaded
with uo-4AM were used to measure intracellular Ca2+ transients. Optical mapping measured Ca2 + handling in puried uo-4AM loaded
hiPSC-CM monolayers. Echocardiograms and surface ECGs in anesthetized mice evaluated the condition of the hearts.
Results: HD mutation increases cytosolic Ca2+ sensitivity with respect to control (EC50: HD-0.730.1 vs.WT-0.150.03 M Ca2+). Cytosolic [Ca2 +] (0.1 M) increased the activity of HD higher than WT
(Po b0.01 WT and 0.236 HD). CaT amplitude (1.5 0.2 vs.2.2 0.4 F/
F0, n=4 and 5) and time-to-peak (10144 vs. 18140ms) were decreased in HD with respect to control and Increased in HD (42024
vs. 28726 ms,p b0.05). The incidence of monolayers with abnormal
Ca2+ release after isoproterenol treatment was HD1 25%, HD2 45%, HC
0% and HNRC 0%. Heart rate and fractional shortening were not signicantly different between WT and HD+/-. Only HD+/- mice developed
PVCs, bigeminy and bidirectional ventricular tachycardia after Epi/Caff
challenge (n=6 for all groups).
Conclusions: HD confers a gain of function to RyR2 channels; Ca2+
handling abnormalities in HD monolayers and arrhythmias in mice are
due to higher Ca2+ sensitivity of RyR2 channels that is linked with the
phenotype of the disease.

WE-092
Refractoriness in human atria: Time and voltage dependence of
sodium channel availability
Lasse Skibsbye1, Thomas Jespersen1, Torsten Christ2, Mary M Maleckar3,
Jussi T Koivumki3,4
1
Department of Biomedical Sciences, Faculty of Health and Medical
Sciences, University of Copenhagen, Copenhagen, Copenhagen, Denmark,
2
Department of Experimental Pharmacology and Toxicology, University
Medical Center Hamburg-Eppendorf, Hamburg, Germany
3
Center for Cardiological Innovation and Center for Biomedical Computing,
Simula Research Laboratory, Oslo, Norway
4
Department of Biotechnology and Molecular Medicine, A.I. Virtanen Institute for Molecular Sciences, Kuopio, Finland

Background: Refractoriness of cardiac cells limits maximum frequency of electrical activity and protects the heart from tonic contractions. Short refractory periods comprise excellent arrhythmogenic
substrates and augmentation of refractoriness is therefore seen as the
main mechanism of antiarrhythmic drugs. Excitability of
cardiomyocytes depends on availability of sodium channels, a function
involving both time- and voltage-dependent release from inactivation.
The aim of this study was to characterize how sodium channel inactivation affects refractoriness and thereby human atrial electrophysiology.
Methods and results: Steady-state activation and inactivation parameters of sodium channels measured in vitro in isolated human atrial
cardiomyocytes were used to parameterise a new mathematical human
atrial cell model. Action potential data were acquired from human atrial
trabeculae muscle strips of patients in either sinus rhythm or chronic
atrial brillation. The ex vivo measurements of action potential duration,
effective refractory period and resting membrane potential were well-

S54

Abstracts

replicated in simulations using this new in silico model. Interestingly,

the voltage threshold potential at which refractoriness was observed
was not different between sinus rhythm and chronic atrial brillation
tissues and was neither affected by changes in frequencies (1 vs. 3 Hz)
nor by variations in action potential duration.
Conclusions: Our results suggest a preferentially voltagedependent, rather than a time-dependent, effect with respect to refractoriness at physiologically relevant rates in healthy human atria. Hence,
as the resting membrane potential is hyperpolarized in chronic atrial brillation, this voltage-dependent dominance of excitability will profoundly increase the risk of re-initiating and maintaining arrhythmia
in brillating atria.

WE-093
Carvedilol and its non--blocking analog VK-II-86 prevent digitalisinduced Ca++ waves in cardiac myocytes.
Luis A Gonano1, Marisa Seplveda1, Tamara Tottef1, Tom G Backs2, S.R
Wayne Chen2, Alicia Mattiazzi1, Martn Vila Petroff1
1

University of La Plata, La Plata, Argentina

University of Calgary, Alberta, Canada

Background: Cardiotonic glycosides inhibit the sarcolemmal Na+/

K+-ATPase and cause an increase in intracellular Na+, which reduces
Ca++ extrusion through the Na+/Ca++ exchanger. The result is an increase in sarcoplasmic reticulum (SR) Ca++ load and cardiac contractility. However, these compounds have associated arrhythmic effects due
to the occurrence of spontaneous SR Ca++ waves as a result of SR Ca++
overload and CaMKII-dependent phosphorylation of RyR2.
Taking into account that Carvedilol and its non--blocking analog
VK-II-86 are able to prevent spontaneous SR Ca++ waves, we hypothesize that Carvedilol and VK-II-86 would be able to prevent digitalisinduced SR Ca++ waves/spontaneous contractile activity without affecting inotropic response.
Methods and results: In rat cardiac myocytes, paced at 0.5 Hz and
perfused in the presence of 50 M Ouabain for 20 minutes, we observed
an increase in cell shortening of 60 5% (n = 15). We also observed
spontaneous contractile activity as an index of SR Ca++ waves after
stopping electrical stimulation. On average, Ouabain-treated cells presents a signicantly higher number of non-stimulated events (NSE)
compared with control cells (69 10 NSE/10min vs 11 4 NSE/
10 min respectively)
In similar experiments performed in the presence of 1 M Carvedilol,
the frequency of NSE was signicantly reduced to 244 events/10min
(n=13). To conrm that the effect of Carvedilol was dependent on its
capacity to reduce RyR2-mediated spontaneous Ca++ release instead
of its -blocking effect, we used Atenolol (a -blocker without effects
on RyR2 function) and VK-II-86. The presence of Atenolol did not significantly alter the frequency of NSE promoted by Ouabain. In contrast, VKII-86 signicantly reduced the frequency of NSE promoted by Ouabain
(399 events/10min; n=14).
Additionally, VK-II-86 did not affect the development of the positive
inotropic response and the increase in SR Ca++ load induced by Ouabain treatment.
Conclusions: We conclude that the combination of cardiac glycosides with VK-II-86 would improve cardiac contractility without increasing the risk of triggered-arrhythmias.

WE-094
Internal Pacemaker Cell Mechanisms Mediating Autonomic Nervous
Regulation of the Heart Rate
Joachim Behar, Yael Yaniv
Technion, Haifa, Israel

Introduction: Sinoatrial nodals pacemaker cells (SANCs) generate

regular and spontaneous action potentials (APs) that control the rate
of cardiac contraction in mammalians. The brain modulates SANC automaticity, via the autonomic-nervous system, by stimulating membrane
receptors that activate (adrenergic) or inactivate (cholinergic) adenylyl
cyclase (AC). However, there is a limited understanding of the underlying ionic and molecular mechanisms involved in the cross talk between
these membrane receptors and the internal intrinsic mechanisms of
SANCs. We hypothesize that AC-cAMP-PKA signaling is the major messenger between the autonomic-nervous system modulation to SANC
function.
Methods: We modied an SANC computational model to include
autonomic receptors stimulation and its resulting modulation of the
level of intracellular AC-cAMP-PKA. We test the SANC function response
to adrenergic receptor stimulation (by isoproterenol, ISO) or cholinergic
stimulation (by carbachol, CCh). In addition, we perform new experiments on spontaneously beating SANC to assess the role of PKA on AP
ring rate modulation in response to ISO and CCh.
Results: Similar to the experimental results, the model simulations
showed a reduction of 26% in AP ring rate in response to CCh (100
nM) and an increase of 22% in response to ISO (100 nM) with respect
to the basal rate. Eliminating AC-cAMP-PKA signaling abolished the
core effect of autonomic-receptor stimulation on the AP ring rate. Specically, disabling the phospholamban modulation of the SERCA activity
resulted in a signicant reduced effect of CCh and a failure to increase
the AP ring rate under ISO stimulation. The experiments on live
SANC demonstrated the association between PKA activity and the AP
ring rate.
Summary: We provide both experimental and theoretical evidences, that the autonomic nervous system mainly regulates SANC
function via AC-cAMP-PKA signaling. The model predicts that the activation of the SERCA pump via phospholamban phosphorylation is a critical player within this regulatory process.

WE-095
An implanted dual-site pacing device mimics pacing-induced
dyssynchrony and cardiac resynchronization therapy in freely moving rats
Wesam Mulla, Sabina Sapunar, Sigal Elyagon, Hovav Gabay, Janet Ozer,
Noah Liel-Cohen, Yoram Etzion
Ben-Gurion University, Beer-Sheva, Israel
Background: Patients with heart failure often exhibit electrical conduction disturbances leading to electromechanical dyssynchrony and
poor outcome. Right ventricular (RV) pacing can also induce
dyssynchrony and worsen outcome in a similar manner. Cardiac
resynchronization therapy (CRT), in the form of biventricular (BIV) pacing, is a potent modality to treat dyssynchrony. However, critical issues
such as a ~ 30% failure of CRT treatment mandate extensive additional
research. Animal models currently relay on large mammals, which are
expensive and not readily available. Our group developed a simple
methodology for dual-site epicardial pacing in conscious freely moving
rats. We previously demonstrated remarkable similarities to large
mammalian ndings by applying speckle-tracking echocardiography
during different pacing modes.
Aims: (1) To precisely characterize the hemodynamic effects of ventricular pacing in the rat model. (2) To determine the electrophysiological and biochemical effects of RV vs. BIV tachypacing in conscious freely
moving rats.
Methods and results: Two bipolar electrodes were implanted in the
RV and LV of adult SD rats. Electrodes were exteriorized through the
back. Following post-operative recovery, pressure-volume loop recordings were performed during pacing and ventricular function was evaluated. BIV pacing acutely enhanced systolic function compared with RV

Abstracts

or LV pacing. As a single site, however, LV pacing improved systolic

function considerably and similarly to BIV pacing. Three days of RV
tachypacing (n= 6), but not BIV tachypacing (n= 6), induced dispersion of ventricular refractoriness between the RV and LV by
10.03.8 ms and prolonged the QT interval by 6.63 3.1 ms relative
to control (pb 0.05 for both). Biochemically, RV tachypacing increased
p-JNK levels in the early-activated septum of the LV relative to the
late-activated free wall.
Conclusions: This model mimics important electromechanical features seen in large mammalian hearts, and is therefore an attractive
new tool to study the complex pathophysiology of ventricular
dyssynchrony and CRT.

WE-096
Human Calmodulin Mutation associated with Idiopathic Ventricular
Fibrillation causes CaMKII-dependent RyR2 Activation
Nieves Gomez-Hurtado 1, Hyun S Hwang1, Christopher N Johnson1,
Walter J Chazin1, Derek Laver2, Bjorn C Knollmann1
1

Vanderbilt University, Nashville, TN, USA

University of Newcastle, Callaghan NSW, Australia

Background: Calmodulin (CaM) mutations have been associated

with an autosomal dominant syndrome of sudden death that can present with clinical features of catecholaminergic polymorphic ventricular
tachycardia (CPVT) or long QT syndrome (LQTS). CPVT-linked CaM mutations activate ryanodine receptor (RyR2) Ca release channels; whereas LQTS-CaMs have no effects on RyR2 channels but prolong the action
potential by impairing L-type Ca current (LTCC) inactivation. A novel
CaM mutation in CALM1 gene (F90L) was recently found in a family
with Idiopathic Ventricular Fibrillation (IVF) but the mechanism by
which this CaM mutation leads to IVF is not known. Here, we studied
the regulation of RyR2 by F90L-CaM.
Methods and results: Recombinant wild-type (WT) and mutant
CaMs (F90L and N54I) were bacterially expressed and puried. Ca
waves and sparks analyses were done in permeabilized murine
cardiomyocytes incubated with WT or mutant CaMs using confocal microscopy. F90L-CaM increased Ca wave and spark frequency analogous
to N54I, a previously reported CPVT-CaM mutant. However, single
RyR2 channel measurements in lipid bilayers showed that unlike
N54I-CaM, F90L-CaM does not activate RyR2 in a direct fashion. CaMKII
inhibition using 1 M AIP completely abolished the effect of F90L-CaM
on Ca waves and sparks but did not prevent RyR2 activation by N54ICaM. Accordingly, ablation of CaMKII phosphorylation site in RyR2
(S2814A) or introduction of a modication in RyR2 that mimics phosphorylated RyR2 (S2814D) also abolished F90L-CaM effect, conrming
that CaMKII phosphorylation of RyR2 is needed for F90L effects. CaMKII
activation by F90L-CaM was also conrmed by immunoblot. Western
blots revealed that F90L-CaM, but not N54I-CaM, induced an increase
in both CaMKII-Thr286 and RyR2-Ser2814 phosphorylation levels compared to WT-CaM.
Conclusion: In contrast to previously reported CPVT CaM mutants,
the novel F90L-CaM evokes arrhythmogenic Ca disturbances by indirect
activation of RyR2 via CaMKII, which may be a molecular mechanism
underlying IVF.

WE-097
Prevailing action potential duration determines the electrical restitution curve
James Winter1, Yang Hsiang-Yu2, Angela W.C. Lee1, Ken T MacCleod2,
Michael J Shattock1
1

King's College London, London, UK

Imperial College London, London, UK

S55

Background: The dynamics of rate-dependent adaptation of action

potential duration (APD), termed electrical restitution, are thought to
be an important determinant of ventricular arrhythmia. APD prolongation is a hallmark of disease, such as heart failure; however, there has
been little recognition of the apparent association between restitution
kinetics and APD. Abnormal QT prolongation is associated with an increased risk of ventricular arrhythmia.
Objective- To test the hypothesis that restitution kinetics are determined by APD.
Methods and results: Experiments were conducted in isolated
hearts and ventricular myocytes from adult guinea pigs. Restitution
curves were measured in control and following interventions that prolong (clolium, veratradine, low [Ca2+]e) and shorten (catecholamines,
rapid pacing) APD. Despite markedly different mechanisms of action,
prolongation of the ventricular action potential was associated with a
steepening of electrical restitution with all interventions (Figure 1A).
By comparison, the slope of the restitution curve was reduced when
APD was shortened. Isolated myocytes from animals with chronic transverse aortic constriction (TAC), a model of hypertrophy and heart failure, demonstrated prolongation of APD and augmented restitution
kinetics (TAC vs. sham, Figure 1B). This phenotype was revered by application of a small outward current sufcient to normalise APD to
sham values (TAC-INJ). The intrinsic geometrical relationship between
the rate of repolarization and APD is shown to underpin these common
effects, rather than specic effects on ion channel conductances.
Conclusions: APD is a major determinant of electrical restitution.
Any factor that prolongs the action potential, irrespective of the underlying mechanism, will increase the slope of the restitution curve. This
nding has implications for understanding of basic mechanisms of arrhythmia in conditions associated with QT prolongation.

WE-098
Effective treatment of atrial brillation in isolated guinea pig hearts
by combining established anti-arrhythmics and small conductance
Ca2+ activated (SK) K+ channel block
Jeppe Kirchhoff1, Jonas G Diness2, Majid Sheykhzade1, Morten Grunnet2,
Thomas Jespersen1
1

University of Copenhagen, Copenhagen, Denmark

Acesion Pharma, Copenhagen, Denmark

Introduction: Atrial brillation (AF) is the most common sustained

tachyarrhythmia. It is associated with increased morbidity and mortality and there is an unmet need in the current pharmacological treatment
of AF due to low efcacy and severe side effects. The small conductance
Ca2+ activated K+ (SK) inhibition have been reported to exhibit anti AF
effect ex vivo and in vivo. In recent years focus has been increasing on
combining different anti-arrhythmia for synergistic antiarrhythmic
effect.
Hypothesis: We hypothesized that the combination of SK channel
block and established antiarrhythmics in sub-efcacious concentrations
may prevent AF and have the possibility for a reduced proarrhythmic
potential.
Method: Guinea pig hearts were placed in the Langendorff perfused
model. AF was induced by addition of acetylcholine and burst pacing of
the right atrium. Sub-efcacious concentrations of the SK channel
blocker were tested alone and in combination with sub-efcacious concentrations of ecainide, ranolazine, amiodarone or dofetilide.
Results: The combination of SK blocker and ecainide, ranolazine,
amiodarone or dofetilide reduced AF durations compared to the compounds as monotherapy. In higher concentrations at which monotherapy of ecainide and dofetilide cardiovert AF, a signicant increase in QT
interval was observed. This was not observed in combination therapy
with SK channel blocker as the effective dose of the compounds could
be reduced 3-fold.

S56

Abstracts

Conclusion: Our data suggest that combination of SK blocker and

ecainide, ranolazine, amiodarone or dofetilide at reduced doses may
be an effective and safer way to treat atrial brillation.

WE-099
Unnatural Amino Acid Photo-Crosslinking of the IKs Channel
Complex Demonstrates a KCNE1:KCNQ1 Stoichiometry of up to 4:4
Christopher Murray, Maartje Westhoff, Emely Thompson, Robert Emes,
Jodene Eldstrom, David Fedida
University of British Columbia, Vancouver, Canada
Background: The slow delayed rectier current (IKs) provides
repolarizing potassium current during the cardiac action potential. It is
composed of KCNQ1, which forms the tetrameric voltage-gated ion
channel, and KCNE1, a single transmembrane domain -subunit.
KCNE1 resides in the channels exterior clefts and dramatically delays
opening. While this channel complex was characterized almost 20
years ago, the stoichiometry between the and -subunits remains
controversial. Several studies have reported either a strict ratio of 2
KCNE1: 4 KCNQ1 or a variable ratio up to 4:4. Here, we sought to clarify
this issue using IKs fusion proteins, where KCNE1 was linked to one
KCNQ1 (EQ) or two KCNQ1 subunits (EQQ), which produce channels
with compulsory 4:4 or 2:4 stoichiometries, respectively.
Results and conclusions: Whole cell and single channel characterization of EQQ in mammalian cells demonstrated a hyperpolarized V1/2
of activation, reduced conductance and shorter rst latency of opening
compared to EQ or wild type IKs. All of these differences were abolished
by co-expression of EQQ with KCNE1-GFP. To conrm that these additional subunits can be integrated into the complex, the UVcrosslinking unnatural amino acid, p-benzoyl-L-phenylalanine (Bpa)
was genetically incorporated into KCNE1-GFP at residue F57 using the
amber stop codon (TAG) suppression system. Application of UV light
to KCNQ1 + F75Bpa KCNE1-GFP complexes held at -90 mV, trapped
channels in the closed state. The same UV-treatment of F57Bpa KCNE1
with EQQ was found to crosslink at half the rate of KCNQ1, which
shows the association of the independent KCNE1 subunits into the unoccupied clefts in the EQQ channel complex. These ndings differentiate
the functionality of 2:4 KCNE1:KCNQ1 from a wild type channel complex and demonstrate that there is no intrinsic mechanism limiting
the association of additional -subunits up to four, conrming a variable
stoichiometry model for IKs.

WE-100
Role of the NBCn1 Na +/HCO-3 Co-transporter in Mitochondria of
Hypertrophic Hearts
Fernanda Carrizo Velsquez, Lorena Vargas, Bernardo Alvarez
Cardiovascular Research Center, La Plata, Buenos Aires, Argentina
NBC Na+/HCO-3 cotransporter and NHE1 Na+/H+ exchanger have
been associated with cardiac disorders and recently located in mitochondria of cardiomyocytes and coronary endothelial cells (CEC), respectively. Mitochondrial NHE1 (mNHE1) blockade delay the
mitochondrial permeability transition pore (MPTP) opening and reduce
mitochondrial-derived superoxide production, two critical events exacerbated in cells of the disease heart. Conversely, activation of the NBC
isoform, NBCn1, prevented apoptosis in CEC subjected to ischemic
stress. We characterize the role of these transporters in heart mitochondria of adult spontaneously hypertensive (SHR) and control (Wistar)
rats. To examine the role of mNHE1 in mitochondria of SHR and Wistar
rats, expression of mNHE1 in ventricular mitochondrial lysates was analyzed by immunoblots. mNHE1 expression increased by ~ 40% in hypertrophic SHR compared to control (P b 0.05, n = 4). To determine if

the increased expression of mNHE1 in cardiac hypertrophy correlates

with an increase transport activity of the exchanger, mitochondria
were loaded with BCECF-AM and the maximal rate of pHm change measured after addition of 50 mM Na+, monitored by epiuorescence. Mitochondria of SHR showed greater changes in pHm compared to Wistar
rats, 0.100.01 vs. 0.060.01 (Pb0.05, n=5). Additionally, mitochondrial suspensions from SHR and control myocardium were exposed to
200 M CaCl2 to induce MPTP opening (light scattering decrease, LSD)
with the consequent mitochondrial swelling (MS). Surprisingly, SHR
rats showed smaller LSD and a reduction in MS, 6710% (n=26), compared to control, 100 8% (n = 23). Blockade of the NBC with 1 M
S0859 signicantly increased the MS in both, control 139 10%
(n=7), and SHR 11510% (n=7) mitochondria. Finally, NBCn1 Na+/
HCO-3 cotransporter increased by ~70% its expression in SHR heart muscle mitochondria, compared to normal (P b0.05, n = 5). Together our
data suggest that the increased NBCn1 activity seem to play a compensatory role in hypertrophic hearts, protecting mitochondria from Ca2+induced MS and MPTP opening.

WE-101
Increased complex I dependent respiration and increased restriction
for ADP in volume overload-induced atrial dilatation
Kalju Paju, Taavi Pdramgi, Nadeda Peet, Margus Eimre, Lumme
Kadaja, Mart Roosimaa, Andres Piirsoo, Enn Seppet, Arno Ruusalepp
University of Tartu, Tartu, Estonia
Background: Atrial dilatation is a typical consequence of cardiac failure caused by hemodynamic overload. The relations between structural, electrical, and contractile remodeling to
oxidative phosphorylation (OXPHOS) and glycolysis are poorly
understood.
Methods and results: The pieces of right atrium from 77 patients,
detached in order to establish extracorporal circulation during heart
surgery, were used for studies. We found that atrial dilatation was associated with impaired mitochondria as indicated by decreased rate of
glutamate-dependent respiration. This decrease occurred at all functional states of mitochondria nonphosphorylating and phosphorylating, either stimulated by excessive ADP or submaximally by
endogenous ADP produced by ATPases of mitochondrial kinases. Functional coupling between the adenylate kinase system and OXPHOS
was not affected in our dilatated atrium bers, but the coupling between
the kreatine kinase system and OXPHOS diminished by 17%. The significant increase of the KmADP in the absence and presence of creatine in
dilatated bers indicated that the diffusion restriction for ADP into the
mitochondrial intermembrane space was due to all ADP transport pathways, including CrP shuttle. On contrary the adenylate kinase
aktivities increased and we observed also overexpression of HK2 in dilated human atria.
Conclusion: Despite inpaired complex I dependent respiration
and increased diffusion restriction for ADP, no changes regarding
adenylate and creatine kinase occurred. Cardiac energy dependence
on glucose is enhanced in volume overload-induced atrial
dilatation by functional coupling of HK2 with OXPHOS system in
mitochondria.

WE-102
The effect of chronic continuous hypoxia on enzyme activities and
membrane permeability of rat heart mitochondria
Martin Kalous1, Zdenek Drahota2, Anna Chytilova2, Jan Neckar2
1

Faculty of Sciences, Charles University, Prague, Czech Republic

Institute of Physiology, Academy of Sciences of the Czech Republic, Prague,
Czech Republic

Abstracts

Introduction: Adaptation to continuous normobaric hypoxia (CNH)

increases cardiac ischemic tolerance. Cardiac mitochondria can play the
role in this protection. We investigated the effects of CNH on the activity
of selected mitochondrial proteins in spontaneously hypertensive rats
(SHR), and in a novel conplastic strain SHR-mtBN. Mitochondrial calcium
retention capacity were measured exploring potential role of mitochondrial permeability transition pore (MPTP) in cardiac protection.
Methods: Rats were kept 21 days at CNH (inspired O2 fraction 0.1).
Left ventricular homogenate were used for determination of the enzyme activity of malate dehydrogenase (MDH), citrate synthase (CS),
NADH-cytochrome c oxidoreductase, succinate-cytochrome c oxidoreductase and cytochrome oxidase (COX). Mitochondrial respiration
were measured as oxygen consumption. Mitochondrial calcium retention capacity was determined uorimetrically.
Results: Only MDH activity decreased in hypoxic SHR (23%). The respiratory pattern and respiratory control index were similar in mitochondria isolated from left ventricles of normoxic and hypoxic rats.
Basic COX activity did not differ between the strains and was not affected by CNH. The reserve COX activity (measured with 0.02% lauryl
maltoside) was signicantly increased after adaptation to CNH in both
strains (by 45 % in SHR and 38 % in SHR-mtBN). We conrmed that the
mitochondrial calcium retention capacity is lower in SHR hearts compared with control Wistar ones. However, we did not nd any difference
in this parameter between SHR and SHR-mtBN strains.
Conclusions: Adaptation to CNH affected myocardial activity of
some mitochondrial proteins in hypertensive strains, and increased
the reserve COX activity in the left ventricle of SHR independently of mitochondrial genome. Heart mitochondria of SHR strain is more sensitive
to calcium concentration than the control Wistar ones, but no difference
between SHR and SHR-mtBN strains suggests no effect of mitochondrial
genome on MPTP properties.
This work was supported by the Czech Science Foundation grant no. 1310267S

WE-103
Mice lacking the mitochondrial calcium uniporter have alterations
in F1F0-ATP synthase
Randi Parks1, Sara Menazza1, Angel Aponte2, Toren Finkel3, Elizabeth
Murphy1
1

Systems Biology Center, NHLBI, NIH, Bethesda, MD, USA

Proteomic Core Facility, Bethesda, MD, USA
3
Center for Molecular Medicine, Bethesda, MD, USA
2

Knockout (KO) of the mitochondrial Ca 2 + uniporter (MCU)

abrogates rapid mitochondrial Ca 2 + uptake and permeability
transition pore (PTP) opening. However, hearts from global
MCU-KO mice were not protected from ischemic injury (Pan
et al,Nat Cell Biol,2013). This study investigates the hypothesis
that the lack of protection in the absence of MCU may be explained by alterations in PTP opening and/or mitochondrial protein complexes. To investigate whether pore opening occurs in
MCU-KO, hearts were Langendorff-perfused in the presence of a
cyclophilin D-independent pore inhibitor. PTP inhibition was
protective against 30-mins of ischemia in WT hearts, but less so
in MCU-KO. This suggests that pore opening is not a signicant
contributor to ischemic injury in MCU-KO. To better understand
adaptations that occur in the MCU-KO heart that may alter ischemic cell death mechanisms, the cardiac proteome of WT and
MCU-KO whole heart homogenates was compared using tandem
mass tags (n = 5 KO, 5 WT). Results indicate that 96 proteins
were decreased and 112 proteins were increased by 1.3-fold or
greater in MCU KO hearts in comparison to WT. Given the report
that F1F0-ATP synthase is a component of the mitochondrial PTP
(Giorgio et al,PNAS,2013), it was of interest that two ATP

S57

synthase proteins were altered in MCU-KO. ATP synthase subunit

s was decreased 2.2-fold and F1 complex assembly factor 1 was
decreased 1.6-fold in comparison to WT. Blue native PAGE experiments were performed to examine levels of ATP synthase monomers and dimers in isolated mitochondria. Interestingly, the ratio
of dimers to monomers was reduced in MCU-KO. Furthermore,
in-gel trypsin digestion of ATP synthase followed by mass spectrometry analysis revealed that the composition of monomers
and dimers differed between WT and MCU-KO. These results suggest that absence of MCU may alter ATP synthase subunit expression, as well as the formation of ATP synthase dimers.

WE-104
Blocking cell surface nucleolin in heart cells prevents uptake of
immunogenic DNA
Lars Henrik Mariero1, Anton Baysa1, Yuchuan Li1, May-Kristin Torp1,
Guro Valen1, Jarle Vaage2, Kre-Olav Stenslkken1
1

University of Oslo, Oslo, Norway

Oslo University Hospital, Oslo, Norway

Rationale: Cellular debris causes sterile inammation after myocardial infarction. The human heart contains 25 per cent mitochondria and
mitochondrial DNA (mtDNA) is a damage-associated molecular pattern
that can trigger the immune system and induce injurious inammation.
It is not known if mtDNA can trigger inammatory signaling pathways
in the cardiomyocyte and how it is internalized to associate with its putative receptor, toll-like receptor 9 (TLR9). A better understanding of the
post-infarction inammatory response holds the promise of new
treatments.
Objective: To understand if and how mtDNA induces inammatory
responses in cardiac cells and whether cell surface nucleolin is implicated in internalization of immunogenic DNA.
Methods and results: The gene expression of the pro-inammatory
cytokines interleukin-1, tumor-necrosis factor and interferon 1
was upregulated by mtDNA, but not nuclear DNA (nDNA) in
cardiomyocytes exposed to 40 minutes of non-lethal hypoxia and two
hours of reoxygenation. In HEK293 cells, mtDNA induced NF-B activity
in normoxia. Furthermore, 40 minutes of hypoxia and 6-12 hours reoxygenation and CpG DNA synergistically induced TLR9-dependent NF-B
activity. In subcellular protein fractions, nucleolin was expressed in cardiomyocyte membranes and inhibition of cell-surface nucleolin with
midkine inhibited the uptake of CpG DNA in cardiomyocytes and cardiac broblasts.
Conclusion: We show for the rst time that isolated cardiomyocytes
respond with an inammatory response to mtDNA, but not nDNA.
Nucleolin on the cell surface is a possible route for DNA internalization
in cardiac cells. Cell-surface nucleolin might be a therapeutic target to
reduce uptake of immunogenic DNA.

WE-103
Increased calpain-1 in cardiomyocyte mitochondria disrupts ATP
synthase and promotes reactive oxygen species generation to induce
dilated heart failure in mice
Ting Cao1, Dong Zheng1,2, Rui Ni1,2, Lulu Zhang1, Tianqing Peng1,2
1

Soochow University, Suzhou, China

Lawson Health Research Institute, London, Ontario, Canada

Background: Calpain-1 has been shown to increase in mitochondria

of the heart under pathological conditions including ischemia/reperfusion, diabetes and sepsis. Our recent study reported that mitochondrial

S58

Abstracts

calpain-1 promotes superoxide generation in cardiomyocytes and it

may be implicated in myocardial injury and dysfunction. This study
was to investigate whether and how increased calpain-1 in mitochondria induces adverse myocardial remodeling and heart failure.
Methods and results: A novel line of transgenic mice overexpressing cardiomyocyte-specic and mitochondria-targeted
calpain-1 was generated. Over-expression of mitochondriatargeted calpain-1 increased mitochondrial reactive oxygen species (ROS) generation in mouse hearts and induced adverse myocardial remodeling including cardiac hypertrophy, brosis and
enlarged ventricular chambers, leading to heart failure and
early death in transgenic mice, characteristic changes of dilated
cardiomyopathy. These effects of mitochondrial calpain-1 upregulation were attenuated by administration of mitochondriatargeted antioxidant mito-TEMPO. Increased mitochondrial
calpain-1 also correlated with decreased ATP synthase activity
in transgenic mouse hearts. In cultured cardiomyocytes, selective
over-expression of calpain-1 in mitochondria induced superoxide
generation, decreased ATP synthase activity and promoted apoptotic cell death, all of which were inhibited by up-regulation
of ATP5A1 or mito-TEMPO.
Conclusions: Mitochondrial calpain-1 induces myocardial injury, remodeling and dysfunction possibly by disrupting ATP synthase and promoting ROS generation in cardiomyocytes. Thus,
mitochondrial calpain-1 may be a therapeutic target for heart
failure.

TH-001
Changes in cardiac adenosine A3 receptor function and expression
associated with essential hypertension
Roselyn Rose'Meyer, Leanne Low, Ming-Fen Ho
Grifth University, Southport, Queensland, Australia
Background: Essential hypertension is considered to be a
multifactorial disorder and if not treated can contribute to the
development of heart failure. As the adenosine receptors have a
signicant role in mediating vasodilation and cardioprotection,
alterations in their structures or signalling pathways may be involved in the development of hypertension. This study measured
the mRNA expression of adenosine A 3 receptors cardiac tissues
and determined whether they could be altered with essential hypertension. We also investigated adenosine selective A3 receptor
agonist mediated vasodilator responses in coronary blood vessels
using the isolated perfused heart preparation.
Methods: Male spontaneously hypertensive rats (SHR, 10 weeks)
and age-gender matched Wistar rats were used in this study. Cardiac
tissues and a range of blood vessels were collected and processed to isolate mRNA and adenosine A3 receptor expression measured using real
time PCR. Rat isolated hearts were set up in Langendorff mode and perfused with Krebs-Henseleit solution containing 8-phenyltheophylline
(10 M) an antagonist of adenosine A1, A2A and A2B receptors to isolate
adenosine A3 receptor mediated coronary vasodilation.
Results: Adenosine A3 receptor agonists APNEA and CL-IB-MECA induced coronary vasodilation in the presence of 8-phenyltheophylline
(10 M). Vasodilator responses to these agonist were attenuated in
hearts from SHR when compared to control tissues (p b 0.05). The
mRNA expression of adenosine A3 receptors was down-regulated in
atria, left ventricle and thoracic aorta from SHR when compared to cardiac tissue from normotensive animals (pb0.05).
Discussion: This study demonstrated decreases in the expression of
adenosine A3 receptors occurred in cardiac tissue and reduced adenosine A3 receptor mediated coronary vasodilation in hearts from spontaneously hypertensive rats. Our ndings with regard to changes in the
adenosine A3 receptor populations in hypertensive hearts suggest that

adenosine A3 receptor could play a role in physiopathology of essential

hypertension.

TH-002
Physiological and pathological left ventricular hypertrophy of comparable degree is associated with characteristic differences of in vivo
hemodynamics associated with distinct expression of mitochondrial
regulators
Attila Olh, Balzs Tams Nmeth, Csaba Mtys, Lszl Hidi, rpd Lux,
Mihly Ruppert, Dalma Kellermayer, Alex Ali Sayour, Lilla Szab,
Marianna Trk, Anna Meltzer, Bla Merkely, Tams Radovits
Heart and Vascular Center, Semmelweis University, Budapest, Hungary
Background: Left ventricular (LV) hypertrophy is a physiological or
pathological response of LV myocardium to increased cardiac load. We
aimed at investigating and comparing hemodynamic alterations in
well established rat models of physiological (PhyH) and pathological
hypertrophy (PaH) by using LV pressure-volume (P-V) analysis.
Methods: PhyH and PaH were induced in rats by swim training and
by abdominal aortic banding, respectively. Morphology of the heart was
investigated by echocardiography. Detailed characterization of cardiac
function was completed by LV P-V analysis. In addition histological
and molecular biological measurements were performed. All data
were normalized to the corresponding control group.
Results: Echocardiography revealed myocardial hypertrophy of similar degree in both models (LV mass index: + 21.7 2.1% PhyH vs.
+ 27.3 3.3% PaH, n.s.), which was conrmed by post-mortem heart
weight data. In aortic-banded rats we detected subendocardial brosis.
Reactivation of fetal gene program could be observed only in PaH
model. PhyH was associated with increased stroke volume, whereas unaltered stroke volume were detected in PaH along with markedly elevated end-systolic pressure values. Sensitive indices of LV contractility
were increased in both models, in parallel with the degree of hypertrophy. Active relaxation was ameliorated in athletes heart, while it
showed marked impairment in PaH (time constant of LV pressure
decay (): -7.72.6% PhyH vs. +37.011.1% PaH, pb0.01). Mechanical efciency and ventriculo-arterial coupling were improved in PhyH,
whereas remained unchanged in PaH. Myocardial gene expression of
mitochondrial regulators showed marked differences between PaH
and PhyH (peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1): + 19.1 10.3% PhyH vs. -37.8 7.2% PaH,
pb0.01).
Conclusions: We provided the rst comparative hemodynamic
characterization of PhyH and compensated PaH in relevant rodent
models. Increased LV contractility could be observed in both types of
LV hypertrophy, characteristic distinction was detected in diastolic
function (active relaxation) and mechanoenergetics (mechanical efciency), which might be explained by mitochondrial differences.
TH-003
Differential expression of plasmalogen lipids following modulation
via dietary supplementation in a mouse model of reduced PI3K
activity
Yow Keat Tham1,2, Natalie A. Mellett1, Peter J. Meikle1,2, Julie R.
McMullen1
1

Baker IDI Heart and Diabetes Institute, Melbourne, Australia

Monash University, Melbourne, Australia

Background: Phosphoinositide 3-kinase p110 (PI3K) is a critical

regulator of physiological cardiac hypertrophy. Cardiac-specic dominant negative PI3K (DnPI3K) transgenic mice have smaller hearts and
are more susceptible to cardiac insults. Increased PI3K activity is

Abstracts

protective in response to various cardiac disease settings in mice.

Plasmalogens are a class of glycerophospholipids enriched in the
heart, previously shown to be protective against reactive oxygen species. A pilot study suggested that altered PI3K activity was associated
with similar changes in plasmalogen levels.
Aim: To determine whether increased plasmalogens could restore
heart size in DnPI3K mice.
Methods: Plasmalogens were increased via dietary supplementation
with 1% batyl alcohol (BA)/ kg of chow. Non-transgenic (Ntg)/DnPI3K
mice began chow/1% BA dietary intervention at approximately 8 weeks
old for 16 weeks (N = 4-6 per group). Lipid analysis was performed
using liquid chromatography electrospray ionisation tandem mass spectrometry. Data was analysed in Multiquant 2.1 software and specic lipid
species normalised to total phosphatidylcholine (PC) levels.
Results: Total cardiac PC and phosphatidylethanolamine (PE)
plasmalogens were decreased in chow fed DnPI3K vs. Ntg mice
(Pb 0.05). PC and PE plasmalogens with C18:0 fatty alcohol side chains
were increased in Ntg and DnPI3K mice on 1% BA diet vs. chow fed mice
(Pb0.05). Interestingly, 1% BA supplementation increased expression of
several C18:0 PC and PE plasmalogens species in the DnPI3K vs. Ntg
mice. Morphological analysis however, revealed no change in heart weight
to tibia length ratio amongst cohorts. The pathological marker, atrial natriuretic peptide increased 25 fold in chow fed DnPI3K vs. Ntg mice (Pb0.05)
and this trend was not altered with supplementation of 1% BA.
Conclusions: Under basal settings, increasing plasmalogens in
DnPI3K mice does not restore cardiac size. However, differential modulation of some lipid species in BA fed DnPI3K mice suggests this approach may provide protection in a setting of cardiac stress.

TH-004
Proliferative and hypertrophic defects contributes to LMNA associated dilated cardiomyopathy
Kenji Onoue1,2, Hiroko Wakimoto2, Jiangming Jiang2, Michael Parfenov2,
Danos Christodoulou2, Steve DePalma2, David Conner2, Joshua
Gorham2, David McKean2, Yoshihiko Saito1, Jonathan Seidman2,
Christine Seidman2
1

Nara Medical University, Kashihara, Nara, Japan

Harvard Medical School, Boston, MA, USA

Background: LMNA is one of the leading causative genes of genetically

inherited dilated cardiomyopathy (DCM). Unlike sarcomere related
genes, LMNA encodes nuclear envelope proteins, lamin A and C, and
does not have direct association with contractile function. However, mutation in this gene also develops DCM. The underlying mechanisms of developing DCM with LMNA mutation still remain obscure.
Methods and results: To characterize Lmna mutant mice, we
assessed cardiomyocyte number, size, nuclei counting and cell cycle activity. Both cell number and cell size were reduced, myocytes were immature and cell cycle activity, assessed by EdU incorporation to nucleus
and phospho-histone H3 staining, was retarded in Lmna mutant mice.
RNA-sequencing and pathway analysis revealed proliferation of cells
had the strongest impact on Lmna mutant mice. Especially, Cdkn1a,
which encodes cell cycle regulating protein p21, had signicant relationship with Lmna mutation. Upregulation of p21 was observed not
only RNA transcription level but also protein level by Western blot
and immunostaining. DNA damage was more robustly detected in
Lmna mutant mice by immunostaining. Furthermore, a repressed cardiomyocyte proliferating response after resecting apex of the neonatal
heart was observed in Lmna mutant mice. In addition, Lmna mutant
mice lacked the ability of compensatory hypertrophic response against
pressure overload after administration of angiotensin II.
Conclusions: These data strongly suggest that Lmna mutation damages
DNA, which induces p53 and p21 activities and contributes to the reduction

S59

of cell proliferation as well as hypertrophic response in Lmna mutant mice.

Inadequate response against cardiac injury and pressure overload stresses
plays important roles in developing DCM with LMNA mutation.

TH-005
Targeting the L-type Ca2 + channel alters mitochondrial function
and attenuates hypertrophic cardiomyopathy in a Troponin I mutant mouse model
Helena Viola1, Victoria Johnstone1, Christopher Semsarian2,3, Livia
Hool1,4
1

The University of Western Australia, Western Australia, Australia

Centenary Institute, University of Sydney, New South Wales, Australia
3
Royal Prince Alfred Hospital, New Soth Wales, Australia
4
Victor Chang Cardiac Research Institute, New South Wales, Australia
2

Hypertrophic cardiomyopathy (HCM) affects 1 in 200 of the general

population. It is characterised by myocyte remodeling, disorganisation of
cytoskeletal proteins and altered metabolic function. Mitochondrial function can be regulated by alterations in L-type Ca2+ channel (LTCC) activity,
and the cytoskeletal network plays an important role in this response. We
have previously demonstrated that the human HCM causing cardiac troponin I mutation Gly203Ser, leads to a faster LTCC inactivation rate and impaired functional communication between the LTCC and mitochondria in
a mouse model of the mutation (cTnI-G203S). This results in a hypermetabolic mitochondrial state, which precedes development of HCM.
Application of a peptide derived against the cardiac alpha interacting
domain (AID-TAT) slows the LTCC inactivation rate and decreases metabolic function in wt cardiac myocytes (Viola et al JMCC 2009, Viola et al
JAHA 2014). Here we examined the efcacy of in vitro and in vivo exposure
of cTnI-G203S to AID-TAT on mitochondrial function by assessing alterations in mitochondrial membrane potential (m, JC-1 uorescence)
and mitochondrial oxygen consumption (avoprotein autouorescence).
We nd that acute in vitro exposure of cTnI-G203S cardiac myocytes
to AID-TAT normalises m in response to activation of the LTCC with
channel agonist BayK(-) (cTnI-G203S+AID-TAT= 4.40.4% increase,
n= 6 versus cTnI-G203S =29.2 1.8% increase, n =15, p b0.05). AIDTAT also attenuated increases in avoprotein autouorescence in response to BayK(-) (cTnI-G203S+ AID-TAT = 4.00.3% increase, n=6
versus cTnI-G203S=24.46.5% increase, n= 8,pb 0.05). In vivo treatment of cTnI-G203S mice with AID-TAT via intraperitoneal (IP) injection
(10M 3x per week for 5wks) also normalised m (cTnI-G203S+AIDTAT=22.12.6% increase, n=13 versus cTnI-G203S=35.84.7% increase, n=3, pb 0.05), and avoprotein autouorescence in response
to BayK(-) compared to untreated mice (cTnI-G203S + AIDTAT=17.92.5% increase, n=24 versus cTnI-G203S=44.010.1% increase, n =7, p b 0.05). Treatment also prevented the development of
HCM, as evidenced by changes on echocardiography. These data suggest
that treatment of cTnI-G203S mice with AID-TAT restores mitochondrial
function and prevents development of HCM.

TH-006
ProteoSeq a proteotranscriptomics approach to decode alternative
isoform expression in cardiac hypertrophy
Maggie PY Lam, T Umut Dincer, Yi Xing, Peipei Ping
UCLA, Los Angeles, CA, USA
Background: Alternative protein isoform expression is a critical feature of the fetal genetic program associated with the early failing heart.
Notable examples include differential expressions of sarco/endoplasmic
reticulum Ca2+-ATPase (SERCA2a/2b), cardiac sodium channel SCN5A,
and titin. Advances in RNA-seq have led to the discovery of novel

S60

Abstracts

isoforms at the transcript level; however, without information on their

physiological functions and disease relevance, the signicance of these
transcripts remains ambiguous and cannot be validated. Therefore, the
characterization of protein isoforms encoded by alternative transcripts
becomes a unique approach to ascertain the discovery of novel isoforms.
To date, information on novel protein isoforms encoded by alternative
transcripts in the cardiac proteome is scarce.
Methods: We developed a proteotranscriptomics approach,
ProteoSeq, which combines transcriptomics datasets, cardiovascular
disease models, mass spectrometry, and computational algorithms to
characterize proteome-wide alternative isoform expression in the
heart. We derived species- and tissue-specic splicing information
from an ENCODE dataset (C57BL/6J mouse heart; ENCSR000BYQ)
using a Bowtie-Tophat-rMATS pipeline, then translated the information
into custom junction peptide databases to guide protein isoform discovery. Matching proteomics data were acquired in-house followed by a
ProLuCID-DTASelect search.
Results: This new approach enabled the discovery of multiple novel
splice junction peptides in the mammalian heart, presenting strong evidence for the corresponding alternative splicing events at the protein
level. These isoforms belong to diverse pre-mRNA processing events
from mutually exclusive exons, skipped exons, alternative 5 and 3
splice sites, to retained introns. Differential expressions of multiple
novel protein isoforms are conrmed in models of cardiac hypertrophy
(e.g., pyruvate kinase M1 to M2).
Conclusion: We demonstrate the utility of our proteotranscriptomics
approach for identifying new alternative protein isoforms at a proteome
scale. ProteoSeq is being implemented as a unied web-based platform
for on-the-cloud multi-omics analysis. ProteoSeq is currently supported
and beta-tested by 20+ laboratories globally.

TH-007
Folic acid reduces doxorubicin-induced cardiomyopathy by modulating endothelial nitric oxide synthase
Yanti Octavia1,2, Georgios Kararigas3, Martine de Boer1, Rinrada
Kietadisorn2, Melissa Swinnen4, Hans Duimel5, Fons Verheyen5, Ihsan
Chri1, Maarten Brandt1, Caroline Cheng1, Stefan Janssens4, Dirk
Duncker1, An Moens1,2
1

Division of Experimental Cardiology, Department of Cardiology,

Thoraxcenter, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands
2
Department of Cardiology, Cardiovascular Research Institute Maastricht,
Maastricht University Medical Center, Maastricht, The Netherlands
3
Institute of Gender in Medicine and Center for Cardiovascular Research,
Charite University Hospital, and DZHK (German Centre for Cardiovascular
Research) Berlin partner site, Berlin, Germany
4
Department of Cardiovascular sciences, University of Leuven, KU Leuven,
Leuven, Belgium
5
Electron Microscopy Unit, CRISP and Department of Molecular Cell Biology, Maastricht University Medical Center, Maastricht, The Netherlands
Aims: The use of doxorubicin (DOXO) as a chemotherapeutic drug has
been hampered by cardiotoxicity leading to cardiomyopathy and heart
failure. Folic acid (FA) is a modulator of endothelial nitric oxide (NO) synthase (eNOS), which in turn is an important player in diseases associated
with NO insufciency or NOS dysregulation, such as pressure overload
and myocardial infarction. However, the role of FA in DOXO-induced cardiomyopathy is poorly understood. The aim of this study was to test the
hypothesis that FA prevents DOXO-induced cardiomyopathy by modulating eNOS and mitochondrial structure and function.
Methods and results: Male C57BL/6 mice were randomised to a single dose of doxorubicin (20 mg/kg intraperitoneal) or sham. FA supplementation (10 mg/day per oral) was started 7 days before injection and

continued thereafter. DOXO resulted in 70% mortality after 10 days,

with the surviving mice demonstrating a 30% reduction in stroke volume compared with sham groups. Pretreatment with FA reduced mortality to 45% and improved stroke volume (both P b0.05 vs. DOXO).
These effects of FA were underlain by blunting of DOXO-induced cardiomyocyte atrophy, apoptosis, interstitial brosis, and impairment of mitochondrial function. Mechanistically, pretreatment with FA prevented
DOXO-induced increases in superoxide production, by reducing the
eNOS monomer:dimer ratio and S-glutathionylation, and attenuated
DOXO-induced decreases in superoxide dismutase, eNOS phosphorylation and NO production. Furthermore, the protection effects of FA were
abolished in eNOS-knockdown human microvascular endothelial cells.
Conclusions: Enhancing eNOS function and subsequently reducing
oxidative stress with FA may be a novel therapeutic approach to attenuate DOXO-induced cardiomyopathy.

TH-008
The cardiopulmonary vascular system and the ventilatory reex;
scientic merits and clinical implications
Anna Faingersh-Klebanov, Amir Landesberg
Technion IIT, Haifa, Israel
Introduction: The ventilatory baroreex is not well explained
phenomenon, where an increase in heart rate and decrease in blood
pressure are associated with an increased tidal volume. We hypothesize
that changes in lung blood pool and capillary pressure directly affect
lung compliance and play a key role in mediating this reex. The
study investigated this hypothesis.
Methods: The pulmonary blood pool was modulated by inducing
slowly progressing pneumothorax in mechanically ventilated rabbits
(n=7), by continuous air injection into the pleural space. Hemodynamic parameters, tidal pressures and ows, EtCO2 and SpO2 were recorded.
Tidal volume and respiratory system compliance were calculated.
Results and discussion: The slowly progressing pneumothorax was
associated with immediate progressive decline in the BP and compensatory increase in HR. A counterintuitive decrease in EtCO2 was observed
at the initial phase, concurrent with a gradual increase in the tidal volume (+14.65.3%) and respiratory compliance (13.75.2%). The respiratory rate and the inspiratory pressure were constant. Therefore,
the increase in tidal volume resulted from a gradual increase in lung
compliance. Only after 28 min the respiratory indices exhibited the reverse responses, when tension pneumothorax developed.
The initial phase mimics the ventilatory reex. However, the counterintuitive increase in tidal volume and decrease in pCO2 were not
due to involvement of the central nervous system, as the rabbits were
mechanically ventilated at constant inspiratory pressure. The effect appears with mild pneumothorax, demonstrating the high sensitivity of
lung compliance to changes in lung circulation. The opposite occurs in
heart failure where the pulmonary capillary blood pool increases, leading to smaller lung compliance and dyspnea.
Conclusions: The ventilatory reex was observed in ventilated animal
(without nervous pathway) and it is determined by a direct effect of the
pulmonary circulation on lung compliance. Lung blood pool and capillary
pressure are important determinants of the cardio-pulmonary "baroreex".

TH-009
Heart failure assessment with a multiscale model
Jorge Negroni1, Edmundo Cabrera Fischer1, Sarah Kosta2, Pierre Dauby2,
Elena Lascano1
1

Favaloro University, Buenos Aires, Argentina

University of Liege, Liege, Belgium

Abstracts

Background: Heart failure (HF) produces mechanical and hemodynamic impairment. Mathematical models have analyzed the impact of
HF on experimentally identied myocyte components, but their integration into a ventricular model forming part of a multiscale circulatory
approach has not been properly addressed.
Objective: The aim of this study was to compare the experimental
and multiscale model hemodynamic and regional contractile response
to acute HF.
Methods: The left ventricle (LV) was based on a validated contractile
human myocyte and the remaining chambers were dened as elastic
structures. Electrically simulated preload and afterload were coupled
to heart chambers, integrating a closed circulatory circuit. HF effect in
the myocyte decreased K1 and Ito channel conductances by 49% and
36% and SERCA2a activity by 24%, and increased sodium-calcium exchanger conductance by 100%. Right ventricular elastance was decreased by 30%. Halothane (H) 3-4% was used to elicit HF in openchest sheep (n=23) instrumented with LV piezoelectric crystals (wall
thickness, WT), Swan Ganz catheter (cardiac output, CO) and ventricular and carotid artery catheters for intraventricular and arterial pressure
(AP) assessment.
Results: The hemodynamic performance of the model in normal conditions was: mean AP (MAP): 82 mmHg, CO: 4.3 L/min
and ejection fraction: 65%. In sheep experiments, MAP, CO and
systo-diastolic WT fraction (WTF) dropped to 74.2 10.2%,
73.0 17.5% and 71.0 27.1%, respectively after 15 min H
(p b0.01 vs. 100% baseline). Model simulated HF gave comparable
results: 75.6%, 73.8% and 80.3% for MAP, CO and WTF, conrming
suitable HF effect on the myocyte.
Conclusion: The model shows adequate coupling between
myocyte-derived left ventricular chamber and the circulatory loop,
and would be useful to predict the contractile and hemodynamic response to changes in myocyte variables.

TH-010
The specic inhibition of the cardiac electrogenic sodium/bicarbonate cotransporter (NBCe1) leads to cardiac hypertrophy
Romina Di Mattia, Mara Carolina Ciancio, Ernesto Alejandro Aiello,
Alejandro Orlowski
Centro de Investigaciones Cardiovasculares, La Plata, Buenos Aires,
Argentina
The Na+/HCO-3 cotransporter (NBC) regulates cardiac intracelular
pH (pHi). There are two isoforms of NBC in the cardiomyocyte, the electrogenic NBCe1 (2 HCO-3: 1 Na+) and the electroneutral NBCn1 (1 HCO-3:
1 Na+). Both isoforms incorporate Na+ into the cell but the NBCe1 does
it more efciently because contributes with half of Na+ per HCO-3. The
increase of Na+ enhances intracellular Ca2+ leading to cardiac hypertrophy (CH). We have previously demonstrated in CH models that
while the activity of NBCe1 is reduced, that of the NBCn1 is increased.
Due to the absence of specic pharmacological inhibitors we were unable to demonstrate if this phenomenon was cause or consequence of
CH. We developed an interference RNA (shNBCe1) cloned in a lentiviral
vector to study the effect of the specic inhibition of NBCe1 in CH. In
western-blot assay we found a reduction of expression of NBCe1 in
cells transduced with the shNBCe1 (cont: 1005 %, n=4 vs shNBCe1:
152 %, n=4, Pb0.05). We used confocal microscopy to study the expression of NBCe1 in transduced neonatal myocytes and we found a signicantly decrease of NBCe1 expression. Furthermore, we found an
increase of cell size (cont: 14330 350 AU, n = 68 vs shNBCe1:
18570,61611 AU, n=66, Pb 0.05). In parallel experiments, the lentivirus was injected into the rat anterolateral wall of the left ventricle.
After 30 days of injection, we obtained the mass ventricle index (MVI)
by echocardiography, showing an increase of MVI in rats hearts injected

S61

with the shNBCe1 (shNBCe1:1.85 0.07 mg/g, n = 2 vs cont:

1.570.03 mg/g, n=2). In addition, NBCe1 activity was investigated
in cardiomyocytes isolated from these rats, using intracellular uorescent measurements of BCECF-AM, to monitor pHi. Membrane potential
depolarizing pulses (potassium pulse) were applied by extracellular addition of 45 mM K+, to study NBCe1 activity in isolation.
Cardiomyocytes transduced with shNBCe1 showed a decrease of
NBCe1 activity, indicating consisting reduction of NBCe1 expression
and function. Overall, these results suggest that the development of
CH involves, at least in part, the decrease of NBCe1 expression and
function.

TH-011
The Role of Prolin-1 in Hypertrophic Signalling of Adult
Cardiomyocytes
Viola Kooij, Peter O'Gara, Sian Harding
Imperial College London, London, UK
Background: Hypertrophy is characterized by altered protein
abundance and increased cell size. Pathological changes generally
associate with increased amounts of natriuretic peptide A (ANP)
and B (BNP). Previously, we found that the actin-binding protein
prolin-1 is an essential component of the hypertrophic signalling
response in neonatal rat ventricular cardiomyocytes and its effects
were mediated by pERK1/2. However, this work was limited by
the use of developing cardiomyocytes, rather than stable adult
cells, and therefore it was difcult to distinguish between the consequences of normal cell growth, and physiological and/or pathological hypertrophy. This study reports the functional and
mechanistic role of prolin-1 in the hypertrophic signalling response of stable adult rat cardiomyocytes.
Methods and results: Overexpression of prolin-1 was accomplished using adenoviral transfection. The functional effect of prolin1 on contractility was measured utilizing a video edge-detection system
and data acquisition software (Ion Optix). Morphologically, increased
levels of prolin-1 resulted in enlarged cell size. However, these changes were not accompanied by increased transcript levels of ANP and BNP.
In addition, utilizing the specic inhibitors PD98059 and rapamycin, we
found that prolin-1 induced cell enlargement was regulated by both
ERK1/2 and mTOR (p=b0.01 and p=b0.001 respectively). Functionally, increased levels of prolin-1 resulted in enhanced contractility without changing relaxation times. However, the increase in contractile
force was regulated by mTOR (p=b0.01) but not ERK1/2.
Conclusion: Our results show that prolin-1 is an essential mediator
of the hypertrophic signalling response in stable adult rat
cardiomyocytes, and inuences cell size through both ERK1/2 and
MTOR, and cell contractility through mTOR. Taken together, these data
suggest that prolin-1 is a key mediator of physiological hypertrophic
signalling.

TH-012
Moderate-intensity physical activity reduces systemic inammation
and maintains cardiorespiratory function following PM2.5 exposure
during exercise in rats
Andrew Fenning1, Alannah van Waveren1, Mitch Duncan2, Fiona
Coulson1
1

CQUniversity, Rockhampton, Qld, Australia

The University of Newcastle, Newcastle, NSW, Australia

Background: Exposure to ne particulate matter (PM) during outdoor activities in populated cities in Asia, Central and South America is

S62

Abstracts

increasing. Following excess PM exposure, the risk of cardiorespiratory

complications and events is signicantly increased.
Aims: The purpose of the current study is to 1) examine the benecial effects of moderate levels of PA on functional and biochemical
markers of the cardiorespiratory system, 2) establish the detrimental effects of a single, daily PM exposure event on cardiorespiratory function
and 3) determine if exercising during daily PM exposure increases the
deleterious effects caused by PM exposure due to increased inhalation
of particulates.
Methods: Four groups were used: control (CON), physical activity
(PA), PM2.5 exposed and PA combined with PM2.5 exposure (PA+PM)
(n=16 per group). Both PA and PM exposure were initiated when the
animals reached 8 weeks of age, for 8 weeks.
Results: PA alone did not alter body weight, markers of inammation or BP compared to control animals. However, there was a signicant decrease in epididymal fat pad mass. The PM exposed rats were
hypertensive, showed increased systemic inammation and oxidative
stress without pathological changes in the cardiac action potential or
impaired vascular function. PA was able to decrease systemic inammation in PM exposed animals, including reduction in IL-6 serum levels,
however this did not translate to an improvement in blood pressure
or vascular reactivity. Smooth muscle relaxation in the trachea from
the PA + PM tissues was not signicantly different to CON and PA
groups but was signicantly higher than the PM group.
Conclusions: The current study showed that while there is an increased CVD risk associated with PM exposure, engaging in PA during
exposure events imposes no increased risk with exercise providing a
protective mechanism against some of the biochemical signaling changes caused by inhaled PM.

TH-013
Osteopontin Regulates the Inammatory and Fibrotic Response of
Transgenic Mice Expressing Cardiac Specic Active Na +/H + Exchanger Isoform 1
Fatima Mraiche1, Nabeel Abdulrahman1, Iman Abdelaziz1, Alain
Gadeau2
1

Qatar University, Doha, Qatar

University of Bordeaux, Pessac, France

Background: Heart failure is increasing in incidence and prevalence

around the world. As a result, the need for new therapeutic advances is
urgent. We have previously shown that elevated cardiac specic NHE1
activity induced cardiac hypertrophy both in vivo and in vitro. This overexpression of active NHE1 elicited modulation of gene expression in
cardiomyocytes including an up regulation of myocardial osteopontin
(OPN) expression. To determine the role of OPN in inducing NHE1 cardiomyocyte hypertrophy, we developed an in vitro and in vivo model
expressing active NHE1 in the presence of silenced OPN.
Methods: In vitro, H9c2 cardiomyocytes were characterized for parameters of cardiomyocyte hypertrophy in the presence of active
NHE1 and OPN siRNA. In vivo, we evaluated by echocardiography, histology and qRT-PCR the cardiac phenotypes and function of the transgenic mice expressing active NHE1 or active NHE1 cross breed with
OPN knockout mice (OPN-/-).
Results: Our data showed that expression of active NHE1 resulted in
a remodeled cardiac phenotype both in vitro and in vivo. In vitro, OPN
siRNA regressed the hypertrophic effect. However, in vivo, the decrease
in FS (%) and EF (%) demonstrated in NHE1 expressing transgenic mice
was not reversed in the presence of OPN-/-. Interestingly, transgenic
mice expressing NHE1 demonstrated an upregulation of brosis and inammatory mediators (CD44 and IL-6), all of which were regressed in
the presence of OPN-/-.
Conclusions: We have developed an interesting comparative model
of active NHE1 transgenic mouse lines which express a dilated

hypertrophic phenotype expressing CD44 and brosis, an effect which

is regressed upon knocking out OPN. Despite the regression CD44 and
the brotic response in NHE1-OPN-/-, the cardiac function as assessed
by echocardiography was not reversed.

TH-014
Characterization of the role of inhibitory G protein, adenylyl cyclase
isoforms and phosphodiesterases to regulate -adrenoceptorevoked inotropic responses.
Marie Victoire Cosson1,2, Halvard Hiis1,2, Finn Olav Levy1,2, Kurt Allen
Krobert1,2
1

Department of Pharmacology, Institute of Clinical Medicine, University of

Oslo and Oslo University Hospital, oslo, Norway
2
K.G. Jebsen Cardiac Research Centre and Center for Heart Failure Research,
Faculty of Medicine, University of Oslo, oslo, Norway
Background: Our data indicate that inhibitory G protein (Gi) exerts
intrinsic receptor-independent inhibitory activity upon adenylyl cyclase
(AC). The two major subtypes of AC in the heart are AC5 and AC6. The
aims were to determine if intrinsic Gi inhibition is AC subtype selective
and whether there is a differential role of AC5 and AC6 to mediate 1adrenoceptor- (1AR) and 2AR-evoked inotropic responses. In addition, To determine if there is an interplay between Gi and phosphodiesterases 3 and 4 (PDE3,4).
Methods: 1AR- and 2AR-mediated changes in contractility were
measured ex vivo in left ventricular myocardium from wild type
(WT), AC5 or AC6 knockout (KO) mice with or without pertussis toxin
(PTX) pretreatment to inactivate Gi.
Results: Adrenaline potency (EC50) to evoke a 1AR-mediated inotropic response (IR) was increased in AC6KO versus WT and AC5KO
and also by PDE4 inhibition only in AC5KO with no change in the maximal IR. Preliminary data suggest PTX increases adrenaline potency in
WT and unveils a 2AR-IR not observed in WT even after PDE3,4 inhibition. A 2AR-IR is also observed after prior PDE4 inhibition only in PTXtreated ventricle. Unlike WT, a 2AR-IR is observed in AC5KO after only
PDE 3,4 inhibition and in AC6KO after only PDE4 inhibition.
Conclusion: These data are consistent with prior data indicating Gi
exerts a tonic inhibition upon AC since both 1AR-IR and 2AR-IR are
enhanced by PTX. Further, neither 1AR-IR nor 2AR-IR appear dependent upon either AC5 or AC6. At least 2AR-IR appears primarily regulated by PDE4. In contrast, the regulatory role of PDE4 seen upon
1AR-IR in AC5KO appears absent in AC6KO. Together, these data indicate a complex interplay amongst Gi, AC isoforms and PDEs.

TH-018
Transferring an in vitro model of pathological cardiac hypertrophy
from rat to human engineered heart tissue
Tessa Werner1,2, Marc N Hirt1,2, Kaja Breckwoldt1,2, Ingra Mannhardt1,2,
Brbel Ulmer1,2, Arne Hansen1,2, Thomas Eschenhagen1,2
1
Department of Experimental Pharmacology and Toxicology, University
Medical Center Hamburg-Eppendorf, Hamburg, Germany
2
DZHK (German Centre for Cardiovascular Research), partner site Hamburg/Kiel/Lbeck, Hamburg, Germany

Background: Previously, we established an in vitro hypertrophy

model based on rat engineered heart tissue (rEHT). In this system,
afterload enhancement (AE) was induced by mechanical reinforcement
of the silicone posts to which the EHTs were attached. We found that AE
resulted in diminished contractile function, cardiomyocyte enlargement, increased brosis, and activation of the fetal (hypertrophic)
gene program. The aim of the current study was to perform similar

Abstracts

experiments on EHTs, made from human induced pluripotent stem cell

derived cardiomyocytes (hiPSC-CMs), in order to more closely model
the situation in patients.
Methods: Three different hiPS-cell lines from healthy donors were
differentiated to cardiomyocytes using a growth factor-based two-step
protocol, which yields up to 90% -actinin-positive cells. Following
the common mesodermal induction, cardiomyocytes were generated
via inhibition of wnt-signaling and in parallel hiPSC-broblasts were
differentiated from the same cell lines using wnt-activation and FGFtreatment. The cardiomyocytes were then used to generate brinbased human EHTs (hEHTs), or were supplemented with 5% hiPSCbroblasts to get isogenic multicellular constructs. After three weeks
of cultivation, the spontaneously beating hEHTs were subjected to AE
for 7 days.
Results: Video-optical analysis of the AE-hEHTs (n =47) revealed
lower contractile forces (-24%) and longer relaxation times (+ 10%)
compared to control hEHTs (n=51), but, in contrast to rat EHTs, no cardiomyocyte hypertrophy, brosis or fetal gene program. Fibroblastcontaining multicellular hEHTs showed faster development, higher
maximal forces (+35%, n=23) and more pronounced AE-induced dysfunction (35% lower forces, n = 11). However, results were not
completely consistent across different cell lines or different hypertrophic stimuli (AE or 50 nM endothelin-1).
Conclusion: AE in pure hiPSC-CM EHTs leads to contractile dysfunction, but without consistent hypertrophy. AE-induced functional impairment was more pronounced in broblast-supplemented
multicellular EHTs. Future experiments are required to investigate
their hypertrophic and brotic response and causes of inconsistencies
between cell lines and stimuli.

TH-019
Tenascin-C promotes brosis and impairs cardiac function under
pressure overload
Max Kreibich, Elda Dzilic, David Santer, Lorenz Frster, Sandra Trojanek,
Dietmar Abraham, Martin Krssak, Attila Kiss, Karola Trescher, Bruno
Podesser
Medical University of Vienna, Vienna, Austria
Background: Extensive reorganization of the extracellular matrix
involving altered activity of matrix metalloproteinases (MMPs) is responsible for an excess of brous connective tissue and cardiac dysfunction in the failing heart. The two extracellular matrix proteins TenascinC (TN-C) and the extracellular matrix metalloproteinase inducer CD147
(EMMPRIN) have been identied as possible regulators for MMPs. However, the roles of TN-C and CD147 levels on cardiac remodelling during
left ventricular hypertrophy (LVH) have not yet been studied. Therefore,
the purpose of this study was to assess the inuence of these two proteins under pressure overload in a TN-C knockout (KO) model of transverse aortic constriction (TAC).
Methods: TAC or sham surgery was performed in TN-C-KO or wild
type (WT) animals, respectively. After four and ten weeks cardiac function was evaluated by magnetic resonance imaging (MRI; Medspec 3T
MR) before animals were sacriced and histologic and immunehistochemistry analyses were made.
Results: After 10 weeks WT-TAC animals showed signicantly more
cardiac hypertrophy: heart weight (1968 vs. 15213mg), ventricular myocytes size (550 25 vs. 300 27m2), septum thickness
(1.590.08 vs. 1.20.04) and brosis (17 3 vs. 5 2% of LV) were
signicantly higher as compared to KO-TAC hearts (all pb 0.01). Similarly MRI evaluation revealed signicantly impaired cardiac function (EF
44.5 3.1 vs. 66.9 4.3; p b 0.01) and signicantly higher expression
of MMP-9 (22.4 3.1 vs. 14.5 1.2 % of LV, p b0.05), MMP-2
(15.5 1.2 vs. 12.2 1.1 % of LV, p b 0.05) in WT-TAC. There is a

S63

tendency towards higher CD147 levels in WT-TAC mice. These ndings

correlated signicantly with the level of brosis (pb0.01).
Conclusion: TN-C is a key regulatory factor during cardiac remodelling in the pressure overloaded heart, leading to increased expression of
MMPs, which results in more ventricular hypertrophy and brosis as
well as impaired cardiac function.

TH-020
Tenascin-C in the murine geriatric heart after myocardial infarction
Felix Nagel1, David Santer1, Elda Dzilic1, Maximilian Kreibich1, Stefan
Stojkovic3, Martin Krssak2, Karola Trescher1, Bruno K Podesser1
1
Ludwig Boltzmann Cluster for Cardiovascular Research, Department for
Biomedical Research, Medical University of Vienna, Vienna, Austria
2
Centre of Excellence High Field MR, Department of Radiology, Medical University of Vienna, Vienna, Austria
3
Department of Internal Medicine II, Medical University of Vienna, Vienna,
Austria

Introduction: Aging is associated with a higher incidence, mortality,

and complication rate of myocardial infarction (MI). Tenascin-C (TNC)
is a glycoprotein produced in the infarction border zone. Previous studies discussed TNC as prognostic marker for outcome after MI.
Methods: In male geriatric (OM, age: 18 months) and young (YM,
age: 11 weeks) OF1 mice MI was induced by permanent LAD ligation.
In SHAM groups the procedure was performed without LAD occlusion.
32 days after MI, cardiac MRI was used for hemodynamic evaluation.
TNC plasma and septum tissue concentrations were assessed by ELISA
(IBL 27767).
Results: In a 2-way ANOVA MRI examination showed signicant effects of age and of MI vs. SHAM on ejection fraction, stroke volume heart
weight ratio, cardiac output heart weight ratio, end-systolic, and enddiastolic left ventricular volumes. Moreover, MI had a signicant effect
on stroke volume. No signicant effects of age and of MI vs. SHAM
were found on heart rate and cardiac output. Furthermore, no signicant interactions between the two factors were found in any parameter.
TNC plasma concentration was signicantly increased in mice with
MI at all time points, and signicantly decreased in geriatric mice 3
and 7 days after MI compared to young mice after MI (3 days: OM:
4.52 0.94g/ml, YM: 11.11 3.46g/ml; 7 days: OM: 4.22 1.92g/
ml, YM: 9.034.09g/ml). Additionally, geriatric mice after MI showed
decreased TNC septum tissue concentrations (7 days: OM:
0.1140.043ng/mg, YM: 0.2170.064ng/mg).
Conclusion: We have successfully implemented a geriatric mouse
model of MI with common signs of heart failure. Conrmed by MRI,
we found signicant hemodynamic differences between MI and SHAM
groups, and also between OM and YM. We could nd rst evidence
for age dependent differences in TNC production. These alterations
should be respected in clinical studies examining the prognostic role
of TNC in MI and heart failure.

TH-022
The Role of Calcium-Sensing Receptor in Human Peripheral T Lymphocytes on the different stages of Acute Myocardial Infarction
Yihua Sun1, Jingya Zeng1, Yong Sun2
1

Department of Clinical Laboratory,The Harbin Medical University Tumor

Hospital, Harbin, China
2
Department of Cardiology, The Afliated Second Hospital of Harbin Medical University, harbin, China
Background: Acute myocardial infarction (AMI) is an inammation
disease which seriously affects the human health. Calcium-sensing

S64

Abstracts

receptor (CaSR)in T lymphocytes is involved in inammation reaction.

But, the relation between AMI and CaSR in T lymphocytes is not very clear.
Methods: In this study, we collected human peripheral blood T lymphocytes from AMI patients in different stages of PCI (percutaneous coronary intervention) (on the onset of AMI, the rst day after PCI, the third
day after PCI, and the fth day after PCI) to identify the expressions of
CaSR and related signal transduction pathway proteins, the levels of
Th-1 type and Th-2 type cytokines in plasma, the lymphocytes apoptosis rate and number. At the same time, related laboratory indicators,
drinking or smoking history and medical history such as hyperlipidemia, hyperglycemia or hypertension were recorded.
Results: The results showed that cTnI, hs-CRP, LDL-C and FBG levels
and the incidence of hypertension, hyperlipidemia and diabetes increased signicantly in AMI group compared with the normal group.
And, the expressions of CaSR, P-ERK1/2, P-JNK (subgroup of MAPKs),
P-p65 (subunit of NF-B), Caspase-12 and the secretions of all the cytokines were increased on the onset of AMI, continued to increase greatly
on the rst day after PCI. But, from the third day after PCI, all the indicators began to decline. Meanwhile, the neutrophils to lymphocytes ratio
(NLR) increased and the apoptosis rate of all the CD3+, CD3+ CD4+ and
CD3+ CD8+ T lymphocytes increased, and the change trend was consistent with the expressions of proteins.
Conclusion: These results indicated that CaSR in the human peripheral
blood T lymphocytes were involved in the AMI onset and progression,
which probably was related with the NF-B and MAPK signaling pathways.
Key Words: Calcium-sensing receptor; Acute myocardial infarction;
Lymphocyte;Signaling pathway; Cytokine

TH-023
Effect and regulation mechanism of exogenous catestatin on blood
pressure and cardiac function in renal hypertensive rats
Xiaofang Fan, Lu Ding, Qingqing Zheng, Xuanying Chen, Xuerui Wang,
Yongsheng Gong
Institute of Hypoxia Medicine,Wenzhou Medical University, Wenzhou,
Zhejiang, China
Aim: To examine the effect and mechanism of catestatin (CST), a small
molecular active peptide, on blood pressure and cardiac function in renal
hypertension induced by the method of 2-kidney 1-clip (2K1C) in rats.
Methods: Forty male SD rats were randomly divided into two groups:
control group (n=10) and 2K1C renal hypertension group (n=30). Six
weeks after 2K1C operation, 2K1C renal hypertension group were randomly subdivided into three groups: 2K1C group, 2K1C+CST group
(80g/100g weight), 2K1C+NS group (0.9%NS). Cardiac function was
measured by left ventricular catheterization and blood pressure was measured by femoral artery catheterization. The ratio of left ventricular
weight/body weight (LVW/BW) was calculated as left ventricular mass
index. The levels of histamine (His), epinephrine (E) and CST in plasma
were measured by ELISA assay. Calcium receptor-like receptor (CRLR)
gene expression level in left ventricular tissue was tested by real-time PCR.
Results: The blood pressure of rats in 2K1C renal hypertension
group was increased gradually from the 3rd week after 2K1C operation,
and reached maximum in the 6th week. Systolic function and diastolic
function parameters in 2K1C group were higher than those in Control
group. The systolic function and diastolic function parameters in
2K1C+CST group were signicantly lower than those in 2K1C+NS
group. Compared with Control group, the pressure volume loop (PVL)
in 2K1C group was to the right and its area was increasingly shifted
under the effect of pressure load. However, the PVL in 2K1C+ CST
group was left and its area was decreasingly shifted, when compared
with those in 2K1C+NS group. The level of CST in 2K1C group was
lower than those in Control group. The content of His and E in 2K1C
group was higher than those in Control group. However, an application

of CST signicantly increased His, when compared with 2K1C+NS

group. The results from real-time PCR showed that levels of CRLR
gene expression in left ventricle in 2K1C group were lower than those
in Control group. However, an application of CST obviously decreased
CRLR gene expression, when compared with those in 2K1C+NS group.
Conclusions: Exogenous catestatin can signicantly lower the blood
pressure of renal hypertensive rats, which may be related to the decrease of left ventricular systolic function and promote the release of
histamine.

TH-024
The effect of genes involved in monogenic human cardiomyopathies
in a polygenic model of cardiac hypertrophy
Priscilla Prestes1, Francine Marques2, Claire Curl3, Paul Lewandowski4,
Lea Delbridge3, Stephen Harrap3, Fadi Charchar1
1

Federation University Australia, Ballarat, Australia

Baker IDI Heart And Diabetes Research Institute, Melbourne, Australia
3
University Of Melbourne, Melbourne, Australia, 4Deakin University, Geelong, Australia
2

Background: Cardiac hypertrophy (CH) is the main risk factor for

heart disease after age. Genetic factors are known to be involved, but
their contribution is still poorly understood. We hypothesise that
genes implicated in monogenic human forms of CH might also be involved in the more common polygenic forms of the disease.
Aims: Our aim was to use the hypertrophic heart rat (HHR), a unique
normotensive polygenic model of CH, to investigate mRNA expression
of genes previously described to be associated with monogenic forms
of dilated and hypertrophic cardiomyopathy in humans.
Methods: We measured the expression of 37 transcripts with the
TruSeq Targeted RNA expression kit using the MiSeq Desktop sequencer
(Illumina) in left ventricles of HHR and its sister control strain, the normal
heart rat (NHR), at ve ages (2 days old, 4-, 13-, 33- and 50 weeks old).
Results: We found only one gene (Ttr) differentially expressed in all
age groups (FDRb0.1; Pb 0.05). Ttr is involved in cardiac amyloidosis, inltrating cardiovascular structures, leading to hypertrophy. However, in
animals older than 13 weeks old, when CH is established in the HHR,
we found four genes upregulated (Actc1, Ankrd1, Cav3 and Fhl2). These
genes are involved in a variety of muscle development pathways, growth
and contractibility. Interestingly, Ankrd1 (fold change 1.3-2.47) has been
described to be upregulated in the failing myocardium of dogs and in
the left ventricles of patients with CH. Fhl2 is associated with cardiomyopathy in rats but seems to not be essential in cardiac development in mice.
Conclusion: Our results show that genes involved in monogenic
forms of human CH may also inuence polygenic forms of the disease
and deserve further investigation.

TH-025
Assessment of miR-669f in the development of pulmonary arterial
hypertension and right ventricular hypertrophy
Li Li2, Sudhiranjan Gupta1
1

Texas A&M University, Temple, TX, USA

Pekiing University, Beijing, China

Background: Pulmonary arterial hypertension (PAH) is a proliferative

vascular disease with a poor prognosis resulting in right ventricular hypertrophy (RVH) and RV failure. The pathology of PAH involves vascular
cell remodeling including pulmonary arterial endothelial cell (PAEC) dysfunction and pulmonary arterial smooth muscle cell (PASMC) proliferation. Recently, (miRNAs have emerged as a new class of posttranscriptional regulators of genes having a key role in vascular

Abstracts

remodeling. However, the function of miRNAs in the development of PAH

and RVH remain elusive. Here, we investigate that miR-466a/-669f-cluster is a pathogenic niche regulating the pulmonary vascular remodeling
and inhibition of miR-669f prevents RVH by restoring BMPRII and
PPAR level.
Method/Result: We identied a panel of novel dysregulated miRNAs
and miRNA clusters in the RV and lungs of MCT treated WT mice. Among
them; we discovered miR-466a/-669f cluster is critical for the development of PAH. We conrmed our nding using MCT and hypoxiainduced mouse models and observed signicant upregulation of miR466a/-669f cluster in the RV and lungs. To screen the potential target
genes for miR-669f in an unbiased fashion, we transfected mouse PAEC
with miR-669f mimic and inhibitor and followed by the stimulation
with TGF 1, separately and conrmed BMPRII and PPAR are the bonade target for miR-669f. The in vitro studies showed that TGF 1 stimulation signicantly enhanced the expression of mature miR-669f and reduced the level of BMPRII and PPAR in rodent PAEC. The in vivo
inhibition of miR-669f showed a promise in attenuating PAH.
Conclusion: Our ndings provide evidence that miR-669f displays a
critical role in the pathogenesis of vascular remodeling leading to the
development of PAH and RVH by directly targeting BMPRII and PPAR ,
and that inhibition of miR-669f reversed the remodeling process. We
conclude that miR-669f could be a triggering factor in PAH and may providing new mechanistic information for therapeutic benet.

TH-026
Cardiac Apoptosis In The Prediabetic Heart: CaMKII, Ca Misshandling
And Mitochondria Dysfunction
Mariln Federico1, Sommese Leandro1, Zanuzzi Carolina2, Portiansky
Enrique2, Dedman John3, Kaetzel Marcia3, Wherens Xander4, Mattiazzi
Alicia1, Palomeque Julieta1
1

Centro de Investigaciones Cardiovasculares, UNLP, CONICET-CCT La Plata,

La Plata, Buenos Aires, Argentina
2
Fac. de Cs. Veterinarias; UNLP, CONICET-CCT La Plata, La Plata, Buenos
Aires, Argentina
3
Department of Genome Science, University of Cincinnati College of Medicine, Cincinnati, OH, USA
4
Cardiovascular Research Institute, Baylor College of Medicine, Houston, TX,
USA
The mitochondria are a well-known intermediate of apoptosis, which
is one of the more important steps leading to heart failure (HF). This disease occurs more frequently in people with type 2 diabetes than in the
general population. However, cardiac apoptosis has not been previously
evaluated at the prediabetic state. Since CaMKII is involved in cardiac apoptosis and Ca2+ mishandling, the aim of the present study was to evaluate the presence of cardiac apoptosis in a prediabetic model (PM)
induced by a fructose-rich diet (FRD) in rats or mice and the putative
link with CaMKII activity and mitochondria dysfunction. FRD rats showed
decreased contractility (echocardiography) and increased CaMKII (PCaMKII 191.618.3%), and ROS (185.428.6%) with respect to control
diet (CD) rats (100%). Moreover, the apoptotic ratio Bax/Bcl2 increased
in FRD vs CD rats (273.639.7%) as well as TUNEL positive nuclei. Mitochondria from FRD rats showed signicant more swelling (DO 0.340.05
CD vs 0.530.03 FRD), enhanced mitochondria membrane depolarization and mitochondria Ca2+ content than CD rats. Moreover, myocytes
from FRD rats signicantly increased sarcoplasmic reticulum (SR) Ca2+
leak vs CD myocytes. In Wild Type (WT) mice, collagen type III increased
in FRD (27.065.24%) with respect to CD (13.331.23%) hearts. FRD SRAIP mice (which express the CaMKII autocamtide inhibitory peptide [AIP]
at the SR membranes) showed less TUNEL positive nuclei and no change
in collagen type III than FRD WT mice. Co-treatment with tempol, a membrane permeable ROS scavenger, decreased apoptosis, collagen type III as

S65

well as SR Ca2+ leak in FRD WT mice. Moreover, mitochondria swelling

could be also prevented in S2814A mice, which ryanodine receptor
(RyR2) cannot be phosphorylated by CaMKII. The results would indicate
a causal link between CaMKII activation by increased ROS, SR Ca2 +
leak produced by CaMKII-dependent phosphorylation of RyR2 and mitochondria damage induced by Ca2+ overload.

TH-027
Glycoprotemics reveals decorin fragments with anti-myostatin
activity in human atrial brillation
Javier Barallobre-Barreiro1, Shashi K Gupta2, Anna Zoccaratto1, Rika
Kitazume-Taneike1, Mei Chong1, Jens W Fischer3, Thomas Thum2,
Joerg Heineke4, Antoine Kichler5, Kinya Otsu1, Manuel Mayr1
1

Kings British Heart Foundation Centre, Kings College London, London, UK

Institute for Molecular and Translational Therapeutic Strategies, MHHannover, Hannover, Germany
3
Institute for Pharmacology and Clinical Pharmacology, Heinrich-HeineUniversity, Dsseldorf, Germany
4
Experimental Cardiology, Department of Cardiology and Angiology, MHHannover, Hannover, Germany
5
Laboratoire Vecteurs: Synthse et Applications Thrapeutiques, UMR 7199
CNRS Universit de Strasbourg, Illkirch, France
2

Background: Myocardial brosis is a feature of many cardiac diseases. We used proteomics to prole glycoproteins in the human cardiac extracellular matrix (ECM).
Methods and Results: Left atrial specimens from patients who developed postoperative atrial brillation (AF) were compared to patients who maintained sinus rhythm (SR). Out of more than 100
ECM proteins identied, the levels of the small leucine-rich proteoglycan (SLRP) decorin were reduced in patients with postoperative AF.
Within its protein core, eighteen different fragmentation sites were
identied using mass specrometry. In contrast, no fragmentation
was observed for biglycan, the most closely related SLRP. Decorin processing differed between human ventricles and atria and was altered
in disease. Atrial appendages from patients in persistent AF had
higher levels of decorin harboring a unique cleavage site not found
in atrial appendages from patients in SR. This cleavage site preceded
the N-terminal domain of decorin that controls muscle growth via altering the binding capacity for myostatin. A synthetic peptide corresponding to this region dose-dependently inhibited the response to
myostatin in cardiac myocytes, where phosphorylation of AMPK and
SMAD2 (i.e. downstream targets of myostatin) resulted affected. The
same effect was observed in and in perfused mouse hearts. Notabily,
myostatin expression was decreased in hearts of decorin null mice.
In contrast, C-terminal fragmentation of decorin, important for the interaction with connective tissue growth factor (CTGF), was reduced in
patients with persistent AF.
Conclusion: This proteomics study is the rst to analyze the human
cardiac ECM. Novel processed forms of decorin core protein uncovered
in human atrial appendages can regulate the local bioavailability of
anti-hypertrophic and pro-brotic growth factors and may impact on
the manifestation or perpetuation of cardiac arrhythmias.

TH-028
Cardioprotective Effect of IGF-1 Upon The Hypertrophied Myocardium Of The Spontaneously Hypertensive Rats (SHR): A Key Role On
Cardiac Na+/H+ Exchanger (NHE-1) Activity And Oxidative Stress
Alejandra Yeves, Juan Burgos, Andrs Medina, Irene Ennis
Centro de Investigaciones Cardiovasculares, La Plata, Buenos Aires,
Argentina

S66

Abstracts

Oxidative stress and NHE-1 hyperactivity are interrelated

phenomena that play a key role in pathological but not in
exercise-induced cardiac hypertrophy (CH). We have demonstrated that IGF-1, released during exercise training, through
AKT inhibits NHE-1 and that a swimming routine transformed
pathological into physiological CH in the SHR. Therefore, we hypothesize that IGF-1 by preventing NHE-1 hyperactivity and oxidative stress could be responsible for the cardioprotective effect
of training in SHR. NHE-1 activity in cardiomyocytes (proton efux mmol/L/min) monitored by BCECF-AM epiuorescence was
signicantly reduced by IGF-1 (2.03 0.47, n = 7), effect
prevented by AG1024, an antagonist of IGF-1 receptor
(3.71 0.9, n = 7) and by the AKT inhibitor MK2206
(4.01 0.65, n = 12). Similarly, IGF-1 signicantly reduced H2O2
production in cardiomyocytes loaded with DCF-DA (IGF-1:
-3.63 1.1; n = 7, IGF-1 + AG1024: 6.06 3.4, n = 7; control:
5.12 2.5, n = 12, AU after 10 min incubation). The antioxidant
action of IGF-1 was accompanied by a signicant increase in the
activity of superoxide dismutase (SOD) catalase (IGF-1: 20 1.5,
n = 7 and 44.9 3.6 N = 5 vs. control: 14.5 1.6, n = 5 and
34 2.3, n = 7, U/mg, respectively). Interestingly the benecial
effects of IGF-1 correlated with higher cardiac contractility revealed by an increase in cardiomyocyte shortening (IGF-1:
145.8 14, n = 5 vs. control: 96.8 5, n = 3, % at 10 min respect
to time 0, p b0.05). Since the bioactive peptide apelin, upregulated by training, may increase cardiac contractility and
was proposed to exert antioxidant effects, we quantied its
mRNA abundance and that of its receptor APJ in our experimental
conditions nding that IGF-1 signicantly increased both (IGF-1:
251 48 and 184 29 vs. control: 100 6.2 and 100 15.9,
apelin and APJ respectively).
In summary, our results suggest that the inhibition of NHE-1 hyperactivity as well as the antioxidant effect of IGF-1, probably by apelinmediated increase in SOD and catalase activity, represent benecial cardiac adaptations leading to the physiological phenotype in the SHR subjected to exercise training.

TH-029
Polycystin-1 regulates L-type calcium channel stabilization during
mechanical stretch in cardiomyocytes
Ivonne Olmedo2, Jaime Riquelme1,3, Diego Varela2, Gina Snchez2,
Paulina Donoso2, Zully Pedrozo2,3
1

Facultad de Ciencias Qumicas y Farmacuticas, Universidad de Chile, Santiago, Chile

2
Facultad de Medicina, Universidad de Chile, Santiago, Chile
3
Advanced Center for Chronic Diseases, Facultad de Medicina and Facultad
de Qumica y Farmacia, Universidad de Chile, Santiago, Chile

Deregulation of LTCC protein levels has been reported in cardiac hypertrophy and ischemic heart disease; however, the underlying mechanisms are unknown. Mechanical stretch is a common factor in both
pathologies. Polycystin-1 (PC1) is a mechanosensor and a G-protein
coupled receptor, GPCR (Gi/o) expressed in cardiomyocytes. We hypothesized that, in cardiomyocytes, PC1 regulates LTCC protein levels
in response to mechanical stretch.
Methods: Mechanical stretch was induced in vitro using cyclic mechanical stretch (MS) or hypo-osmotic solution (HS) in neonatal rat
cardiomyocytes control or with siRNA against PC1 (siPC1). We measured the protein levels of CaV1C LTCC subunit and p-AKT in the presence of AKT inhibitor, pertussis toxin, ARk. Also, we overexpressed a
mutated c-terminal of PC1 (mct-PC1) in order to avoid the interaction
between the Gi protein and the ct-PC1.

Results: CaV1C protein levels increased after MS or HS and

these increments were blunted in polycystin-1 knockdown
(siPC1) cardiomyocytes. Changes in CaV1C mRNA were not detected, suggesting that PC1 stabilizes LTCC during mechanical stretch.
AKT, necessary to CaV1C and CaV2 binding, was activated after
HS but blunted in siPC1. CaV1C protein increment also was
prevented by AKT inhibitor (10 M). AKT activation and CaV1C increment induced by HS were blunted in presence of pertussis toxin
(Gi/o inhibitor) or G subunit inhibitor (ARk). Finally, overexpression of mct-PC1 inhibited the increased of Ca V1C protein
levels and AKT activation by HS.
Conclusion: PC1 is involved in LTCC stabilization during mechanical
stretch in cardiomyocytes possibly through its GPCR (Gi) activity.
Grant support: Fondecyt 1150887 and FONDAP 15130011 to ZP,
Postdoctoral Fondecyt 3140449 and 3160298 to IO and JR respectively,
Fondecyt 1120240 to DV and Fondecyt 1130407 to GS.

TH-030
A mechanism of calmodulation of the human cardiac sodium
channel
Christopher Johnson, Matthew Thompson, Markus Voehler, Walter
Chazin
Vanderbilt School of Medicine, Nashville, TN, USA
The human cardiac sodium channel (NaV1.5) is responsible for the
initial upstroke of the action potential and essential to heart function.
Genetic mutations causing channel dysfunction are associated with
the life threatening cardiac conditions Brugada and Long QT syndromes.
Despite much investigation, successful treatment options for patients
suffering from NaV1.5 dysfunction are lacking. In-depth understanding
of the molecular mechanisms of channel function and regulation provides a powerful means to identify and develop novel therapeutic targets and improvements to existing treatments. To this end, we have
undertaken studies of the binding of the Ca2+ sensing regulatory protein calmodulin (CaM) to the NaV1.5 channel inactivation gate. We discovered a previously unrecognized high afnity interaction and
generated a high-resolution structural model using a combination of
X-ray crystallography, NMR spectroscopy and small angle X-ray scattering. Ca2+-activated CaM is found to bind to two independent sites on
the channel inactivation gate in an unanticipated domain conguration.
The structure enabled predictions of the mechanism of mal-function for
cetain disease associated mutations contained within the NaV1.5 inactivation gate. Our predictions were tested using NMR analyses, which
conrmed perturbations of the interaction with CaM. Our results combined with data from previous studies provides a rationale and molecular mechanism for Ca2 + CaM modulation (Calmodulation) of NaV1.5,
and sets the stage for evaluating the therapeutic potential of targeting
this key regulatory interaction.

TH-031
Tenascin-C deciency attenuates abdominal aortic aneurysm
progression
Felix Nagel, Anne K Schaefer, Philipp Kaiser, David Santer, Attila Kiss,
Karola Trescher, Bruno K Podesser
Ludwig Boltzmann Cluster for Cardiovascular Research, Department for
Biomedical Research, Medical University of Vienna, Vienna, Austria
Purpose: Tenascin-C (TNC) is a matricellular protein produced by
vascular smooth muscle cells and broblasts in various remodeling processes. In numerous cardiovascular pathologies high TNC levels are associated with unfavorable outcomes. TNC production has also been

Abstracts

found in abdominal aortic aneurysms (AAA). The aim of the study is to

evaluate whether TNC deciency could attenuate AAA formation.
Methods: We compared male AJ TNC -/- and AJ wildtype (WT) mice.
After laparotomy and preparation of the infrarenal aorta, AAA were induced by periaortal CaCl2 0,5M application for 15 minutes. In the
sham-operated groups the same procedure was performed, however
aortas were incubated with saline solution. The aortic diameter was
measured before AAA induction and before organ harvesting after 3
and 10 weeks. The main parameter was the ratio of the diameters.
Results: TNC knockout (KO) mice with AAA showed signicantly lower
diameter ratios than the wildtype group 3 weeks (TNC KO: 1.390.25, WT:
1.670.22 pb0.05) and 10 weeks (TNC KO: 1.510.47, WT: 1.980.55
pb 0.05) after AAA induction. No signicant changes in diameter ratios
were found in sham groups (3 weeks: TNC KO: 0.920.08, WT:
0.960.22, n.s., 10 weeks: TNC KO: 1.050.16, WT: 0.940.10, n.s.). Additionally, WT mice with AAA showed a more disrupted Elastin structure
than TNC KO mice 10 weeks after AAA induction.
Conclusions: In our study we found rst evidence that TNC deciency is associated with reduced AAA formation. To identify possible causal
pathways immunohistological and molecular biological assessments
will be conducted.

TH-032
Critical transcriptional regulation of stress-response kinase JNK2 in
CaMKII gene expression in the aging atrium
Xianlong gao, Xiaomin wu, Weiwei Zhao, Xun Ai
Loyola University Chicago, Maywood, IL, USA
Introduction: Stress-response c-Jun N-terminal kinase (JNK) is implicated in a wide range of physiological and pathological cellular processes. We recently revealed that JNK isoform 2 directly activates
CaMKII, a pro-arrhythmic molecule, which enhances atrial
arrhythmogenicity in the aged heart. Cardiac CaMKII delta isoform
(CaMKII) is known to regulate Ca handling proteins and promotes
pathogenesis of cardiac arrhythmias. Here, we assess the role of JNK2
in CaMKII gene expression in the aged atrium.
Methods and results: We found that CaMKII protein expression
(immuoblotting) markedly increased in human atria with increasing age
as well as in HL-1 atrial myocytes treated with JNK activator anisomycin.
However, either a JNK2 specic inhibitor JNK2I-IX or overexpression of
inactivated dominant-negative JNK2 (Adeno-JNK2dn) completely attenuated this anisomycin-induced CaMKII up-regulation (compared to
Adeno-LacZ-infected controls), whereas overexpression of AdenoJNK1dn did not. JNK2-induced up-regulation of CaMKII was further conrmed in HL-1 atrial myocytes co-infected with Adeno-MKK7D-JNK2,
but not in the cells co-infected with Adeno-MKK7D-JNK1. Moreover, dramatically up-regulated CaMKII mRNA (quantitative qPCR) was exhibited
in human atria with increasing age and in HL-1 atrial myocytes treated
with anisomycin. It is known that JNK regulates target gene expression
via its downstream transcriptional factors including c-Jun and ATF2. We
found that activated JNK was associated with a substantially increased
phosphorylation of c-Jun but unchanged ATF2 in both aged atrium and
anisomycin-treated HL-1 atrial myocytes. Cross-linked chromatinimmunoprecipitation (XChIP) assay showed signicantly increased binding of c-Jun to CaMKII promoter in the presence of anisomycin. Moreover,
transcriptional activity of CaMKII promoter in CaMKII promoter vector
transfected HEK293 cells was signicantly elevated in response to
anisomycin challenge assessed by luciferase reporter assay.
Conclusion: We discovered a critical role of JNK2 in up-regulating
CaMKII expression. This JNK2 isoform-specic regulation occurs
through the activation of CaMKII promoter, which is modulated by
JNK downstream transcriptional factor c-jun in atrial myocytes.

S67

TH-033
Up-regulation of 5-Hydroxytryptamine receptor signaling in coronary arteries after organ culture
Chun Yu Deng, Hui Yang, Su Juan Kuang
Department of Medical Research, Guangdong Cardiovascular Institute,
Guangdong General Hospital, Guangdong Academy of Medical Sciences,
Guangzhou, Guangdong, China
Background: 5-Hydroxytryptamine (5-HT) is a powerful constrictor
of coronary arteries and is considered to be involved in the pathophysiological mechanisms of coronary artery spasm. But mechanism of enhancement of coronary artery contraction to 5-HT is unclear during the
development of coronary artery disease. Organ culture of intact blood vessel segments has been suggested as a model for the phenotypic changes of
the smooth muscle cells in cardiovascular disease in recent studies.
Methods: The objectives of the present study were to characterise
the 5-HT receptor-induced vasoconstriction and quantify the 5-HT receptor signaling expression levels in cultured rat coronary arteries.
Results: The results demonstrated that the cumulative application of
5-HT produced a concentration-dependent vasoconstriction in fresh
and 24 h-cultured rat coronary arteries without endothelium. 5-HT induced markedly higher contractions in cultured coronary arteries than
in fresh coronary arteries. U46619- and CaCl2-induced contractions
were comparable in two groups. 5-HT stimulates 5-HT2A receptor and
PLC cascade to induce coronary vasoconstriction. Calcium inux
through L-type calcium channels (LCC) and non L-type calcium channels contributed the coronary artery constrictions induced by 5-HT.
Vasoconstraction induced by thapsigargin was augmented in cultured
coronary arteries compared with fresh coronary arteries. The decrease
in Orai1 expression signicantly inhibited 5-HT-evoked coronary arterial cell Ca2+ entry. 5-HT2A receptor, Orai-1 and Stim1 expression levels
were augmented in cultured coronary arteries compared with fresh coronary arteries.
Conclusions: Upregulation of 5-HT2A receptor signaling pathway
elicits the enhancement of vasoconstriction induced by 5-HT in cultured
coronary arteries.

TH-034
Target identication of curcumin on ischemic blood ow and anticancer activities by network analysis and biological approaches
Xuejun LI
School of Basic Medical Sciences, Peking University, Beijing, China
We investigated the angiogenic effects of curcumin on an ischemia
and lung cancer model. Unilateral femoral arteries of C57BL/6 mice
were disconnected on one side of the mouse and LLC cells were
xenografted on the opposite side. Angiogenic effects and underlying
mechanisms associated with curcumin were investigated. Molecular
targets, signaling cascades and binding afnities were detected by
Western blot, 2-DE, computer simulations and SPR techniques.
Curcumin promoted post-ischemic blood recirculation and suppressed
lung cancer progression in inbred C57BL/6 mice via regulation of the
HIF1alpha/mTOR/VEGF/VEGFR cascade oppositely. Inammatory stimulation induced by neutrophil elastase (NE) promoted angiogenesis in
lung cancer tissues, but these changes were reversed by curcumin
through directly reducing NE secretion and stimulating alpha1-AT and
IRS-1 production. Curcumin had opposite effects on blood vessel regeneration under physiological and pathological angiogenesis, which was
effected through negative or positive regulation of the HIF1alpha/
mTOR/VEGF/VEGFR cascade. Curcumin had the promise as a new treatment modality for both ischemic conditions and lung cancer simultaneously in the clinic.

S68

Abstracts

TH-035
The role of mast cell tryptase in the progress of atherosclerosis
Xiuling Zhi1, Xiaobo Li2, Pohsheng Yeong2, Hao Zhang2, Hongxia Shao1,
Luanfeng Pan1, Lianhua Yin1,2
1

Training Center of Medical Experiments, School of Basic Medical Sciences,

Fudan University, Shanghai, China
2
Department of Physiology & Pathophysiology, School of Basic Medical Sciences, Fudan University, Shanghai, China
Atherosclerosis is by far the most frequent underlying cause of coronary artery disease and is associated with high morbidity and mortality.
Accumulated mast cells in atherosclerotic plaques secrete a high level of
tryptase that may participate in the pathogenesis of atherosclerotic disease by diverse pathways. In our study, we found that tryptase might
promote foam cell formation by suppressing LXRalpha activation via
PAR-2/LXRalpha/LXRalpha target genes signaling pathway. The addition of tryptase into THP-1-derived macrophages increased both intracellular lipid accumulation and total cholesterol level. These effects
were resisted by APC366, a selective inhibitor of mast cell tryptase.
Tryptase dramatically resisted 22RHC induced activation of LXRalpha
protein expression, which can be reversed by SAM-11 (a PAR-2specic neutralizing antibody) and reduced LXRalpha, ABCG1, ABCA1
and SREBP-1c mRNA levels and ABCG1 protein level, which were all
blocked by APC366. PAR-2 agonist also redeemed 22RHC stimulation
to activate LXRalpha, ABCG1 protein expression, and mRNA levels of
LXRalpha and its target genes in THP-1-derived macrophages. In addition, tryptase promotes plaque haemorrhage distinctively because 50%
of the ApoE-/- mice in the tryptase overexpression group had plaque
haemorrhage, while only 10% in the siRNA group did. Hematoxylin
and eosin(HE) staining showed that the mouse cervical artery plaque
area was much larger in the tryptase overexpression group and there
was greater artery stenosis. The immunohistochemistry of the cervical
artery plaque showed that plasminogen activator inhibitor-1 (PAI-1)
expression was the lowest while tissue plasminogen activator (tPA),
CD31, CD34 and VEGF was the highest in the tryptase overexpression
groups. This observation was completely contrary to what was observed
in the siRNA group. Thus, regulating tryptase expression in MCs may
provide a potential target for atherosclerosis treatment.

TH-036
Intermedin 1-53 attenuates vascular calcication in rats with chronic kidney disease by upregulation of alpha-Klotho.
JinRui Chang1, Jun Guo1, Yue Wang1, YueLong Hou1, WeiWei Lu1,
JinSheng Zhang1, Yanrong Yu1, XiuYing Liu1,2, XiuJie Wang1,2, YouFei
Guan1, Yi Zhu1, Jie Du1,2, ChaoShu Tang1, YongFen Qi1
1

Peking University Health Science Center, Beijing, China

The Key Laboratory of Remodeling-related Cardiovascular Diseases, Capital Medical University, Ministry of Education, Beijing, China

Deciency in -Klotho is involved in the pathogenesis of vascular

calcication. Since intermedin1-53 (a calcitonin/calcitonin gene related
peptide) protects against vascular calcication, we studied whether
intermedin1-53 inhibits vascular calcication by upregulatingKlotho. A rat model of chronic kidney disease (CKD) with vascular
calcicationinduced by the 5/6 nephrectomy plus vitamin D3 was
used for study. The aortas of rats with CKD showed reducedintermedin
content but an increase of its receptor, calcitonin receptor-likereceptor,
and its receptor modier, receptor activity-modifying protein 3.
Intermedin1-53 treatment reducedvascular calcication. The expression of -Klotho was greatly decreased in the aortas of rats with CKD
but increased in the aortas of intermedin1-53-treated rats with CKD.
In vitro, intermedin1-53increased -Klotho protein level in calcied

vascular smooth muscle cells. -Klothoknockdown blocked the inhibitory effect of intermedin1-53 on vascular smooth musclecell calcication and their transformation intoosteoblast-like cells. The effect of
intermedin1-53 to upregulate -Klotho andinhibit vascular smooth
muscle cell calcication was abolished by knockdown of its receptor
or its modier protein, or treatmentwith the protein kinase Ainhibitor
H89. Thus, intermedin1-53 may attenuate vascularcalcication by upregulating -Klotho via the calcitonin receptor/modifying protein complex and protein kinase Asignaling.

TH-037
Impact of High Salt Independent of Blood Pressure on PRMT/ADMA/
DDAH Pathway in the Aorta of Dahl Salt-Sensitive Rats
Jianjun Mu, Yu Chao, Chao Chu, Tongshua Guo, Zuyi Yuan
Department of Cardiovascular Medicine, First Afliated Hospital of Xian
Jiaotong University, Xian, China
Objectives: The objectives of this study were to investigate the impact of a high salt diet on the PRMT/ADMA/DDAH (protein arginine
methyltransferases; dimethylarginine dimethylaminohydrolase) pathway in Dahl salt-sensitive (DS) rats and SS-13BN consomic (DR) rats,
and to explore the mechanisms that regulate ADMA metabolism independent of blood pressure reduction.
Methods: 8-weeks-old male Dahl salt-sensitive (SS) rats and SS13BN (13BN) rats were randomly divided into ve groups: SS normal
diet group (NaCl 0.3%, SN group), SS high-salt diet group (NaCl 8%, SH
group), high salt diet (8% NaCl) and hydralazine (10 mg/kg/d)
intragastric administration (SH + HYD group), 13BN normal diet
group (containing NaCl 0.3%, BN group), 13BN high-salt diet group
(containing NaCl 8%, BH group). The plasma concentration of ADMA
and NOx were determined, mRNA and protein expression of PRMT-1,
mRNA expression and activity of DDAH, mRNA and protein expression
of eNOS in aortic tissue were detected with RT-qPCR and Western blot.
Results: Plasma levels of nitric oxide (NO) in DS rats given a high salt
diet and subjected to intragastric administration of hydralazine (SH +
HYD group) were lower than those given a normal salt diet (SN
group). There were signicant decreases in expression and activity of
dimethylarginine dimethylaminohydrolase (DDAH) and endothelial
NO synthase (eNOS) in DS rats given a high diet (SH group) in comparison to the SN group. The activity of DDAH and expression of eNOS in the
SH + HYD group decreased more signicantly than SN group. The
mRNA expression of DDAH-1 and DDAH-2 were lowest in the SH
group. The results suggest that salt, independent of blood pressure,
can affect the PRMT-1/ADMA/DDAH system to a certain degree and
lead to endothelial dysfunction in Dahl salt-sensitive rats.
Keywords: endothelial dysfunction; asymmetric dimethylarginine;
dimethylarginine; dimethylaminohydrolase; endothelial nitrite oxide
synthase; oxidative stress

TH-038
Ji-Cheng Chen, Hao-Yu Cai, Yan Wang, Jian Lu
Department of Pathophysiology, the Second Military Medical University,
shanghai, China
Stomatin is an important lipid raft-associated protein which interacts with membrane proteins and plays a role in the membrane organization. In order to know the effect of glucocorticoid (GC) on the
expression of stomatin in vivo and in vitro, and the mechanism and signicance of regulation of stomatin by GC, in this study, we at rst examined the mRNA levels of stomatin in heart, lung and cerebral cortex of
rat underwent sham surgery or adrenalectomy(ADX) with or without

Abstracts

supplementation of Dex, a synthetic GC. We found that adrenalectomy

resulted in signicant decrease of stomatin mRNA in all above tissues,
and treatment of ADX rats with Dex signicantly increased the levels
of stomatin mRNA of heart and lung, but did not in cerebral cortex.
These results indicate that GCs up-regulate the expression of stomatin
in vivo in a tissue-specic manner. Dex also up-regulated expression
of stomatin in A549 cells, which was mediated by its receptor(GR).
The reporter gene activity determined by luciferase assay showed that
up-regulation of stomatin expression by Dex occured at transcriptional
level . furter deletion and mutational studies demonstrated that a GC response element (GRE) within the promoter region mainly contributed
to the induction of stomatin by Dex. Moreover, we found that Inhibiting
stomatin expression by stomatin siRNA signicantly decreased dense of
peripheral actin ring in dex treated A549 cells. Taken all together, these
data indicated that GC signicantly up-regulated the expression of
stomatin in vivo and in vitro, which could stabilize membraneassociated actin in A549 cells.

TH-040
LncRNA Hand2-AS1, Hand2, and MiR-138-5p Crosstalk to Participate
in VSMC Phenotypic Switch
Shaoguang Sun, Mei Han

S69

30 min global ischemia and 2 h reperfusion. Cardiac tissue, H9C2 myoblasts and isolated cardiomyocytes were used to optimise conditions
and validate changes in PKA & Epac expression and activity. The effect
of cell-permeable cAMP analogue, an activator of both PKA and Epac
(8-Br-cAMP-AM; 8-Br), on haemodynamic function was investigated
in the presence or absence of an inhibitor of PKA (H-89) or Epac (ESI09). In cardioprotection studies, 8-Br was introduced to the heart prior
to ischaemia and compared to the effect of activation of either PKA (6Bnz-cAMP-AM; 6-Bnz) or Epac (CPT-2-O-Me-cAMP-AM; CPT). Functional recovery, lactate dehydrogenase (LDH) release and infarct size
were used to assess I/R injury.
Results: Simultaneous inhibition of PKA and Epac by 8-Br increased
baseline haemodynamic function and induced a marked
cardioprotective effect (complete recovery of haemodynamic function,
3.5-fold reduction of infarct size and 3-fold reduction of LDH release
vs. control). These effects were abolished by selectively inhibiting PKA
and Epac using H-89 and ESI-09. Both PKA activation alone (6-Bnz) or
Epac activation alone (CPT) increased baseline haemodynamic function
but could not confer signicant protection. However, the
cardioprotective effect of 8-Br could be mimicked by using a mixture
of PKA and Epac activators.
Conclusion: Cell permeable cAMP analogues that simultaneously
activate both PKA and Epac confer marked protection against I/R injury.
Activation of either PKA or Epac alone has little cardioprotective effect.

Hebei Medical University, shijiazhuang, China

Vascular smooth muscle cell (VSMC) phenotypic switch is a common pathological feature of vascular remodeling diseases. Long noncoding RNAs (lncRNAs) have many important regulatory functions,
but the functions in VSMC phenotypic switch are largely unkown.
Here, we identied that Hand2 (heart and neural crest derivatives
expressed 2) gene and lncRNA Hand2 antisense RNA 1 (Hand2-AS1)
are co-expressed, and their expression levels are signicant decreased
in dedifferentiated VSMC by RNA-seq and qRT-PCR analysis. By using
both gain-of-function and loss-of-function approaches, we found
Hand2 promote VSMC phenotypic switch by regulating SM22, a differentiated VSMC maker gene. Furthermore, we demonstrated that
lncRNA Hand2-AS1 binds to the Hand2 gene promoter, and increases
Hand2 expression at transcriptional level. MiR-138-5p inhibits Hand2
expression by targeting its 3-untranslated region. LncRNA Hand2-AS1
is a competitive endogenous RNA, blocks miR-138-5p to targeting
Hand2, and increases Hand2 expression at post-transcriptional level.
In summary, our ndings provide a novel mechanism that one lncRNA
can regulate one target gene from both transcriptional and posttranscriptional level, our results indicate lncRNA Hand2-AS1, Hand2,
and miR-138-5p can form a regulation loop to participate in VSMC phenotypic switch.

TH-041
Epac is an essential component of the cAMP-mediated
cardioprotection and acts synergically with PKA
Igor Khaliulin, Mark Bond, Zara Dyar, Raheleh Amini, Jason Johnson, MSaadeh Suleiman
University of Bristol, Bristol, UK
Background: Acute -adrenergic stimulation and subsequent elevation of cAMP level are implicated in cardioprotection against ischaemia/
reperfusion (I/R) induced by heart conditioning. However, cAMP signalling involves activation of both protein kinase A (PKA) and guanine nucleotide exchange protein (Epac). In this study, we aimed at identifying
the involvement of PKA and Epac in cardioprotection.
Methods: Langendorff perfused adult rat hearts were used either for
protein determination, isolation of cardiomyocytes or subjected to

TH-042
Temporal
Phosphoproteomics
to
Investigate
the
Mechanotransduction of Vascular Smooth Muscle Cells in Response
to Cyclic Stretch
Ying-Xin Qi, Yu-Chen Yang, Xiao-Dong Wang
Institute of Mechanobiology & Medical Engineering, Shanghai Jiao Tong
University, Shanghai, China
Background: Vascular smooth muscle cells (VSMCs) are exposed to
mechanical cyclic stretch in vivo, which play important roles in maintenance of vascular homeostasis and regulation of pathological vascular
remodeling. Reversible protein phosphorylation is crucial for intracellular signaling transduction. However, the dynamic phosphorylated prole induced by cyclic stretch in VSMCs is still unclear.
Methods and results: Using the stable isotope labelling by amino
acid in cell culture, VSMCs were labeled and exposed to 10% physiological cyclic stretch in vitro at 1.25 Hz for 0 min, 15 min, 30 min, 1 hr and 6
hr, respectively. Using TiO2 beads and liquid chromatography tandem
mass spectrometry, the temporal phosphoproteomic proles in response to cyclic stretch were then detected. Bioinformatics analysis including fuzzy c-means clustering, functional classications, and
Ingenuity Pathway Analysis were applied to further reveal the potential
mechanotranduction networks. The results indicated that protein kinase C (PKCs) family, Rho-associated coiled-coil containing protein kinase 1 (ROCK1) and Akt may participate in cyclic-stretch induced
VSMC functions. Cyclic stretch repressed the expression of ROCK1,
while it had no signicant effect on the phosphorylation of PKC/II,
PKC/ and PKC/. PKC was activated rst at short time-phase
(15 min and 30 min), and again at long time-phase (6 hr, 12 hr and
24 hr). The activation of p-PKC was immediate and short-term, similar
to p-Akt.
Concultion: Our present in vitro work hence revealed that cyclic
stretch activates complex mechanotransduction networks, suggesting
that novel mechanoresponsive molecules, i.e., PKC, PKC, and ROCK1,
may participate in the mechanotransduction and modulation VSMC
functions.
(This research was supported by grants from the National Natural
Science Foundation of China, No. 11222223).

S70

Abstracts

TH-043
Involvement of BK Channel in Differentiation of Vascular Smooth
Muscle Cells Induced by Mechanical Stretch
Xue-Jiao Wang1, Hu-Cheng Zhao2, Bo Huo3, Ying-Xin Qi1, Zong-Lai
Jiang1
1
Institute of Mechanobiology & Medical Engineering, Shanghai Jiao Tong
University, Shanghai, China
2
Lab of Biomechanics, Department of Engineering Mechanics, Tsinghua
University, Beijing, China
3
School of Aerospace Engineering, Beijing Institute of Technology, Beijing,
China

Background: The differentiation of vascular smooth muscle

cells (VSMCs), which are exposed to mechanical stretch in vivo,
plays an important role in vascular remodeling during hypertension. Here, we demonstrated the mechanobiological roles of large
conductance calcium and voltage-activated potassium (BK) channels in this process.
Methods and results: In comparison with 5% stretch (physiological), 15% stretch (pathological) induced the de-differentiation of
VSMCs, resulting in signicantly decreased expressions of VSMC
markers, i.e., -actin, calponin and SM22. The activity of BK channels,
assessed by patch clamp recording, was signicantly increased by 15%
stretch and was accompanied by an increased alternative splicing of
BK channel -subunit at the stress axisregulated exons (STREX). Furthermore, transfection of whole BK or STREX-deleted BK plasmids revealed that STREX was important for BK channels to sense mechanical
stretch. Using thapsigargin (TG) which induces endoplasmic reticulum
(ER) stress, and xbp1-targeted siRNA transfection which blocks ER
stress, the results revealed that ER stress was contribute to stretchinduced alternative splicing of STREX.
Conclusion: Our results suggested that during hypertension, pathological stretch may induce the ER stress in VSMCs, which affects the alternative splicing and activity of BK channels, and subsequently
modulates VSMC differentiation.
(This research was supported by grants from the National Natural
Science Foundation of China, Nos. 11232010 and 11229202).

TH-044
Functional and morphological improvements mediated by longterm -arrestin biased agonism of the AT1R in familial dilated
cardiomyopathy
David M. Ryba1, Jieli Li1, Conrad L. Cowan2, Brenda Russell1, Beata M.
Wolska1, R. John Solaro1
1

University of Illinois at Chicago, Chicago, IL, USA

Trevena, Inc., King of Prussia, PA, USA

Background: Biased agonism of the angiotensin II type 1 receptor

(AT1R) has been shown to improve cardiac contractility and promote
cardioprotection. Recent work by our laboratory has indicated that
these effects may be due to changes at the level of the myolaments.
We hypothesized that -arrestin signaling would increase myolament
Ca2+-response and may be of therapeutic value in the context of familial dilated cardiomyopathy (DCM).
Methods: We tested a DCM-linked mouse model expressing a mutant form of sarcomeric tropomyosin (Tm-E54K). We treated these
mice for three months with either TRV120067 (TRV067), an angiotensin
receptor modulator that blocks G-protein responses while stimulating
-arrestin mediated responses, or losartan, an angiotensin receptor
blocker. At the end of the treatment protocol, we assessed cardiac function using echocardiography, the myolament Ca2 +-response of
detergent-extracted ber bundles, and proteomic approaches to

understand changes in post-translational modications of proteins

that may explain functional changes.
Results: We found that Tm-E54K mice treated with TRV067
had improved cardiac function and morphology whereas
losartan-treated mice had no functional improvements but did
have some improvement in left-ventricular wall dimension. Myolaments of TRV067-treated Tm-E54K mice had an improved
Ca2 +-sensitivity of tension and normalized maximal tension generation, which were depressed in untreated controls. We attributed these changes to an increase in myosin light chain
(MLC2v) and MYPT1/2 phosphorylation that was seen only in
TRV067-treated mice. Western blots revealed these functional
changes were due to an activation of ERK1/2-RSK3 signaling,
which we show, for the rst time, directly increases MLC2v phosphorylation. Morphological improvements were attributed to
downregulation of -catenin signaling, which was also found in
losartan-treated Tm-E54K mice.
Conclusions: Improvements in cardiac function due to biased
agonism of the AT1R are due to changes in the myolament Ca2 +response and long-term biased ligand therapy may be a viable approach
for the treatment of familial DCM.

TH-045
TOR pathway regulates calcium handling in heart tissue through
eIF-4E and 4E-BP
Manuela Santalla1,2, Carlos Valverde1, Greco Hernndez3, Alicia
Mattiazzi1, Paola Ferrero1,2
1

Cardiovascular Research Center, La Plata, Buenos Aires, Argentina

Department of Basic Sciences, University of Northwest of Buenos Aires,
Pergamino, Buenos Aires, Argentina
3
Division of Basic Research, National Institute of Cancer (INCan), Mxico
City, Mexico
2

The target of rapamycin (TOR) pathway regulates growth, survival

and aging. It senses environmental cues to control metabolism, protein
synthesis and autophagy, and its dysregulation has been implicated in
cardiac diseases. Protein synthesis is the best characterized process controlled by TOR. Initiation of translation occurs when the eukaryotic initiation factor (eIF4E) promotes mRNA recruitment to the ribosome. This
step takes place when eIF4E recognizes the cap structure of mRNAs. The
eIF4E-binding protein (4E-BP) inhibits cap recognition by associating
with eIF4E. TOR phosphorylates and inhibits 4E-BP, thus promoting
translation. Changes in expression of eIF4E and 4E-BP alter cardiac
stress-response during aging, but the molecular mechanisms associated
to cardiac calcium handling remains not understood. In this report, we
studied the effect of genetic up and downregulation of eIF4E and 4EBP on cardiac calcium handling using Drosophila melanogaster as genetic
model. We assessed the intracellular calcium level by registering the
uorescent signal of a cardiac reporter system (TinC-Gal4-UASGCaMP3) in semi-intact preparation of 7 days-old ies. Overexpression
of 4E-BP incremented the Ca2+-transient amplitude (125%) and relaxation (100%), and the sarcoplasmic reticulum (SR) calcium load (20%).
These effects were linked to a higher SR Ca2 + reuptake through the
Ca+2-ATPase pump (SERCA). Downregulation of 4E-BP prevented
these changes. Accordingly, interference of eIF4E mimicked the effects
of 4E-BP overexpression on cardiac performance. Likewise, a 48 hs period of starvation provoked an increment in the amplitude of Ca2 +transients and SR-Ca 2+ load. TOR inactivation, and therefore 4EBP derepression, on ies overexpressing eIF4E is consistent with the phenotypes observed in ies overexpressing 4E-BP. eIF4E downregulation
and TOR inactivation mimicked these effects. Altogether, our results
provide evidence for a critical role of the TOR pathway, via eIF4E and
E-BP,on cardiac Ca2+ handling, SERCA activity and contractility.

Abstracts

TH-046
NOX2 activity induces lateralization, S-nitrosylation and opening of
connexin/pannexin hemichannels, causing arhythmogenesis and
apoptosis in dystrophic cardiomyopathy
Alejandra Vielma1, Mauricio Boric1, Daniel Gonzalez2
1

Pontica Universidad Catolica de Chile, Santiago, Chile

Universidad de Talca, Talca, Chile

Background: Duchenne dystrophy is a fatal progressive genetic disease that causes cardiomyopathy. One of the features of this disease is
oxidative stress, which derives mainly from NADPH oxidase (NOX) in
the dystrophic heart. It has been shown that oxidative stress interferes
with connexin 43 (Cx43) location to the intercalated discs; and hemichannels formed by connexins (Cx) or pannexins (Px) constitute a potential pathway for dissipation of ionic gradients and tissue damage.
Aims: Here we tested the hypothesis that increased oxidative stress
due to increased NOX activity causes S-nitrosylation, lateralization and
deregulation of Cxs and/or Pxs, increasing cell permeability, causing
myocytes
apoptosis,
decreased
inotropism
increased
arrhythmogenicity in mdx mice, a model of Duchenne disease.
Results: Hearts from 2 and 10 months of age mdx mice presented increased NOX activity and oxidative stress, reduced contractility and
higher number of arrhythmic episodes. At the cellular level, mdx hearts
presented a larger number of apoptotic cells and increased degree of brosis, as compared with controls. All these conditions were more severe at 10 month of age, and were reversed to control when mdx
animals were treated chronically 1 month with NOX inhibitor apocynin.
While total cardiac Cx43 content was unchanged, dystrophic hearts
showed higher presence of Cx43 at lateral membranes in 2- and 10month mdx mice. Hemichannels opening, evaluated using ethidium
permeability was substantially higher in mdx hearts and this condition
was normalized when mice were treated by apocynin or acutely,
using hemichannel blockers carbenoxolone (for Cx) and probenecid
(for Px). In addition, mdx hearts exhibit increased S-nitrosylation of
Cx43 and Px1 that was reversed by apocynin.
Conclusions: These results suggest that, in Duchenne disease, increased NOX activity deregulates Cx43 distribution and Snitrosylation, causing hemichannels formation and/or activation,
which may contribute to increased apoptosis and cardiac dysfunction.

TH-047
HGF/Met tyrosine kinase receptor in heart physiology and
pathophysiology
Tiziana Crepaldi1, Simona Gallo1, Stefano Gatti1, Valentina Sala1,
Alessandro Bonzano2, Paolo Maria Comoglio2

S71

approach get closer to the clinic, with the development of original

tools for therapeutic application.

TH-048
Translocase of the outer mitochondrial membrane 22 is a novel substrate for p38 alpha Mitogen Activated Protein Kinase
Eva Denise Martin1, Sharwari Verma1, Nicholas T. Hertz2, Rebecca S.
Levin2, Alma L. Burlingame2, Kevan M. Shokat2, Andrew Gilmore3,
Goncalo C. Pereira4, Nicolas Rognant4, Andrew P. Halestrap4, Michael
S. Marber1
1

King's College London, London, UK

University of California San Francisco, California, USA
3
University of Manchester, Manchester, UK
4
University of Bristol, Bristol, UK
2

TOM22 is a key component of the outer mitochondrial membrane

pore complex responsible for the import of precursor proteins from
the cytosol into their nal position in the mitochondrial matrix. Little
is known about its regulation and phosphorylation in mammals. p38
alpha MAPK is a stress activated kinase and a member of the MAP kinase
family. We identied TOM22 as a novel substrate of p38 alpha MAPK.
We identied the p38 alpha phosphorylation site and conrmed it by
mutation of the serine 15 residue to alanine in a recombinant protein
and tested in an in vitro kinase reaction. Wildtype TOM22 was phosphorylated by p38 alpha MAPK but the mutant lacking the phosphorylation site was not. Isolated perfused rat hearts were subjected to either
10 min ischemia or were perfused as control hearts. Following homogenisation, both sample and control hearts were phosphorylated with an
analogue sensitive form of p38 alpha MAPK, mutated to allow use of an
ATP analogue, to label substrates of the kinase. Substrates were isolated
by the covalent capture method. TOM22 was phosphorylated by p38
alpha MAPK in both ischaemic and control heart samples. A rabbit polyclonal antibody raised against the phosphorylation site, did not detect
differences in the phosphorylation levels between control, ischaemic
or ischemic and p38alpha inhibitor SB203580 treated Langendorff perfused mouse hearts. There was a reduction in phosphorylation of
TOM22 detected by western blotting in lysates from mitochondria expressing non-active p38 alpha MAPK compared to wildtype expressing
p38 alpha MAPK. Further work is underway to investigate the functional
signicance of this phosphorylation.

TH-049
NFAT and MEF-2 control the Expression of Calsequestrin-2 in rat
Cardiomyocytes
Rafael Estrada-Avils, Gabriela Rodrguez, ngel Zarain-Herzberg

University of Turin, Turin, Italy

FPO/IRCCS, Turin, Italy

Universidad Nacional Autnoma de Mxico, Mexico city, Mexico

Our work has mainly investigated the role of HGF/Met tyrosine kinase receptor signaling in the heart during physiological and pathological conditions. Targeting HGF/Met activation in neonatal heart in vivo
modulates the gene expression program involved in cardiomyogenesis.
Furthermore, sustained activation of Met pathways in postnatal
cardiomyocytes in vivo strongly increases the heart growth. Our research has also extended to the inuence of Met activation in the
heart protection against injury. Importantly, we have shown that Met
stimulation by HGF protects cardiac cells from hypoxic damage both
in vivo, in a mouse model of myocardial infarction, and in vitro, in
cardiomyoblast cells cultured in low oxygen tension. Recently, we
have shown that HGF protects cardiac cells from antracyclinemediated cardiotoxicity, showing that the induction of ROS-triggered
apoptosis and autophagy is attenuated by HGF. In addition, our

Calsequestrin-2 (CASQ2) is the main Ca2+-binding protein inside the

sarcoplasmic reticulum of cardiomyocytes. The proximal CASQ2 gene promoter is highly conserved, containing a TATA-Box, and binding sites for
MEF-2 (Myocyte Enhancer Factor-2) and SRF (Serum Response Factor)
transcription factors. Previously, we demonstrated that MEF-2 and SRF
binding sites within this region are functional in neonatal rat
cardiomyocytes. The calcineurin/NFAT pathway is functional in
cardiomyocytes. NFAT (nuclear factor of activated T-cells) transcription
factor regulates the expression of muscle specic proteins, such the myosin heavy chain gene. In this work, we investigated if NFAT regulates
CASQ2 gene expression. Sequence analysis of the human CASQ2 gene promoter revealed potential NFAT binding sites at -1869 bp and -230 bp.
Functional assays in neonatal rat cardiomyocytes with two hCASQ2 promoter constructs (-3102/+176 and -288/+176) showed that the

S72

Abstracts

inhibition of NFAT dephosphorylation with Cyclosporine A (CsA) or with

INCA-6 reduced the luciferase activity of both hCASQ2 promoter constructs
up to 50%. CsA and INCA-6 also reduced the CASQ2 mRNA levels. Additionally, NFATc1 and NFATc3 over-expressing cardiomyocytes showed a 2-3fold increase in luciferase activity of both hCASQ2 promoter constructs
that was prevented by CsA treatment. However, EMSA and site-directed
mutagenesis experiments failed to demonstrate a direct interaction between NFAT and CASQ2 gene promoter. Mutation of the -133bp MEF2
site prevented trans-activation by NFAT overexpression. Chromatin Immunoprecipitation assays revealed NFAT and MEF-2 enrichment within the
-288 bp to +76 bp of the hCASQ2 gene promoter, suggesting that NFAT interacts with MEF2 at the -133 bp site. Taken together, these data demonstrate that the Ca2+-calcineurin/NFAT pathway modulates expression of
the CASQ2 gene in cardiomyocytes. Funded by CONACyT grant 164413 to
A.Z.-H., and doctoral scholarship 57838 to R.E-A.

TH-050
Proteins Secreted Preferentially in Response to ER Calcium Dysregulation Protect Cardiac Myocytes from ER Stress-induced Cell Death
Shirin Doroudgar1,2, Donna J. Thuerauf1, Mirka Stastna3, Haley
Stephens1, Erik A. Blackwood1, Jennifer E. Van Eyk4, Christopher C.
Glembotski1
1

San Diego State University, San Diego, USA

Department of Cardiology, Angiology, and Pneumology Heidelberg University Hospital and DZHK (German Centre for Cardiovascular Research), Partner Site Heidelberg/Mannheim, Heidelberg, Germany
3
Institute of Analytical Chemistry of the Academy of Sciences of the Czech
Republic, Brno, Czech Republic
4
Advanced Clinical Biosystems Research Institute, Heart Institute and Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, USA
2

Protein secretion is important for normal cell-cell communication.

Many secreted proteins are synthesized and folded in the endoplasmic reticulum (ER). A number of diseases, including neurodegenerative and
heart disease, are thought to alter the ER in ways that impair ER protein
folding, which causes ER stress. However, the impact of ER stress on secreted proteins, i.e. the secretome, has not been examined. Accordingly,
we studied how ER stress affects the secretome of neonatal rat ventricular
myocytes (NRVM), a well-established model system for studies of cardiac
myocyte protein secretion. To mimic the effect of heart disease on ER protein folding, NRVM were treated with either tunicamycin (TM) or
thapsigargin (TG), which inhibit ER protein glycosylation or decrease ER
calcium, respectively. The identities of proteins in NRVM-conditioned medium (CM) were determined using proteomics. Twenty-four different
proteins known to be synthesized in the ER were identied in control
NRVM-CM. The levels of most of these proteins, none of which are ER
stress-inducible, were decreased in response to TG or TM. Interestingly,
three ER-resident, ER stress-inducible chaperones, Grp94, Grp78 and Crt
were secreted only in response to TG. Moreover, TG was a potent mediator of cardiac myocyte death in high culture media volumes, but not in
low volumes. Addition of recombinant Grp94, Grp78 and Crt to high culture media volumes decreased TG-mediated cardiac myocyte death. Thus,
TG, which mimics the effects of heart disease on ER calcium in cardiac
myocytes causes the secretion of select ER stress-inducible chaperones,
which protect against cell death upon ER calcium dysregulation.

TH-051
Proximal Endoplasmic Reticulum Stress Response Element is essential for SERCA2 gene basal and Thapsigargin-induced Transcription
Jorge Fragoso-Medina, Gabriela Rodrguez, ngel Zarain-Herzberg
Universidad Nacional Autnoma de Mxico, Mexico city, Mexico

The cardiac sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2a) is

vital for the proper contractile function in the heart. Decreased levels of
SERCA2a mRNA and protein are found in animal models of cardiac hypertrophy and patients with heart failure; however, the molecular mechanisms mediating its altered expression have not been fully elucidated.
The SERCA2 specic inhibitor thapsigargin (Tg) increases cytoplasmic calcium concentration, induces endoplasmic reticulum stress (ERS) and has
been associated with increased SERCA2a expression in cardiomyocytes. In
this work, we show that Tg increased 2-3-fold SERCA2a mRNA, protein, as
well as the transcriptional activity of a human SERCA2 gene promoter construct containing the proximal 259 bp of the promoter sequence and
323 bp of 5-UT region in primary cultures of neonatal rat cardiomyocytes.
Since Tg induces ERS, we mutated three conserved DNA binding sites
(CCAAT, GGC and CCACA) present within the ERS response element
(ERSE) located in the proximal SERCA2 promoter (-60 to -78 bp) and
assessed the response of the mutated constructs to Tg. The CCAAT and
CCACA mutated constructs showed lower basal activity compared to the
wild-type construct and did not respond to Tg treatment, whereas the activity of the GGC mutant did not show any change. Furthermore, by EMSA
and super-shift assays, we showed the interaction of the CCAAT sequence
with NF-Y transcription factor present in nuclear extracts from neonatal
rat cardiomyocytes and observed that Tg treatment decreased DNA interaction with this factor. These results demonstrate that the ERSE present in
the proximal SERCA2 gene promoter is essential for basal transcriptional
activity and also necessary for the response to ERS. Funded by CONACyT
grant 164413 to A.Z.-H., and doctoral scholarship to J.F.-M.

TH-052
Hyperosmotic Stress Promotes No Release in the Rat Myocardium
Malena Morell, Luis Gonano, Juan Ignacio Burgos, Martin G Vila Petroff
Centro de Investigaciones Cardiovasculares Dr Horacio E Cingolani, La
Plata, Argentina
Tissue osmolarity is tightly regulated under physiological conditions.
However, in different pathological situations as states of severe dehydration, hyperglycemia, hyperlipidemia and diabetes, cardiomyocytes
undergo osmotic shrinkage and it is associated with alterations in calcium handling, negative inotropic effects (NIE) and apoptosis.
Nitric oxide (NO) synthesized by the nitric oxide synthase (NOS) has
been well dened as a second messenger and as a regulator of cardiac
function. In a previous study we showed that hyposmotic swelling promotes NO release and that this NO provides contractile support.
The aim of this study is to evaluate whether membrane deformation
produced by hyperosmotic stress also promotes NO release and to examine the underlying mechanisms involved.
We observed that superfusing rat cardiac myocytes, loaded with the NO
sensor (DAF-FM), with a hyperosmotic solution (HS:440 mOsm) results in
a decrease of cell volume (26%1.95; n=21) and a signicant increase in
uorescence of DAF-FM (10%2.55; n=22) compared to myocytes
superfused with an isosmotic solution (IS: 309 mOsm; n=10). When
cells are superfused with HS+L-NAME (inhibitor of NOS),
HS+Nitroguanidine (NG: inhibitor of NOS1) or HS+Wortmaninn (WT:
inhibitor of NOS3) cell volume decreases in absence of NO release suggesting that NOS1 and NOS3 are responsible for NO release during
hyperosmotic stress.
Supporting the involvement of NOS1 and NOS3 in hyperosmotic stressinduced NO release, Western blot analysis showed an increase in NOS1 and
NOS3 activity (pNOS1 and pNOS3) in hearts perfused with hyperosmotic
solution compared with hearts perfused with isosmotic solution.
These results suggest that NOS1 and NOS3 promote NO release during hyperosmotic stress. This NO release could impact on altered cell
function observed in pathological situations associated with
hyperosmotic stress.

Abstracts

TH-053
Role of DBC1 protein in the regulation of hypertension
Maria Caggiani1,2, Adriana Carlomagno2, Carlos Batthyany1,2, Paola
Contreras1,2, Carlos Escande2
1

Facultad de Medicina, Universidad de la Repblica, Montevideo, Uruguay

Institut Pasteur Montevideo, Montevideo, Uruguay

Lifestyle changes have determined an increase in the incidence of

noncommunicable diseases which have become the rst cause of
death worldwide. Among them is arterial hypertension, a silent and invisible killer. In Uruguay the prevalence of hypertension in the adult
population has increased from 30 % in 2006 to 39 % in 2015.
Vascular injury is one of the main consequences of maintained hypertension. The cellular and metabolic mechanisms involved in vascular
injury are complex. The renin-angiotensin-aldosterone system plays a
main role since angiotensin II (ANGII) is involved in the generation of
inammation, brosis and apoptosis; all of them observed in arterial hypertension. However, the molecular mechanisms that underlay these
processes are not completely elucidated.
Our group has been working in the role of Deleted in Breast Cancer -1
(DBC1) protein, a sirtuin 1 inhibitor, in the physiopathology of cardiovascular diseases. DBC1 knock out (KO) mice are protected against atherosclerosis in experimental obesity. Within this context, we aim to
evaluate whether DBC1 plays a relevant role in hypertension. We treated
C57Bl6 mice with ANGII to induce hypertension by means of an osmotic
pump (1mg/kg/day). We measured blood pressure non-invasively twice
a week and isolated the tissues after 28 days of treatment for molecular
biology analysis. Our preliminary results show that DBC1 expression in
renal and vascular tissues is induced by treatment with ANGII in vivo. Surprisingly, DBC1KO mice are more sensitive to ANGII, reaching higher
values of systolic blood pressure than wild type mice. Our results suggest
that DBC1 plays a main role in cardiovascular diseases, although we still
have to understand deeply the mechanisms involved.

TH-054
The adenosine signalosome requires ROS activation to mediate
cardioprotection
Anders O. Garlid1, Keith D. Garlid2, Peipei Ping1
1
Departments of Physiology, Medicine, and Bioinformatics, University of
California, Los Angeles, Los Angeles, CA, USA
2
Department of Biology, Portland State University, Portland, OR, USA

Background: Mitochondria are central actors in cardioprotection

against ischemia-reperfusion injury (IRI), which depends upon activation
of the mitochondrial ATP-sensitive potassium (mitoKATP) channel and inhibition of the mitochondrial permeability transition (MPT) to prevent
cell death and reduce myocardial infarct. MitoKATP opening is mediated
by signalosome, a multi-protein complex that buds off from the cell membrane as a lipid raft comprised of the internalized Gi-protein coupled receptor (GPCR), its attached ligand agonist, and the entire downstream
signaling pathway. Cardioprotection by ischemic preconditioning (IPC)
occurs by way of endogenous adenosine signaling. As ATP decreases during ischemia, it is degraded to adenosine, which moves to the extracellular space to activate adenosine receptors (ADOR) and trigger the GPCR
signaling cascade. This process can be mimicked pharmacologically.
Aims: To characterize the signaling components and activation requirements of the ADOR signalosome.
Methods: The ex vivo, Langendorff-perfused rat heart was used to
generate signalosomes, which can readily be isolated and puried from
the perfused heart and which cause mitoKATP opening and MPT inhibition
in mitochondria isolated from untreated hearts. This permits study of a
signaling unit in its naturally organized state with preserved functionality.

S73

Results: We show that ADOR activation forms a unique signalosome that

contains the expected PI3K-to-PKG pathway but is initially inactive and requires reactive oxygen species (ROS) before it can accomplish its downstream
signaling effects. The ROS signal activates a signalosomal PKC that is upstream of ADOR and triggers the PI3K-to-PKG pathway to confer
cardioprotection by ischemic preconditioning, ischemic postconditioning,
KATP channel openers (e.g., diazoxide), and GSK-3 inhibition.
Conclusions: ADOR signaling is identical to signaling by other GPCR,
but the ADOR signalosome requires both receptor activation and ROS to
complete the signal. The signalosome mechanism is a general mechanism
of cell signaling that is probably utilized by all receptors and all cell types.

TH-061
Switchable Cardiac L-Type Ca2+ Channel Transcript By Mineralocorticoid Pathway.
Thassio Mesquita1, Gaelle Auguste1, Jessica Sabourin1, Gema Ruiz
Hurtado1, Valrie Roufac2, Florian Le-Billan3, Jrme Fagart3, Florence
Lefebvre1, Say Viengchareun3, Eric Morel1, Ana Maria Gomez1, Marc
Lombs3, Jean-Pierre Benitah1
1

UMR-S 1180, Inserm, Univ. Paris-Sud, Universit Paris-Saclay, ChtenayMalabry, France

2
Imaging and Cytometry Platform, UMR 8081 IR4M, Gustave Roussy Institute, Villejuif, France
3
UMR-S 1185, Inserm, Univ. Paris-Sud, Universit Paris-Saclay, Le KremlinBictre, France
Regulation of expression of the ubiquitous L-type Cav1.2 Ca2+ channels (encoded by the CaCNA1C gene) is critically involved in the pathogenesis of serious neurological, retinal, cardiac, vascular and metabolic
disorders, in which the mineralocorticoid hormone, aldosterone, via its
cognate receptor (MR), plays pivotal, yet elusive, roles. MR-related
extrarenal actions can be attributed to Cav1.2 deregulation, notably in cardiac and vascular cells that express this transcriptional factor. However,
underlying molecular mechanisms remain unexplained. Here, we show
that aldosterone induces expression of the cardiac long N-terminal
Cav1.2 isoform (Cav1.2-LNT) through MR transactivation on the most
proximal CaCNA1C gene promoter (P1). In cardiomyocytes aldosterone
increased in dose-dependent manner Cav1.2-LNT expression at both
mRNA and protein levels, correlating with enhanced dose-, time- and
MR-dependent P1-promoter activity, through MR recruitment to specic
DNA binding elements. The in vivo relevance of this regulation is conrmed in transgenic mice harbouring the luciferase reporter gene under
the control of the P1-promoter. Moreover, aldosterone enhanced the
functional expression of the Cav1.2-LNT in rat coronary smooth muscle
cells increasing vascular tone. These results identify cardiac CaCNA1C
gene as a new specic mineralocorticoid target gene, unravelling new insights into the molecular mechanisms associated with MR activation.

TH-062
Meis1 regulates sympathetic target-eld innervation: consequences
for autonomic nervous system induced sudden cardiac death
Jerome Thireau1, Fabrice Bouilloux2, Charlotte Farah1, Sarah Karam1,
Yves Dauvilliers3, Sylvain Richard1, Frederic Marmigre2
1

INSERM U1046 -CNRS UMR 9214, Montpellier, France

INSERM U1051, Institute for Neurosciences of Montpellier, Montpellier,
France
3
Sleep Unit, Department of Neurology, Gui-de-Chauliac hospital, Montpellier, France
2

Background: Sudden cardiac death (SCD) are among the leading

causes of premature death in the general population. Genome-wide

S74

Abstracts

association studies have recently identied the transcription factor Meis1

as a risk factor for SCD. Recent studies demonstrated a function of Meis1
in shaping heart morphology and in cardiomyocytes proliferation. The autonomic nervous system is a major regulator of cardiac functions and its
imbalance is a source of dysrhythmias. Here, we hypothesise that Meis1
is implicated in cardiac nervous system development.
Methods and results: We report that specic Meis1 inactivation in
mouse sympathetic neurons (HtPACRE/Meis1LoxP/LoxP) leads to SCD independently of cardiac structural defect. We showed that Meis1 is implicated in the development of cardiac sympathetic neurons, in particular in
NGF/TRK1 trafcking. Using telemetric system, we record electrocardiograms in baseline condition, and after either pharmacological testing of
autonomic nervous system or treadmill exercise. By heart rate variability
analysis, we show that mice developed impaired sympatho-vagal regulation of cardiac rhythm. Mice exhibited atrial and/or atrioventricular conduction defects that led to spontaneous bradycardia and
desynchronization, concomitant with a high occurrence of sinus arrests.
Pharmacological testing revealed that mutant mice were intolerant to
carbamylcholine injection which induces death in 40% of HtPACRE/
Meis1LoxP/LoxP mice and, as well as to exercise tests on treadmill. During
exercise, the RR decreased by 45% in WT mice (pb0.01, n=8), whereas a
non-signicant and delayed 13% decrease in the RR interval was observed
in mutant mice. The maximal RR decrease in WT mice was 778
vs.1043ms in mutant mice. During the recovery phase, 3 out of 4 mutant mice developed ventricular brillations and died.
Conclusion: Mutant mice presented profound alterations in the
sympatho-vagal regulation of cardiac functions that are independent
of cardiac structural phenotype, arguing for an essential role of the transcription factor Meis1 in the sympathetic nervous system development
and function.

TH-063
Extracellular matrix metalloproteinase 9 (MMP-9) and Tissue
endogenous inhibitor (TIMP-1) has signicantly associated with
cardiovascular dysfunction (CVD) dened by echocardiography
Diego Torres Dueas1, Maria Eugenia Nio1, Edilberto Eduardo2, Manuel
Guillermo Hernndez2, Sergio Serrano Gmez1, Daniela Camila Nio
Vargas1
1
Universidad Autnoma dde Bucaramanga, Bucaramanga, Santander,
Colombia
2
Instituto del corazn de Bucaramanga, Bucaramanga, Santander,
Colombia

Sepsis is a pathophysiological interaction complex of different processes (infectious, inammatory, hemodynamic, organ dysfunction, impaired tissue perfusion). Recent data suggest that the annual cost of
hospital care for patients with septicemia is $ 14 billion in the United
States. MMPs have been involved in the CVD in animal models of sepsis.
However its role in humans has not been clearly dened.
Objective: Establish the association between MMP-9 and TIMP-1
with the CVD from the echocardiographic context in septic patients.
Methodology: An analytic observational study of prospective
cohort was performed, that include 5 health Centers of the city of
Bucaramanga. Sepsis was dened according to the International
Conference on Sepsis of 2001, MMP-9 and TIMPs- 1 was quantied
by immunoassay of systemic blood samples, echocardiograms
were performed within the rst 24 hours of study entry. A bivariate
analysis was performed to dene the association between MMP-9,
TIMPs-1 with echocardiographic variables.
Results: A signicant relationship between MMP-9 with left ventricular diastolic diameter LVDD (P b 0.03) and left ventricular systolic diameter LVSD (P b 0.001) and cardiac ejection fraction of the left
ventricle LVEF (P b0.01) was found. The TIMP-1 was signicantly

related to left atrial volume LAV (P b0.01), the E / A ratio (P b0.05)

and LVEF (P b0.001). Tables 1 and 2.
Conclusion: MMP-9 and its endogenous inhibitor seems to be important as CVD biomarkers on the stage of sepsis. More studies to dene
the true extent of these markers and its prognostic, diagnostic and monitoring value are missing.

TH-064
Interpretation of arrhythmia generation induced by sarcoplasmic
reticulum Ca2+ loss using a human myocyte mathematical model
Juan Ignacio Felice1, Carlos Valverde1, Alicia Mattiazzi1, Elena Catalina
Lascano2, Jorge Antonio Negroni2
1
Centro de Investigaciones Cardiovasculares, CONICET-UNLP, La Plata,
Buenos Aires, Argentina
2
Universidad Favaloro, Ciudad Autnoma de Buenos Aires, Buenos Aires,
Argentina

Background: Contraction in cardiac myocytes is produced by the release of Ca2+ from the sarcoplasmic reticulum (SR) through ryanodine
receptor channels (RyR2) by Ca2 +-induced Ca2 + release (CICR)1.
There are also spontaneous diastolic Ca2+ discharges that are increased
when RyR2 are altered and this situation may trigger arrhythmias2. Experimental data showed that transgenic mice carrying a mutation that
represents a constitutive pseudophosphorylation of RyR2 (S2814D) exhibit spontaneous action potentials (SAP) and that the intensity of these
events decreased until reaching the level of delayed
afterdepolarizations (DAD) when Ca2 + reuptake by the SR-Ca2 +ATPase (SERCA2a) was increased in mice with mutated RyR2 and phospholamban (PLN, a SERCA2a inhibitory protein) ablation (SDKO).
Methods: To analyze the mechanisms involved in these arrhythmic
events, a human myocyte mathematical model3 was used to represent
both experimental conditions. Basal conditions and a proarrhythmogenic
stress were simulated. The model was developed in MATLAB, and ODE15s
solver was used to solve the system of differential equations.
Results and conclusions: The model reproduced the arrhythmic
events. Simulations showed that in S2814D conditions, the enhancement in diastolic Ca2+ leak increased Ca2+ concentration in the dyadic
cleft (DC) that surrounds RyR2 which is exchanged by Na+ through the
Na+-Ca2+ exchanger (NCX) working in forward mode. Na+ entrance
depolarizes the membrane to the threshold level of Na+ channels giving
rise to an action potential. In SDKO conditions, the increased Ca2+ reuptake produces lower NCX activity resulting in membrane depolarization
below the threshold needed to generate SAP; in this situation only DAD
appeared. Simultaneous representation of ionic uxes in the myocyte
using model-derived data allowed us to explain the differences in the
arrhythmic events observed in both experimental conditions.
[1] Bers DM. Nature 415:198-205, 2002.
[2] Priori SG, et al. Circ Res 108:871-883, 2011.
[3] Lascano EC, et al. J Mol Cell Cardiol 60:172-183, 2013.

TH-065
Molecular and Functional Characterization of Novel Mutation in the
Cardiac Ryanodine Receptor Gene (RYR2) in a Patient With Long QT
Syndrome
Carmen Valdivia1, Erika Antunez2, Jonathan Hernandez1, Todd Herron1,
Teresa Villareal2, Pedro Iturralde4, Argelia Mereidos-Domingos3, Hector
Valdivia1
1

University of Michigan, Ann Arbor, MI, USA

Universidad Autonoma de Mexico, Mexico City, Mexico
3
University Hospital of Bern, Bern, Switzerland
4
Instituto Nacional de Cardiologia Ignacio Chavez, Mexico City, Mexico
2

Abstracts

Background: Long QT syndrome is characterized by prolongation of

the QT interval in the ECG, syncope and sudden death. Mutations in 16
genes that encode ion channels or associated proteins account for ~80%
of all cases, however 20% of the cases still remain genetically unknown.
Further, mutations in cardiac ryanodine receptor (RYR2) have been implicated in arrhythmia syndromes such as cathecolaminergic ventricular tachycardia (CPVT).
Methods and results: We identied a novel RyR2 mutation in the
cardiac ryanodine receptor (RyR2) R2920Q, in a patient with family history of sudden death, syncope and prolongation of the QT interval (QTc,
525 ms). 80 cardiogenes were simultaneously sequenced using
Haloplex design on a MiSeq device (Illumina). All novel or low frequency variants predicted to be damaging were conrmed by Sanger sequencing. To study the mechanism by which RyR2 might cause RYR
dysfunction to lead LQT, we engineer the R2920Q and the in the
mRYR and expressed in HEK-293 cells and human IPS-derived
cardiomyocytes (hIPS-CM). The recombinant protein obtained from
HEK-293 cells showed that [3H]ryanodine binding of RyR2-R2920Q
has increased Ca2 + sensitivity compared to RyR2-WT with no difference in protein expression. Monolayers of hIPS-CM expressing
mRYR2-R2920Q or -WT were loaded with a voltage sensitive dye and
subjected optical recordings of action potential at 1 Hz pacing. hIPSCM expressing mRYR2-R2920Q showed an AP90 of 232 compared to
197 or 185 ms in hIPS-CM expressing mRYR-WT and non-transfected,
respectively. Monolayers of hIPS-CM expressing the mRYR- constructs
were conrmed by RT-PCR using mouse primers and immunolabeling.
Conclusion: We identied novel mutation in RyR2 in a patient with
LQT syndrome that resulted in abnormal RYR function; these results are
not surprising since abnormal Ca2+ handling has been reported in animal model of LQT. To elucidate the mechanisms by which RyR2R2920Q is linked to LQT and not CPVT remain unknown.

TH-066
C543 is the reactive cysteine responsible for increased human L-type
calcium channel protein function following glutathionylation
Padmapriya Muralidharan1, Henrietta Cserne Szappanos1, Evan Ingley2,
Livia Hool1,3
1
School of Anatomy, Physiology and Human Biology, The University of
Western Australia, Crawley, WA, Australia
2
Cell Signalling Research, Harry Perkins Institute of Research, Perth, WA,
Australia
3
Victor Chang Cardiac Research Institute, Sydney, NSW, Australia

The development of cardiac hypertrophy is associated with oxidative stress and altered calcium homeostasis. The L-type calcium channel
(LTCC) is the major route for calcium inux into cardiac myocytes. We
have previously demonstrated that oxidative stress is associated with
persistent glutathionylation of the LTCC that results in an increase in intracellular calcium and protein synthesis consistent with the development of myocyte hypertrophy. We searched for the reactive cysteine
on the Cav1.2 (alpha) subunit of the channel responsible for modulating
channel function during oxidative stress. Human long and short N terminal (NT) isoforms of Cav1.2 were expressed in HEK cells. Cysteines
were mutated to a serine or an alanine. The channel protein was puried by histidine tag purication and incorporated in liposomes for functional analysis by patch-clamp technique.
Exposing the long NT isoform to 2mM oxidised glutathione increased Po from 0.026 0.008 to 0.088 0.014 without altering the
magnitude of the current or the currentvoltage relationship (n = 6)
while1mM reduced glutathione decreased Po from 0.029 0.007 to
0.010 0.007 (n = 5; p b 0.05). Similarly oxidised glutathione signicantly increased Po of the short NT isoform that lacks the rst 46
amino acids of the N terminus (n = 16) and following truncation of

S75

the C terminus (n=7). However mutation of 3 cysteines in cytoplasmic

loop I-II attenuated the effect of glutathione on open probability and altered protein folding assessed by thermal shift assay. Specically we
nd that C543 is critical for conferring sensitivity of Cav1.2 to glutathione and is responsible for modifying channel function and posttranslational folding.

TH-067
Voltage and Calcium Dynamics in Atrial-like and Ventricular-like
Cardiomyocytes derived from Human Embryonic Stem Cells by Optical Mapping
Sanam Shafaattalab1, Eric Lin1, Stephanie Protze2, Jeehoon Lee2, Mark
Gagliardi2, Yulia Nartiss2, Peter Backx2, Zachary Laksman3, Gordon
Keller2, Glen Tibbits1,4
1

Simon Fraser University, Burnaby, Canada

University of Toronto, Toronto, Canada
3
Univeristy of British Columbia, Vancouver, Canada
4
Child and Family Institute, Vancouver, Canada
2

Human embryonic stem cell-derived cardiomyocytes (hESC-CMs)

are important in vitro models of human cardiac physiology due to
their ability to recapitulate the corresponding electrical phenotype.
Using simultaneous voltage and calcium optical mapping, the relationship between this electrical activity and the subsequent calcium response was investigated.
We have generated atrial- and ventricular-like cardiomyocytes from
hESCs using established differentiation protocols that employ activin A
and BMP4 signaling for mesoderm induction followed by Wnt inhibition
for cardiac specication. The atrial-like cardiomyocytes (CMs) were generated with a differentiation protocol that also added retinoic acid.
Atrial- and ventricular-like CMs were labeled using the voltagesensitive dye RH-237 and the calcium indicator dye Rhod-2, which
were imaged concurrently on a single CMOS camera. Clusters of CMs
were spontaneously active at various independent rates, and responded
uniformly to electrical eld stimulation. This facilitated an examination
into the rate dependencies of the action-potential (AP) proles and calcium transient dynamics. As expected, atrial voltage dynamics were signicantly faster than the ventricular dynamics, in which ventricular
durations were twice that of the atria. Atrial- and ventricular-like CMs
had distinct calcium dynamics, with atrial-like CMs demonstrating
more rapid repolarization, which was associated with elevated calcium
levels at the end of the AP. In ventricular-like CMs, the more prolonged
AP was associated with a correspondingly prolonged calcium transient.
Voltage and calcium dynamics in both atrial- and ventricular-like CMs
were slowed by the addition of 100 nM dofetilide, which also resulted
in the development of early and late-after depolarizations.
Our preliminary data have demonstrated clear differences in the
atrial-and ventricular like CM populations when generated by targeted
differentiation strategies. The patterns and responses observed are consistent with those seen and expected in vivo.

TH-068
Isolation of cardiac myocytes from human heart
Caroline Pascarel-Auclerc1, Caroline Cros1, Sbastien Chaigne1, David
Benoist1, Richard Walton1, Philippe Pasdois1, Marine Martinez1, Yunbo
Guo1, Bruno Stuyvers1, Sbastien Dupuis1, Marion Constantin1, Dominique Dtaille1, Thomas Desplantez1, Josselin Duchateau2, Louis
Labrousse2, Julien Rogier2, Michel Hassaguerre1,2, Mlze Hocini1,2,
Olivier Bernus1, Fabien Brette1
1

IHU-LIRYC, INSERM U1045, Universit de Bordeaux, Bordeaux, France

CHU Bordeaux, Bordeaux, France

S76

Abstracts

Background: The investigation of single cardiac myocytes from

healthy and diseased hearts of various species is a valuable tool to explore cardiac physio/pathophysiology. The application of cell isolation
to human donor tissue has been proofed to be difcult due to the limited
amount of human tissue (mainly human right atrial appendages during
cardiac surgery). Another limitation is the low viability of
cardiomyocytes after isolation. In this study, we present a method to obtain single cardiac myocytes from different regions of human heart.
Methods and results: Human hearts rejected for transplantation
were obtained from Bordeaux hospital. This protocol was approved by
the Agence de la Biomdecine. Left atrial (LA) and ventricular (LV)
myocytes were obtained by enzymatic dissociation. The ventricles and
right atrium were removed and used for other studies (e.g. high resolution optical mapping). LA was cannulated by the circumex artery and
mounted into a Langendorff perfusion system after suture of the leaky
atrial branches. LA was perfused with a Ca2+-free solution (~10 min),
then collagenase and protease solution (0.08 mM Ca2 +) and
recirculated for ~ 25 min. Enzymes were washed out with a 0.2 mM
Ca2+ solution. LA was separated into 4 regions: Endocardium, Epicardium, roof and pulmonary vein; LV myocytes were also obtained. Cells
were re-suspended into a 1.8 mM Ca2 + solution by steps. Ca2+ transients were recorded (Fura-2, eld stimulation) using an IonOptix system and cell membrane was stained with di-8 ANEPPS and visualized
under confocal microscopy. Ca2 + tolerant myocytes were obtained
from the 4 LA regions and LV. Human cardiac myocytes respond to electrical stimulation and Ca2+ transient can be recorded. Analysis of functional and structural data will be presented.
Conclusion: Isolation of single cardiac myocytes from human samples is a tedious task, but we present data showing reliable method to
obtain functional and structural insights.

TH-069
Characterization of electrophysiological properties of right ventricular tissue in human using optical mapping
Caroline Cros1, Caroline Pascarel-Auclerc1, Richard Walton1, David
Benoist1, Marine Martinez1, Sebastien Chaigne1, Yunbo Guo1, Bruno
Stuyvers1, Philippe Pasdois1, Sebastien Dupuis1, Marion Constantin1,
Thomas Desplantez1, Line Pourteau1, Josselin Duchateau2, Louis
Labrousse2, Julien Rogier2, Michel Haissaguerre1,2, Meleze Hocini1,2,
Olivier Bernus1, Fabien Brette1
1

IHU-LIRYC, INSERM U1045, Univeriste de Bordeaux, Bordeaux, France

CHU de Bordeaux, Bordeaux, France

Introduction: Spatial dispersion of action potential (AP) repolarization plays an important role in arrhythmogenesis. Although the mechanisms underlying tissue-dependent electrotonic modulation have been
studied in various animal species there is limited information in humans.
In this study, we investigated electrotonic modulation by the activation
sequence and the site of pacing in human right ventricular tissues.
Methods: Three human hearts rejected for transplantation were obtained from Bordeaux hospital. This protocol was approved by the University ethic committee. High-resolution optical mapping experiments
were performed in coronary-perfused right ventricle (RV). Potentiometric dye was dissolved in DMSO and further diluted in Tyrode solution (95% O2-5% CO2). RV were paced at 1Hz on 4 different sites of
the endocardial (Endo) or epicardial (Epi) surfaces (base, apex, right
or left) and action potential duration at 80% repolarization (APD), activation time (AT) and repolarization time (RT) were calculated.
Results: APD range from 225 to 300 msec in the three human RV.
Changing pacing site induced signicant differences in APD, with the
longest APD observed when stimulation originated from the base of
the RV. Similar results were observed for AT and RT. In addition,
transmural (Epi vs Endo) APD heterogeneity was observed. Linear

correlation analysis showed no relation between APD and AT in all 3

preparations.
Conclusion: We have demonstrated that optical mapping of human
heart will provide opportunities for elucidation of arrhythmia mechanisms in human. Analysis revealed a pronounced heterogeneity of the
APD in RV, which is strongly modulated by the activation sequence
and pacing site. Such heterogeneity and dispersion of electrophysiological characteristics are crucial to reveal understanding and treatment of
cardiac arrhythmia.

TH-070
IL-1 production induces cardiac arrhythmias in diabetic mice
Emiliano Medei1,5, Gustavo Monnerat Cahli1,5, Micaela LopezAlarcon1,5, Oscar Casis3, Martin Vila-Petroff4, Juan Ignacio Burgos4,
Marisa Seplvera4, Marcelo Bozza6, Claudia Paiva6, Rosana Bassani2,
Luiz Vasconcellos6, Antonio Carlos Campos de Carvalho1,5
1
Institute of Biophysics Carlos Chagas Filho, Federal University of Rio de
Janeiro, Rio de Janeiro/Rio de Janeiro, Brazil
2
Center for Biomedical Engineering, University of Campinas, Campinas/So
Paulo, Brazil
3
Facultad de Farmacia, Universidad del Pas Vasco UPV/EHU, Vitoria, Spain
4
Centro de Investigaciones Cardiovasculares, Conicet, La Plata/Buenos
Aires, Argentina
5
National Center for Structural Biology and Bioimaging CENABIO/UFRJ,
Rio de Janeiro/Rio de Janeiro, Brazil
6
Instituto de Microbiologia, Federal University of Rio de Janeiro, Rio de
Janeiro/Rio de Janeiro, Brazil

Diabetes causes a multitude of secondary disorders, which have

a severe prognostic impact in heart disease and arrhythmias. The
latter are probably due to inammation and pathologic signaling.
Herein, we investigated origin and mechanisms underlying the
onset of these arrhythmias using a combination of genetic and
pharmacological tools. We demonstrate that TLR2 mediates the
production of IL-1, which in turn induces arrhythmias. In fact,
IL-1 induces longer action potential as a consequence of a decrease in potassium current (Ito). Additionally, IL-1 increase calcium sparks in cardiomyocytes. Thus, our study assigns a critical
role to the diabetes-induced inammation process in one of the
major secondary fatalities associated with this widespread disease.
We further demonstrate that blocking the IL-1 receptor can therapeutically treat diabetes-induced ventricular arrhythmias.

TH-071
Cardiac electrical remodelling study on a type 2 diabetes experimental model
Ainhoa Rodriguez de Yurre Guirao1,2, Oscar Casis Senz2, Emiliano
Medei1
1

Universidade de Rio de Janeiro, Rio de Janeiro, Brazil

Universidad del Pas Vasco, Vitoria-Gasteiz, Spain

Aim: The aim of the present study was to investigate the mechanisms underling the cardiac electrical remodeling on type 2 diabetes
(T2D) mice model.
Background: T2D is the most prevalent form of diabetes and it represents about 90% of the diabetic cases all over the world. As a consequence
of the lifestyle and feeding, this syndrome has turned into one of the largest health problem worldwide and it is associated with an increase of premature appearance of several disorders such as cardiovascular
complications which; can evoke cardiac electrical disturbances, as
arrhythmias.

Abstracts

Methods: c57bl/6 adult mice were fed with a high fat diet (HFD)
(45% energy from fat) and on the second week its were injected
intreperitoneally (2 doses of 40mg/kg) of streptozotocin separated
24h each other to induce T2D model. Control group received a standard
chow (4,15% energy from fat) and a vehicle (citrate buffer pH 4.5).
Weight and blood glucose levels and electrocardiogram recording,
were measured weekly. Intraperitoneal glucose tolerance test (IPGTT)
and insulin tolerance test (IPITT) were carried out at the end of the
study (6 weeks).
Results: After 6 week a T2D was established, observing an average
value of 158.77 mg/dl and 108.3 mg/dl of glucose in the T2D and control
group respectively. Additionally the metabolic tests of glucose homeostasis were different between studied groups. Both groups showed similar body weight. However, both QT and QTc intervals of T2D group
were longer than control group.
Conclusion: In the present work we showed that the combination of
HFD (45%) with low dose of streptozotocin (40 mg/Kg/2 times) was
able to induce a T2D mice model. It reproduced the typical metabolic
and cardiac electrical disturbance of this disease.

TH-072
Modeling CPVT1 through patient-specic induced pluripotent stem
cell-derived cardiomyocytes reveals aberrant mechano-biological
and intracellular calcium handling properties associated with
beta-blocker resistance
Ivana Acimovic1, Marwan M. Refaat2, Anton Salykin1, Franck Aimond3,
Jan Pribyl4, Valerie Scheuermann3, Melvin M. Scheinman5, Petr
Dvorak1,6, Vladimir Rotrekl1, Alain Lacampagne3, Albano C. Meli1,3

S77

mechano-biological properties were observed under stress in

agreement with the arrhythmias only induced under stress. Furthermore, we revealed that the CPVT hiPSC-CMs exhibit partial
resistance to the beta-blocker drug metoprolol similarly to the
clinical observations of the CPVT proband.
Conclusions: Our results indicate that hiPSC-CMs can provide a suitable tool for CPVT disease modeling including resistance to betaadrenergic receptor inhibition. In stress conditions, the novel RyR2D3638A mutation may cause sarcoplasmic reticulum Ca2 + leak that
cannot be fully prevented by standard beta-adrenergic receptor
blockade.
Grants: GACR GA13-19910S, SoMoPro 2SGA2744, ESC R12042FF,
PHC Barrande 28379TE, AFM MNM2 2012, FRM SPF20130526710 and
INSERM.

TH-073
Melatonin protects against low potassium induced ventricular brillation: role of melatonin receptors activation and connexin-43
Emiliano Diez1,3, Tamara Beova2, Natalia Prado3, Boris Liptk4, Vladimr Knezl4, Roberto Miatello1,3, Barbara Baov2, Narcisa Tribulov2
1

Instituto de Fisiologa, Facultad de Ciencias Mdicas, Universidad Nacional

de Cuyo, Mendoza, Argentina
2
Institute for Heart Research, Slovak Academy of Sciences, Bratislava,
Slovakia
3
Instituto de Medicina y Biologa Experimental de Cuyo, CONICET, Mendoza, Argentina
4
Institute of Experimental Pharmacology and Toxicology, Slovak Academy
of Sciences, Bratislava, Slovakia

Department of Biology, Faculty of Medicine, Masaryk University, Brno,

Czech Republic
2
Cardiology Division, Cardiac Electrophysiology Section, American University of Beirut Medical Center, Beirut, Lebanon
3
PhyMedExp, University of Montpellier, INSERM U1046, CNRS UMR9214,
Montpellier, France
4
CEITEC, Masaryk University, Brno, Czech Republic
5
University of California, San Francisco Medical Center, San Francisco, CA,
USA
6
ICRC, St. Annes University Hospital, Brno, Czech Republic
Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a
highly lethal inherited arrhythmogenic disorder predominantly caused
by mutations in cardiac ryanodine receptor gene (RYR2). Human induced pluripotent stem cells (hiPSCs) offer a unique opportunity for disease modeling.
Aims: The goals were to derive functional cardiomyocytes (CMs)
from CPVT patient via hiPSCs and test whether the novel CPVT1 mutation is associated with abnormal intracellular Ca2+ handling properties
in CMs.
Methods: Human iPSCs were generated from dermal broblasts
from a young athletic female diagnosed CPVT and carrying a novel heterozygous point mutation RyR2-D3638A. Following molecular characterization, healthy control (HC)- and CPVT-hiPSCs were differentiated
into CMs. Using confocal microscopy and atomic force microscopy,
their intracellular Ca2 + handling and mechano-biological properties
were studied in resting and stress conditions.
Results: HC- and CPVT-hiPSCs expressed pluripotency
markers (OCT4, NANOG, SSEA4) and had normal karyotype. Derived CMs via embryoid body (EB) formation showed typical cardiac markers such as cardiac troponin T and I, and -actinin. At
rest, there was no signicant difference in any property of the
spontaneous Ca2 + transients between HC- and CPVT-hiPSC CMs
while CPVT-EBs exhibit higher beat rate. Signicant differences
in the kinetic properties of Ca 2 + transients as well as in the

Background: Hypokalemia, a common electrolyte abnormality in

clinical practice, enhances the propensity for ventricular brillation
(VF). Melatonin up-regulates the gap junction channels protein,
connexin-43 (Cx43), rendering the heart more resistant to electricallyinduced VF. We hypothesized that melatonin may protect against low
potassium induced VF in part by affecting Cx43 through the activation
of its membrane receptors.
Methods: Isolated rat hearts underwent 10 min of KrebsHenseleit perfusion (4.5 mEq/L K +) followed by K +-decient (1
mEq/L) perfusion in presence of 100 M melatonin, a melatonin
receptor blocker (luzindole 5 M), luzindole + melatonin or the
vehicle of the drugs. Low K + perfusion was maintained 25 min
unless VF occurred earlier. Two min VF was followed by normokalemic perfusion aimed to restore sinus rhythm. Incidence of arrhythmias and heart function were registered and analyzed using
BiolabF software. Ventricular tissue analysis was performed for
Cx43 expression and distribution.
Results: Melatonin was the only treatment that reduced the incidence of low K +-induced VF from 100% (vehicle 15/15;
luzindole + melatonin 10/10; and luzindole 8/8) to 69% (9/13)
(P = 0.0349 vs vehicle by Fisher test) and delayed the occurrence of
VF to 12 min (9-25 IQR) from 7 min (5-12 IQR) in vehicle group
(P = 0.041). Luzindole and luzindole + melatonina developed VF at a
median of 6 min (4-11 IQR) and 8 min (6-14 IQR), respectively. resulted
in a faster recovery of sinus rhythm restitution (P=0.047). Melatonin,
luzindole or luzindole+melatonin did not affect heart rate, PR and QT
intervals as well as the incidence of transient arrhythmias. The levels
of total Cx43 was not changed by any treatment, however, melatonin
prevented dephosphorylation and abnormal topology (lateralization)
of Cx43.
Conclusions: Our results suggest that acute treatment with melatonin protects against low potassium induced VF in part due to prevention
of abnormal expression and distribution of myocardial Cx43 mediated
by melatonin receptors activation.

S78

Abstracts

TH-074
Restoration of p21-activated Kinase Activity Attenuates Atrial Arrhythmia in a Dog Model of Atrial Fibrillation
Jaime DeSantiago1, Dan J. Bare1, R. John Solaro2, Rishi Arora3, Kathrin
Banach1
1

Rush University, Chicago, IL, USA

University of Illinois at Chicago, Chicago, IL, USA
3
Northwestern University, Chicago, IL, USA
2

Introduction: The mechanisms underlying the genesis of atrial brillation (AF) are not fully understood. Activation of the small GTPase
Rac1 through production of reactive oxygen species (ROS) is believed
to contribute to the development of an AF substrate. We identied
Pak1 as an endogenous negative regulator of Rac1 and hypothesized
that stimulation of Pak1 activity attenuates atrial arrhythmia by antagonizing ROS induced changes in Ca handling.
Method: Tissue and isolated myocytes (left superior pulmonary
vein, LSPV) were obtained from dogs with persistent AF (atrial
tachypacing, 600bpm, 3 weeks) or sinus rhythm (SR) and changes in
Pak1 expression were quantied by western blotting. Changes in [Ca]i
(uo-4/AM) or ROS (uorescent2',7'-dichlorouorescein, DCF) were
monitored by confocal microscopy in isolated atrial myocytes (AMs).
AMs from WT and Pak1-/- mice were used to determine the mechanism
by which a decrease in Pak1 enhances arrhythmic activity.
Results: For the rst time we demonstrate that Pak1 is down regulated in the atria of the canine AF model (adjusted density: SR:
85.6 7.2 % vs. AF 50 8.1%, n = 3; p b 0.05) and that this decrease is
mimicked in an in vitro AF model (HL-1 + AngII(24h); Ctrl:
109 5.7% vs. AngII: 78.96.6%; pb 0.05). ECGs in conscious mice revealed increased atrial arrhythmic events in Pak1-/- mice and an increased number of delayed after depolarizations during Ctrl and AngII
stimulation in isolated Pak1-/- AMs. On a cellular level Rac1 stimulation
by AngII (1microM) induced exaggerated ROS production in Pak1-/AMs (DCF(au): WTAngII: 4572 487, n = 20 vs. Pak1-/-AngII:
11231838, n=16, pb 0.05) and an enhanced increase in [Ca]i (F/F0:
WTAngII: 3.4 0.4 n = 6 vs. Pak1-/-AngII: 4.10.4 n = 9, p b0.05). In
isolated WT AMs the AngII induced increase in ROS and DADs were attenuated by stimulation of Pak1 activity with the sphingosine 1 phosphate receptor agonist FTY720 (200 nM) and in canine AMAFs
prevented the AngII induced increase in DADs that was based on spontaneous Ca release.
Conclusion: In AF ROS production is enhanced by down-regulation
of Pak1, an endogenous negative regulator of Rac1. Restoring Pak1 activity could be a therapeutic strategy to attenuate ROS induced arrhythmia and remodeling.

TH-076
The Mitochondrial Calcium Uniporter is a therapeutic target in the
hypoxia/reoxygenation injury
Yuriana Oropeza-Almazn1,2, Christian Silva-Platas1,2, Keith A. Youker3,
Guillermo Torre-Amione1,3, Gerardo Garca-Rivas1,2
1

Ctedra de Cardiologa y Medicina Vascular, Escuela de MedicinaTecnolgico de Monterrey, Monterrey, Nuevo Len, Mexico
2
Centro de Investigacin Biomdica-Hospital Zambrano Hellion,
Tecnolgico de Monterrey, San Pedro Garza Garca, Nuevo Len, Mexico
3
Methodist DeBakey Heart & Vascular Center, The Methodist Hospital,
Houston, Texas, USA
Introduction: The alteration of the intracellular Ca2+ homeostasis
and energy production are important pathogenic mechanisms in HF.
These mechanisms are leading by mitochondrial Ca2+ overload carried
out by the MCU. For a long time, the molecular characterization of the

MCU was limited, but this situation changed recently and allows to reveal in greater detail their involvement in the pathophysiology of
myocardiac diseases.
Methods: Specic siRNA targeting MCU was used to transiently silence the MCU expression in cardiac myoblast. The MCU mRNA expression was measured using qRT- PCR and the protein levels of the MCU
and its regulatory proteins were determined by W. blot analysis. Later,
MCU silenced cells was exposed to hypoxia/reoxygenation protocol. Necrosis, apoptosis, m and mPTP were determined by ow cytometry
and confocal microscopy. In addition, MCU, MICU1 expression was measured from samples of human HF-LV tissues at the time of heart
orthotopic transplantation or the LVAD insertion in patients with HF.
Results: MCU expression decreased by 65% with a consequent decrease in mitochondrial Ca2+ transport. MCU silencing effects reduced
the hypoxia/reoxygenation injury in myoblasts decreasing necrosis
and apoptosis by 30 and 20%, respectively vs control after 3 hours of reoxygenation, with a reduction in caspases 3, 7 activity. In the human tissue, MCU expression was signicantly elevated in HF compared with
non-failing left ventricular samples. In addition, the mitochondrial protein MICU1 witch interacts with the uniporter pore-forming subunit
MCU was 2-fold over-expressed.
Conclusions: The hypoxia/reoxygenation injury reduction suggest
that MCU has a main role in post-ischemic cardiac dysfunction.
Morover, the overexpression of MCU and MICU1 could be mediated mitochondrial calcium overload and cardiac dysfunction in HF. Overall, the
pharmacological inhibition of MCU or MCU knockdown could be a therapeutic approach used to prevent calcium overload, which induces injury in several pathologies such ischemia/reperfusion, cardiac
arrhythmias and HF.

TH-077
Carbonic anhydrase inhibition by benzolamide attenuates myocardial ischemia/reperfusion injury via p38MAPK-dependent
mechanism
Alejandro Ciocci Pardo, Luisa F Gonzlez Arbelez, Juliana C Fantinelli,
Romina G Diaz, Bernardo Alvarez, Susana M Mosca
Dr Horacio E Cingolani Cardiovascular Research Center, National University
of La Plata., La Plata, Argentina
Carbonic anhydrase (CA) catalyze the hydration of CO2 to H+ and
HCO-3. During ischemia-reperfusion CO3H--dependent transporters participate of the intracellular pH (pHi) regulation, leading to Ca2+ overload. The involvement of CA in reperfusion injury has not been
elucidated yet. Isolated rat hearts were submitted after 20-min stabilization to the following protocols: 1.-Ischemic control (IC): 30 min of
global ischemia (GI) and 60 min of reperfusion (R); 2.- BZ: the CA inhibitor benzolamide (5 M) was administered during the initial 10 min of
R. To examine the participation of p38MAPK, SB202190 (10 M) was
perfused simultaneously to BZ. Infarct size (IS) was measured by TTC
staining technique. Left ventricular developed pressure (LVDP), + dP/
dtmax, left ventricular end diastolic pressure (LVEDP) and -dP/dtmax
served to assess myocardial function. The p38MAPK expression was
measured. The changes of pHi in papillary muscle by immunouorescence were also determined. BZ decreased the IS (6.3 0.6 % vs 32
2 %, p b 0.05) and improved postischemic recovery of myocardial function. At the end of R LVDP was 69 4 % vs. 15 4 %; +dP/dtmax: 75
5 % vs. 19 5 %; LVEDP: 23 3 vs. 52 5 mmHg; -dP/dtmax: 72 5 %
vs. 17 5 %, p b 0.05). The p38MAPK level increased after BZ treatment
(189 3 % vs. 53 1 %, p b 0.05). BZ annulled pHi recovery from
sustained intracellular acidosis (JH+ at pHi 6.8 in control was 0.102
0.004 mmol/L x min-1). SB attenuated all the effects detected by BZ.
The present data demonstrate that CA inhibition by BZ protects the
heart against reperfusion injury through a p38MAPK-dependent

Abstracts

pathway and suggest that an attenuation of Ca2+ overload could be the

responsible mechanism.

TH-078
Phospholamban ablation rescues reperfusin arrhythmias in hearts
with Ca/calmodulin kinase II constitutive phosphorylation of
ryanodine receptors, but not myocardium infarction
Gabriela Mazzocchi1, Mariano Di Carlo1, Carlos Valverde1, Evangelia
Kranias2, Xander Wehrens3, Alicia Mattiazzi1
1

Centro de Investigaciones Cardiovasculares. Fac Cs Mdicas. UNLP, La

Plata, Argentina
2
Department of Pharmacology and Cell Biophysics, Cincinnati, Ohio, USA
3
Departments of Molecular Physiology and Biophysics, Medicine (in Cardiology), and Pediatrics, Baylor College of Medicine, Cardiovascular Research
Q3 Q4 Institute, Houston, USA
CaMKII-dependent phosphorylation of ryanodine receptors (RyR2)
at the onset of reperfusion has been previously associated with an increase in cardiac damage and Ca-triggered arrhythmias (Di Carlo et al.,
2014, Said et al., 2011). However, whether increasing SR Ca2+ uptake/
load would also protect against cardiac damage and Ca2+-triggered arrhythmias or exacerbate them, is unknown and difcult to predict, since
the decrease in SR Ca2+ uptake was associated with a decrease in cytosolic Ca2+ but produced an increase in SR Ca2+ leak.
Hypothesis: Increasing SR-Ca2+ uptake by ablation of phospholamban (PLN) rescues reperfusion arrhythmias but fails to protect against
cardiac damage in a mice model with constitutive CaMKII pseudophosphorylation of RyR2 (S2814D mice), linked to reperfusion arrhythmias and exacerbated infarct size. We developed SDKO mice by crossbreeding PLNKO with S2814D mice. At baseline, S2814D and SDKO
mice have structurally normal hearts without arrhythmias. However,
after a period of global ischemia (15 minutes) only S2814D mice developed ectopic beats (6/7 vs. 1/7 in SDKO mice, P b0.05). In contrast,
hearts from SDKO exacerbate infarct size (23.2 0.9 % of risk area,
n=5) after a short ischemic period (15 min), not only with respect to
S2814D hearts (10.8 2.2%, n = 5), but also when compared to
PLNKO hearts (14.3 2.0 %, n=6).
Conclusions: Improving SR Ca2+ uptake by PLN ablation prevents
the arrhythmic events triggered by CaMKII-dependent increase in SR
Ca2+ leak but exacerbates cardiac damage. The results underscore the
benets of increasing SERCA2a activity on reperfusion arrhythmias but
reveal a detrimental effect of increasing SR Ca2 + uptake on cardiac
injury.
*similar contribution to the present work.

TH-079
The use of synthetic wine to delineate the cardioprotective components in red wine
Sandrine Lecour
1

University of Cape Town, Cape Town, South Africa

University of Stellenbosch, Stellenbosch, South Africa

Background: Moderate and chronic consumption of red wine protects against cardiovascular disease. Wine is a complex matrix containing multiple molecules whose concentrations can vary from one bottle
to another. Therefore, the delineation of the cardioprotective components in wine such as alcohol, resveratrol and melatonin is very challenging when using commercially available red wine.
Aim: We used synthetic wine whose composition is well characterized to explore whether the presence of alcohol, resveratrol and melatonin (as found in commercial wine) contributes to the cardioprotective

S79

effect of chronic moderate ( 2 glasses wine/day) consumption of red

wine.
Methods: The drinking water of male Long Evans rats was supplemented with synthetic wine (12% v/v) with/without resveratrol
(100g/L) and/or melatonin (0.075g/L) to a nal concentration corresponding to the concentration found in 2 glasses of wine per day.
After 14 days of treatment, hearts were perfused on the Langendorff
system and subjected to 30 minutes global ischemia (I) followed by
60 minutes of reperfusion (R). Functional parameters were recorded
throughout the experiments and infarct size was measured at the end
of the protocol. Functional recovery (heart rate x left ventricular developed pressure) was expressed as a percentage of baseline value.
Results: Control hearts subjected to I/R presented a functional recovery of 11 2%. Pre-treatment with synthetic wine with/without
melatonin or resveratrol did not improve functional recovery
(156%,121%, 194% respectively, n.s. vs control). However, addition of both melatonin and resveratrol in synthetic wine improved functional recovery to 325% (pb0.01 vs control). Additionally, synthetic
wine enriched with both melatonin and resveratrol signicantly reduced the plasma total antioxidant capacity compared synthetic wine
only
(p b0.01,
Trolox
equivalent:
1.1 2.9mol/mL
vs.
15.23.6mol/mL).
Conclusion: In conclusion, our data strongly suggest that the presence of melatonin and resveratrol in wine is required for
cardioprotection with chronic moderate consumption of wine.

TH-080
Simulated ischemia does not mimic stop ow ischemia in perfused
mouse hearts
Nehun Salas1, Yuriana Aguilar Sanchez2, Alicia Mattiazzi1, Ariel
Escobar2, Carlos Valverde1
1

Centro de Investigaciones Cardiovasculares Dr. Horacio E. Cingolani, La

Plata, Buenos Aires, Argentina
2
School of Engineering and of Natural Sciences, University of California,
Merced, CA, USA
Introduction: Cardiac ischemia is a pathological condition in which
the blood supply to the myocardium is interrupted. This loss of circulation leads to the impairment of cardiac mechanical and electrical function. Still, the role of Ca2 + underlying these dysfunctions is not fully
understood. To identify the ionic alterations that occurred during ischemia, several laboratories appealed to the use of ischemia-like conditions
(hypoxia-metabolic inhibition-acidication), in isolated myocytes.
However, whether simulated ischemia (SI) actually mimics stop-ow
ischemia (SFI) at the cellular level, has not been previously explored
and is the aim of the present work.
Methods: Hearts from Balb/c mice were perfused (Langendorff technique), at constant ow/temperature. Left-ventricle developed pressure, LVDP, and LV-end-diastolic pressure, LVEDP, were assessed with
a latex balloon connected to a pressure transducer. Cytosolic Ca2 +
was assessed in Rhod-2-loaded hearts in a pulsed-local-eld uorescence microscope. Action potentials (AP) were registered with microelectrodes. Phosphorylation of phospholamban (PLN), known to occur
at the onset of reperfusion, was assessed by western blot. Hearts were
submitted to 15min of either SFI or SI (pH 6.2, absence of glucose, N2 instead of O2).
Results: SI produced a milder mechanical dysfunction than SFI. Similarly, Ca2+ transient and AP amplitude were lower during ischemia in
SFI than in SI. Upon reperfusion, the mechanical recovery of LVDP was
signicantly more pronounced in SI than in FR-hearts (LVDP:
48.5 6.2 vs. 11.9 6.6% of preischemic value; LVEDP: 29.0 5.5 vs.
56.6 3.1mmHg, respectively), whereas PLN phosphorylation by
CaMKII at early reperfusion was higher in SFI than in SI hearts

S80

Abstracts

(812 46 vs. 155 26%). No changes were observed in PLN-Ser16phosphorylation in any of these conditions.
Conclusion: SI generates a milder alteration of mechanical
and Ca2 + handling when compared to SFI. The ndings indicate
that SI results have to be interpreted with caution and underscore the use of SFI to assess intracellular Ca2 + during ischemia/reperfusion.

TH-081
Reversible redox modications of ryanodine receptor ameliorate
ventricular arrhythmias in the ischemic-reperfused heart
Romina Becerra1, Brbara Romn1, Mariano N Di Carlo1, Juan IE
Mariangelo1, Margarita Salas1, Gina Sanchez2, Paulina Donoso3,
Guillermo Schinella4, Leticia Vittone1, Xander H Wehrens5, Cecilia
Mundia-Weilenmann1, Matilde Said1
1

Centro de Investigaciones Cardiovasculares, CCT-CONICET La Plata,

Facultad de Ciencias Mdicas, UNLP, La Plata, Argentina
2
Programa de Fisiopatologa, Instituto de Ciencias Biomdicas, Facultad de
Medicina, Universidad de Chile, Santiago de Chile, Chile
3
Programa de Fisiologa y Biofsica, Instituto de Ciencias Biomdicas,
Facultad de Medicina, Universidad de Chile, Santiago de Chile, Chile
4
Facultad de Ciencias Mdicas, Universidad Nacional de La Plata, CIC-PBA,
La Plata, Argentina
5
Cardiovascular Research Institute, Department of Molecular Physiology
and Biophysics, Department of Medicine (in Cardiology), Pediatrics, Baylor
College of Medicine, Houston, USA
Background: Previous results from our laboratory showed that
phosphorylation of ryanodine receptor-2 (RyR2) by Cacalmodulin-dependent kinase II (CaMKII) was critical but not the
unique event responsible for ventricular arrhythmias following ischemia/reperfusion (I/R). Oxidative stress is a prominent feature
of I/R injury and both, CaMKII and RyR2 are subject to oxidation.
Aim: The present study was designed to elucidate the contribution
of redox changes in CaMKII and RyR2 on reperfusion arrhythmias.
Methods: Isolated perfused rat hearts were subjected to I/R (20/
30min) in the presence or absence of inhibitors of NADPH oxidase
(Apocynin) and nitric oxide synthase (L-Name) and a free radical scavenger (MPG). Contractile and electrical parameters were recorded to
detect ventricular premature beats (VPBs). CaMKII oxidation and Snitrosylation, S-glutathionylation and free thiol levels (mBB) of RyR2
were assessed together with glutathione content (GSH) and ROS production (DHE).
Results: CaMKII was oxidized in early reperfusion but this modication had no consequences in enzyme activity or RyR2 phosphorylation.
In addition, I/R induced an increase in the reversible RyR2 oxidations: Sglutathionylation and S-nitrosylation. Accordingly, free thiols on RyR2
decreased. Pre-treatment with Apocynin and L-Name selectively
abolished S-glutathionylation and S-nitrosylation of RyR2, respectively,
and increased VPBs in I/R: (I/R: 4414, Apocynin: 586 and L-Name:
6416 VPBs, pb0.05). Conversely, MPG diminished VPBs (245) with
the simultaneous prevention of both reversible RyR2 oxidations. The
discrepancy in the effects of the drugs could be explained by their differential ability to inuence the nitroso/redox balance: only MPG was effective to preclude the I/R-induced reduction in GSH levels. This result
was conrmed by the decreased I/R-induced ROS production and the
recovery of RyR2 free thiol level towards pre-ischemic values elicited
by the scavenger.
Conclusions: Oxidation of RyR2 contributes to arrhythmogenesis in
I/R. The selective suppression of S-glutathionylation and S-nitrosylation
of RyR2 in an oxidative environment allowed us to unmask a protective
role of these redox alterations which counterbalanced proarrhythmogenic oxidations of RyR2.

TH-082
Extracellular HSP27 and TLR4 exaggerate functional injury in aging
hearts following ischemia
Lihua Ao, Yufeng Zhai, Joseph Cleveland, David Fullerton, Xianzhong
Meng
University of Colorado Denver, Aurora, Colorado, USA
Background: While cardiac functional recovery is poor in the elderly
following cardiac surgery with obligatory global myocardial ischemia/
reperfusion (I/R), the underlying mechanism remains incompletely understood. We found recently that human and mouse myocardium releases HSP27 during global I/R, and extracellular HSP27 plays a role in
post-ischemic inammatory response in adult mouse hearts.
Objectives: The aim of this study was to determine the role of extracellular heat shock protein (HSP) 27 and Toll-like receptors (TLRs) in cytokine
production and functional injury caused by global I/R in aging hearts.
Methods and results: Isolated hearts from aging (18-24 months) and
adult (4-6 months) mice were perfused by the Langendorff system and
subjected to global normothermic I/R (20 min/120 min). Augmented release of HSP27 in aging hearts preceded greater production of cytokines
(MCP-1, KC, IL-6 and TNF-alpha) and worse functional recovery. AntiHSP27 suppressed the inammatory response and markedly improved
functional recovery in aging hearts. Perfusion of recombinant HSP27 to
aging hearts resulted in greater cytokine production and contractile depression. TLR2 KO and TLR4 deciency, particularly the latter, markedly
reduced cytokine production and contractile dysfunction in aging hearts
exposed to recombinant HSP27. Interestingly, aging hearts displayed enhanced NF-kappaB activation following TLR4 stimulation.
Conclusion: Enhanced myocardial inammatory response to global
I/R in aging mouse hearts is due, at least in part, to augmented myocardial release of HSP27. Extracellular HSP27 up-regulates myocardial cytokine production and depresses cardiac contractility through TLR2
and TLR4. Augmented HSP27 release and enhanced myocardial TLR4 responsiveness jointly play an important role in the greater inammatory
response and worse post-ischemic functional recovery in aging hearts.

TH-083
Non-nuclear estrogen receptor activation reduces cardiac ischemicreperfusion injury in mice with cardiac specic ablation of ER-alpha
Sara Menazza1, Swathi Appachi1, Junhui Sun1, John Katzenellenbogen2,
Benita Katzenellenbogen3, Philip W. Shaul4, Elizabeth Murphy1
1

Systems Biology Center, National Heart Lung and Blood Institute, NIH, Bethesda, MD, USA
2
Department of Molecular and Integrative Physiology, Univ. of Illinois at
Urbana-Champaign, Urbana, IL, USA
3
Department of Chemistry, Univ. of Illinois at Urbana-Champaign, Urbana,
IL, USA
4
Department of Pediatrics, UT Southwestern Medical Center, Dallas, TX, USA
Introduction: Steroid hormone receptors, ER and ER, function as
regulated transcription factors. However, recent data indicate that estrogen can also elicit effects through binding to estrogen receptors (ER,
ER and GPR30) at the plasma membrane and initiate kinase signaling.
We investigated the hypothesis that non-nuclear ER activation reduces
cardiac ischemia-reperfusion injury in mice. We also addressed the role
of cardiac ER signaling using a cardiac-specic ER knock out mouse.
Results: We treated ovariectomized wild type mice with estrogen or
an estrogen-dendrimer conjugate (EDC), which has been demonstrated
in mice to be a non-nuclear selective ER modulator, or dendrimer control for two weeks. Ischemia-reperfusion injury was evaluated in isolated Langendorff perfused hearts. Two weeks of treatment with estradiol
signicantly decreased infarct size and improved post-ischemic

Abstracts

contractile dysfunction (40.4 2.5% vs. 62.9 5.8% for infarct and
44.74.0% vs. 27.02.7% for post-ischemic functional recovery). Similarly, EDC treatment signicantly decreased infarct size (40.93.6% for
EDC vs 63.84.7% vehicle) and increased post-ischemic functional recovery (48.83.0% EDC vs. 28.6 2.5% vehicle) compared to vehicletreated hearts. To test if ER was involved in cardioprotection, we generated cardiac-specic ER knockout mice. In these mice, EDC treatment signicantly decreased infarct size (20.1 1.9% vs. 51.2 7.8%
dendrimer) and improved functional recovery (65.8 4.2% vs.
36.8 5.2% dendrimer) compared to vehicle-treated ER knockout
mice. Interestingly, EDC protection was signicantly higher in ER
knockout compare to the wild-type hearts. Moreover, treatment with
a ICI 182 780, a selective inhibitor of ER and ER and an activator for
GPR30 signicantly blocked the EDC mediated cardioprotection.
Conclusion: These results indicate that EDC is effective in providing
cardioprotection during ischemia-reperfusion injury in mice, by a mechanism that does not require cardiac ER or GPR30. Thus, EDC could be
utilized clinically to provide cardiovascular benet without the classical
steroid hormone side effects, such as uterine and breast cancer.

TH-084
Xenon administration at reperfusion protects against myocardial
infarction in the in vivo mouse heart: insight into the mechanism
Tiziana Rosa1, Marleen Forkink1, Victoria Pell1, Michael P Murphy2,
Thomas Krieg1
1

University of Cambridge, Cambridge, Cambridgeshire, UK

Medical Research Council Mitochondrial Biology Unit, Cambridge, Cambridgeshire, UK

S81

unclear. This study was to investigate the effects of the articial synthetic natriuretic peptide vasonatrin peptide (VNP) on MI/R injury in diabetic rats, and underlying mechanisms.
Methods: The high-fat diet-fed streptozotocin (HFD-STZ) induced
diabetic rats were subjected to MI/R (30 min/4 h) and VNP treatment
(100 g/kg, i.v., 10 min before R). In vitro study was performed using
H9c2 cardiomyocytes subjected to hypoxia/reoxygenation (H/R, 3 h/6
h) and incubated with or without VNP (10-8mol/L).
Results: The diabetic state aggravated MI/R injury and showed more
severe myocardial functional impairment than normal state. VNP treatment (100 g/kg, i.v., 10 min before R) signicantly improved LV dP/
dtmax and LVSP, and decreased infarct size, apoptosis index, caspase-3
activity, serum CK and LDH levels (n = 8, P b 0.05). Moreover, VNP
inhibited endoplasmic reticulum (ER) stress by suppressing GRP78
and CHOP, and consequently increased Akt and ERK1/2 expression
and phosphorylation levels (n = 3, P b0.05). These effects were mimicked by 8-Br-cGMP (1 mg/kg, i.p., 20 min before R), a cGMP analogue,
whereas inhibited by KT-5823 (0.5 mg/kg, i.p.), the selective inhibitor
of PKG (Pb0.05). Pretreated DM rats with TUDCA (50 mg/kg, i.p.), an inhibitor of ER stress, couldnt further promote the VNPs cardioprotective
effect. Additionally, gene knockdown of PKG1 with siRNA blunted
VNPs inhibition of ER stress and apoptosis, while overexpression of
PKG1 resulted in signicantly decreased ER stress and apoptosis in
H/R H9c2 cardiomyocytes (n=6, Pb 0.05).
Conclusions: We demonstrated that VNP protects diabetic heart against
MI/R injury by inhibiting ER stress via cGMP-PKG signaling pathway.
Keywords: Natriuretic peptide; Diabetes; Myocardial ischemia/reperfusion; Vasonatrin peptide

Xenon is a noble gas with favourable physical, chemical, and pharmacological properties to serve as an ideal anaesthetic. In previous studies, it was demonstrated that volatile anaesthetics offer specic
protection against myocardial reperfusion injury. We investigated
whether xenon, administered at the onset of reperfusion, protects the
mouse heart from reperfusion injury in vivo. Moreover, as its mechanisms of action are still uncertain, we are exploring whether xenon protection works by preventing ROS production.
C57BL6/J male mice (8-10 weeks) were subjected to 30 min occlusion of the left anterior coronary artery followed by 120 min reperfusion.
During the last 15 min of ischaemia and the rst 10 min of reperfusion,
mice were treated with inhaled 70% xenon/30% oxygen, whilst control
mice inhaled 70% nitrogen/30% oxygen. Infarct size was determined at
the end of the reperfusion period by using triphenyltetrazolium chloride
staining. Xenon reduced infarct size from 40.8% 3.3% of the area at risk
in controls to 27% 1.5% (**pb 0.01). Further work is being carried out
to assess changes in the levels of hydrogen peroxide within mitochondria by using the well-established hydrogen peroxide probe, MitoB,
in vivo and Amplex Red assay in vitro. The effect of xenon on the activity
of mitochondrial Complex I in vitro is also being examined.

TH-085
PKG-dependent inhibition of endoplasmic reticulum stress contributes to protective effects of vasonatrin peptide against myocardial
ischemia/reperfusion injury in diabetic rats
Wenjuan Xing, Qianqian Dong, Haifeng Zhang
Fourth Military Medical University, Xi'an, China
Aims: Diabetes mellitus (DM) increases morbidity/mortality of ischemic heart disease. Although the ability of the natriuretic peptides
to modulate cardiac function and cell proliferation has been recognized,
their effects on myocardial ischemia/reperfusion (MI/R) injury is still

TH-086
Ischaemic preconditioning protects the heart against ischaemiareperfusion injury without affecting ischaemic succinate accumulation and metabolism
Victoria Pell1, Ana S.H Costa2, Angela Logan3, Tiziana Rosa1, John
Mulvey1, Christian Frezza2, Michael Murphy3, Thomas Krieg1
1

Department of Medicine, University of Cambridge, Cambridge, UK

MRC Cancer Unit, Cambridge, UK
3
MRC Mitochondrial Biology Unit, Cambridge, UK
2

Ischaemia-reperfusion (IR) injury occurs when blood supply to an

organ is disrupted and then restored, and underlies many disorders, notably myocardial infarction and stroke. While reperfusion of ischaemic tissue is essential for survival, it also initiates cell death through
generation of mitochondrial reactive oxygen species (ROS). Recent work
has revealed a novel pathway underlying ROS production at reperfusion
in vivo in which the accumulation of succinate during ischaemia and its
subsequent rapid oxidation at reperfusion drives ROS production at complex I by reverse electron transport (RET). Pharmacologically inhibiting
ischaemic succinate accumulation or slowing succinate metabolism at reperfusion has been shown to be cardioprotective against IR injury. Here,
we aimed to establish if ischaemic preconditioning (IPC), as part of its
cardioprotective mechanism, acts via manipulating succinate kinetics
during IR in an in vivo mouse model. Mice were subjected to 30 min occlusion of the left anterior descending coronary artery followed by 1 min reperfusion with or without an IPC protocol of 3 cycles of 5 min ischaemia,
5 min reperfusion prior to sustained ischaemia. The left ventricle was then
rapidly isolated and analysed by mass spectrometry based-metabolomics
to determine the effect of IPC on myocardial succinate levels. Data revealed that IPC had no effect on ischaemic succinate accumulation with
both control and IPC mice having increases in succinate of 2.79 and 2.54
fold respectively compared to normoxia. Analysis of hearts after only
1 min reperfusion revealed that succinate was rapidly metabolised
returning to near pre-ischaemic levels. IPC had no signicant effect on
succinate metabolism at reperfusion. These ndings suggest that IPC

S82

Abstracts

does not affect ischaemic succinate accumulation or its metabolism at reperfusion. Further work is being carried out to determine if IPC affects
RET-mediated ROS production downstream of succinate by inhibiting
the re-activation of complex I at reperfusion.
TH-087
Hypothyroidism reduces cardiac stunning with a mitochondrial regulation of sarcoreticular Ca2+ leak: a mechano-energetical study
Mara Ins Ragone1,2, Mara Lara Lazarte1, Alicia E. Consolini1
1

Universidad Nacional de La Plata, Facultad de Ciencias Exactas, Depto de

Ciencias Biolgicas, Farmacologa, La Plata, Argentina
2
Consejo Nacional de Investigaciones Cientcas y Tcnicas (CONICET), La
Plata, Argentina

Hypothyroidism (HypoT) may be a cardiac risk for angor. In this work,

its inuence on the energetics during cardiac stunning by ischemiareperfusion (I/R) was studied in rats. HypoT was induced by drinking
methimazole (0.02%) for 15 days. Isolated hearts were perfused inside a calorimeter at 37C to measure left ventricular pressure (LVP, in mmHg) and
total heat ow (Ht, in mW) during exposition to 20 minutes I and 45 minutes R. Protocols were done in HypoT and euthyroid (EuT) rats. HypoT improved the postischemic contractile recovery (PICR) to 925% vs 696%
in EuT (pb0.05) and reduced diastolic contracture (LVEDP) without
changing muscle economy (P/Ht). When ischemic hearts were reperfused
with Krebs-36 mM Na+-caffeine 10 mM (to release SR Ca2+ minimizing
the NCX efux) the initial rise of contracture was decreased in HypoT
(+27.31.6) vs EuT (44.14.8 mmHg) without changing the area
under curves (AUC-LVP and AUC-Ht). When hearts were pretreated
with 10 M clonazepam (Clzp, mNCX inhibitor), PICR and P/Ht were reduced to 36.76.4% and 38.47.4% respectively, and LVEDP was increased to 8615 mmHg in HypoT. Contrarily, Clzp cardioprotected EuT
hearts. Cyclosporine A (Cys-A, 0.2 M) slightly improved PICR and P/Ht
but increased LVEDP in HypoT hearts pretreated with Clzp.
Isolated cardiomyocytes loaded with Fluo-4 (free cytosolic Ca2+) or
with Rhod-2 (free mitochondrial Ca2+) were exposed to Krebs-36 mM
Na+-caffeine 10 mM with or without Clzp. F/Fo-Fluo-4 increased and
fell more quickly in HypoT than EuT cells. F/Fo-Rhod-2 increased and
decreased more quickly in HypoT than in EuT, and Clzp raised it.
Results suggest that: a) HypoT reduced the stunning, b) mitochondria
were more sensitive in HypoT than in EuT, contributing through the mNCX,
to regulate the SR store and leak to cytosol, c) blocking the mNCX in HypoT
becomes in contractile dysfunction (not in EuT) with mPTP opening.

TH-088
The way of administration makes a difference in the effects of genistein on cardiac stunning: mechano-energetical study
Germn A. Colareda, Alicia E. Consolini
Universidad Nacional de La Plata, Facultad de Ciencias Exactas, Depto de
Ciencias Biolgicas, Farmacologa, La Plata, Argentina
Although genistein (Gen) could prevent cardiovascular diseases, its effects on cardiac ischemia are contradictory. A previous work showed sex
and temperature-dependence on Gen effects, participating the inhibition
of tyrosin-kinases (TK), blockade of Ca2+ inux and mitochondrial uptake, and increase of SERCA activity (Colareda et al. 2016). Now, we compared the effects of administering 5 mg/kg Gen via IP 24 h before the
experiment (Gen-IP), with those of perfusing 20 M Gen before stunning
(Gen-BS). Two models of stunning were assessed: no-ow ischemia/reperfusion (I/R) and hypoperfusion/reperfusion (Hip/R). In both cases, isolated rat hearts were perfused at 6 ml/min inside a calorimeter at 37C to
measure left ventricular pressure (LVP, in mmHg) and total heat ow (Ht,
in mW) throughout the experiment.

During Hip/R Gen-BS did not change the post-ischemic contractile

recovery (PICR, 23.5 8.3%), reduced total muscle economy (P/Ht)
and increased the diastolic contracture (LVEDP). Contrarily, in male
rats Gen-IP improved PICR (86.412.6%) and P/Ht, but also increased
LVEDP during Hip/R. Perfusing 10 M clonazepam (Clzp, mNCX inhibitor) improved PICR (55.511.1%) and P/Ht during Hip, and reduced
LVEDP. Nevertheless, addition of Gen to Clzp before Hip/R reduced
PICR (19.92.7%) and increased LVEDP (+41.48.4 mmHg during
R). Ciclosporine-A (Cys-A) reduced this dysfunction except the diastolic
contracture, suggesting that Clzp+ Gen induced a Ca2+ overload that
triggered the mPTP activation.
In the severe stunning by I/R, administration of Gen-IP also improved PICR (50,8 4,9%) and P/Ht, more in male than in female rat
hearts, although it increased LVEDP in both. These effects were not
modied by ortho-vanadate 15 mg/kg.
Results suggest that there is an agonistic interaction between Gen
and endogenous estrogen on receptors: Gen is more cardioprotective
in males, which had low exposition to estradiol, than in females. The inhibition of TK dont participate in the in vivo cardioprotection, but Gen
increased the SR leak.

TH-089
Depression And Risk Of Cardiovascular Diseases In Men Aged 25-64
Years: Who Program Monica Psychosocial
Valery Gafarov1,2, Elena Gromova1,2, Dmitriy Panov1,2, Igor Gagulin1,2,
Almira Gafarova1,2
1

Research Institute of Internal and Preventive Medicine, Novosibirsk, Russia

Collaborative laboratory of Cardiovascular Diseases Epidemiology, Novosibirsk, Russia

Objectives: To examine the relationship between depression symptoms and the risk development of arterial hypertension (AH), myocardial infarction (MI) among men aged 25-64 years.
Methods: Within the framework of program WHO MONICA-MOPSY
representative sample of male population aged 25-64 years one of Novosibirsk district was examined in 1994. Total sample was 657 persons.
Depression symptoms were measured with the use of the MONICA psychosocial Interview Depression scale. The incidence of new cases of
AH, MI was revealed over 14-year of follow-up. Cox - proportional regression model was used for an estimation of hazard ratio (HR).
Results: Prevalence of depression in cohort of men with AH was 28.9%, with MI- 65.8%. The risk of AH within 5 years in group of men
with high level of depressive symptoms, in compared with those with
low depressive symptoms was 6.7 times higher, 10 years HR=4.2, 14
years HR = 2.1.The risk of MI within 5 years HR = 2.26, 10 years
HR=2.4, 14 years HR=2.6 (p for all b0.05). Most frequently of cardiovascular diseases occurred in men with higher negative psychosocial
factors, i.e. widowers, divorced, those with primary and notcompleted secondary school education and those engaged in hard and
moderate manual labor as well as pensioners.
Conclusion: Depression is a predictor of cardiovascular diseases in middle-age men. The risk of development of cardiovascular diseases in group of men with depression was 2.5- 6 times
higher than without it.

TH-090
Ticagrelor prevented reperfusion arrhythmias in dysmetabolic rats
Nicolas Renna1,2, Emiliano Diez2, Amira Ponce Zumino2, Roberto
Miatello1,2
1
rea de Fisiopatologa, Facultad de Ciencias Mdicas, Universidad
Nacional de Cuyo, Mendoza, Argentina

Abstracts
2

Instituto de Medicina y Biologa Experimental de Cuyo, CONICET, Mendoza, Argentina

Background: Ticagrelor (TICA) is a potent inhibitor of the P2Y12 receptor. In addition, TICA increases adenosine concentration by interfering with its cellular reuptake. Higher tissular levels of adenosine during
ischemia and reperfusion protects against ischemia-reperfusion injury.
In this study, we used TICA to prevent cardiovascular remodeling and
ventricular arrhythmias in Langendorff-perfused hearts from SHR rats
fed with fructose to induce metabolic syndrome.
Methods: Male WKY and SHR were separated into two groups
whether receiving tap water or 10% (w/v) fructose solution during all
6 weeks and designated as fructose-fed rat (FFR) or fructose-fed hypertensive rat (FFHR), respectively. These four groups were divided into vehicle or TICA: (30 mg/kg intraesophageal for 6 weeks) (n = 8 each
group). Metabolic variables and systolic blood pressure were measured
weekly through the 12 weeks. Cardiac and vascular remodeling were
evaluated by macroscopic and microscopic measurements. Reperfusion
ventricular arrhythmias were determined in Langendorff-perfused
hearts after 10 min of regional ischemia. Connexin-43 and PKCepsilon expression was determinate by western blot.
Results: The FFHR experimental model presented metabolic syndrome criteria, vascular and cardiac remodeling. Chronic treatment
with TICA reduced ventricular brillation incidence in all groups.
Connexin 43 and its phosphorylated form by PKC-epsilon were reduced
in the pathological models. TICA reversed changes in this pathway.
Conclusions: We conclude that FFHR model increases
arrhythmogenesis and TICA protects against these changes. TICA preserve phosphorylated connexin 43, possibly stabilizing the intercellular
communication and this effect is mediated by PKC.

TH-091
Administration of anabolic steroid during adolescent phase promote long-term increase in the susceptibility to myocardial ischemia/reperfusion injury: involvement of cardiac renin-angiotensin
system and katp channel
Fernando Seara1,2, Dahienne Oliveira1, Raiana Barbosa1, Jos Hamilton
Nascimento1, Emerson Olivares2
1

Federal University of Rio de Janeiro, Rio de Janeiro, Brazil

Federal Rural University of Rio de Janeiro, Seropedica, Brazil

Anabolic steroids (AS) abuse between adolescents has been raising

among occidental nations. Given that myocardial infarction is the
most frequent cardiovascular report associated with AS abuse, we hypothesized whether administration of AS during pre/pubertal phase of
Wistar rats promotes long-term increase in the susceptibility to ischemia/reperfusion (IR) injury in adulthood. Rats were treated with testosterone propionate (TP) (AS group, 5 mg.kg-1, starting in the 26
postnatal day [PND], 5 days per week for 5 weeks) or vehicle (control
group, peanut oil 10%, v:v). Rats were euthanized (82 PND) and
heart, liver, lung, kidneys and testis were collected. Isolated-perfused
hearts were submitted to IR protocol. Left Ventricle (LV) end diastolic
pressure (LVEDP), LV systolic pressure (LVSP), LV developed pressure
(LVDP), maximal (+) and minimal (-) rst derivatives of pressure
(dP/dt) were measured. Area of infarct was delimited with triphenyl
tetrazolium. The expression of key genes associated with cardiac hypertrophy was analyzed through quantitative real time polymerase-chain
reaction. Protein expression of Angiotensin-II type 1 receptor (AT1R)
and Kir6.1 was analyzed via Western Blot. NADPH oxidase-dependent
hydrogen peroxide production was analyzed through spectrouorometry. TP signicantly increased cardiac weight (P b0,001) and index
(P b0,001), whereas testicular weight was reduced (Pb 0,001). Infarct
size was increased by TP (Pb 0,05). TP impaired the recovery of LVDP,
LVEDP, + dP/dt and dP/dt in the reperfusion period. Myosin heavy

S83

chain (MHC) mRNA expression was enhanced in the AS group

(P b 0,01), likewise MHC/MHC ratio (P b 0,001). AT1R expression
was up-regulated (Pb0,05) by TP, whereas Kir6.1 was down-regulated
(Pb0,01). Nox activity did not change between groups. Chronic administration of AS promotes long-term increase in the susceptibility to IR injury with abnormalities in the expression of cardiac AT1R and ATPsensitive potassium channel.

TH-092
Novel software tools for crowdsourcing cardiac protein knowledge
in Gene Wiki
Anders O. Garlid1,2, Jessica M. Lee1,2, Jennifer S. Polson1,2, Tevk Umut
Dincer1,2, Sarah B. Scruggs1,2, Ding Wang1,2, Andrew I. Su1,3, Peipei
Ping1,2
1

NIH BD2K Center of Excellence at UCLA, Los Angeles, CA, USA

Departments of Physiology, Medicine, and Bioinformatics, University of
California, Los Angeles, Los Angeles, CA, USA
3
Department of Molecular and Experimental Medicine, The Scripps Research Institute, San Diego, CA, USA
2

Background: Mitochondrial and sarcomeric biology are integral to

our understanding of cardiac physiology and pathophysiology. A wealth
of knowledge is available to experienced biomedical scientists, but its
access and comprehension remain elusive to many, particularly the
general public. The Gene Wiki project, an effort within Wikipedia, was
established to bridge this gap and transform scientic knowledge
from esoteric to common knowledge. However, many mitochondrial
and sarcomeric proteins remain poorly represented and inadequately
annotated.
Aims: The Cardiac Gene Wiki team at UCLA aims to build highquality, interconnected Gene Wiki pages for genes expressed in cardiac
muscle in order to inspire and facilitate crowdsourced annotation by experts in the scientic community as well as citizen scientists.
Methods: Cardiac-expressed genes were clustered into functional subproteomes, with an emphasis on core cardiac mitochondrial and sarcomeric proteins, comprising a total of 620 gene
pages. Two tools were developed to enhance productivity and prioritize crowdsourcing efforts. The article assessment tool reports
the quality of gene pages within Wikipedia by examining the presence of biologically relevant content and the number of semantic
web links and peer-reviewed references. Secondly, a curation tool
was designed to streamline PubMed database searches, a timeintensive aspect of scientic writing, by conducting simultaneous
searches of user-dened keyword combinations.
Results: At the outset of this effort, the assessment tool revealed that
only 5 of the 556 core mitochondrial proteins and 5 of the 64 sarcomeric
proteins had relatively complete pages, while two-thirds were either
missing pages or were grossly incomplete. To date, the Cardiac Gene
Wiki team has improved over 400 articles and added 3,813 references,
11,163 Wiki links, and 2,682kB of content.
Conclusions: Together, these tools and crowdsourcing initiatives
support the aggregation of unstructured knowledge from the biomedical literature and its organization into a structured, user-friendly format
for a broad community of users.

TH-094
Cystathionine-gamma-lyase/hydrogen sulde inhibitinging smooth
muscle cells proliferation through regulating mitochondrial morphology in diabetic rat
Weihua Zhang, Jichao Wu, Fan Yang, Changqing Xu, Fanghao Lu
Harbin Medical University, Harbin, China

S84

Abstracts

Background: Molecular gas hydrogen sulde (HS) reduces the proliferation of vascular smooth muscle cells (VSMCs). Reactive oxygen
species (ROS) overproduction induced by hyperglycemia and high glucose is involved in VSMC proliferation, which may cause mitochondrial
fragmentation. Whether exogenous H2S reduces ROS production, inhibits mitochondrial fragmentation, and decreases VSMC proliferation
is unclear.
Methods and results: The morphological and ultrastructural alterations of the mesenteric secondary artery loop in diabetic rats, changes
in the HS concentration and the relaxation were determined. Additionally, the expression levels of CSE and Cyclin D1 in the mesenteric arteries of rats were examined by western blotting. The intracellular calcium
concentration, the expression of p-CaMK II (phospho-calmodulin kinases II), CSE activity, the concentration of endogenous HS and the proliferation of cultured VSMCs from rat thoracic aortic smooth muscle
cells(RASMCs) were measured by using confocal microscope, western
blotting, MTT and BrdU, respectively. The VSMC layer thickened, the
HS concentration dropped, the relaxation of the mesenteric secondary
artery rings weakened, and the expression of CSE decreased whereas
the expression of Cyclin D1 increased in diabetic rats compared with
the control group. Exogenous H2S (100 M NaHS) reduces ROS production in the cytoplasm and mitochondria. Higher mitochondrial fusionssion protein expression levels for dynamin-related protein 1 (Drp
1) in diabetic rats. When RASMCs proliferate with a high glucose treatment, the mitochondria become small spheres with a short rod-shaped
structure, whereas NaHS, a mitochondrial division inhibitor and
DrpsiRNA prevent VSMC proliferation and maintain mitochondria as
stationary and randomly dispersed with xed structures.
Conclusions: Exogenous H2S aid in inhibiting mitochondrial fragmentation and affect VSMCs proliferation by decreasing Drp 1
expression.

TH-095
Mitochondrial DAMPs in sterile inammation after acute myocardial infarction
May-Kristin Torp1, Yuchuan Li1, Trine Ranheim2, Torun Flateb1, Arne
Yndestad2, Kre-Olav Stenslkken1
1
Division of Physiology, Institute of Basic Medical Sciences, University of
Oslo, Oslo, Norway
2
Research institute of internal medicine, Oslo University Hospital
Rikshospitalet, Oslo, Norway

Background: Acute myocardial infarction results in necrosis and initiation of a local sterile inammation activated by Damage-Associated Molecular Patterns (DAMPs). Based on the endosymbiotic theory,
mitochondria are of bacterial origin, displaying bacterial traits in their
DNA and proteins. Moreover, the cardiomyocyte volume consists of 30%
mitochondria. Our research group has recently shown that mitochondrial
DNA induces cell death and activates the innate immune system in
cardiomyocytes. In this study, we hypothesize that mitochondrial constituents in general, or N-formyl-peptides are detrimental for cardiac cells.
Methods: Cardiac mitochondria were isolated from C57BI6 male
mice and this debris was utilized as agonists for isolated adult mouse
cardiomyocytes and cardiac broblasts. The cardiomyocytes were stimulated with increasing concentrations of mitochondrial debris or the Nformyl-peptide receptor (Fpr) agonist fMLP. Cardiomyocytes and cardiac broblast were also exposed to 40 minutes hypoxia (1% O2) and 2h
re-oxygenation. Cell death was investigated with High-Throughput microscopy. The cardiac broblasts were exposed to mitochondrial debris
and sampled in time intervals, and expression of cytokines was measured by qPCR.
Results: Cardiomyocytes exposed to normoxic conditions showed a
signicant increase in cell death when stimulated with 100g/ml mitochondrial debris compared to control. In addition, hypoxic conditions

increased cell death of cardiomyocytes at lower concentrations of mitochondrial debris (1 and 10g/ml). Cardiac broblasts exposed to 10g/
ml mitochondrial debris showed a signicant increase in Interleukin-6
mRNA expression after 1h compared to control.
Absolute quantication of Fpr genes revealed no expression in
cardiomyocytes or cardiac broblasts. However, the receptors were
expressed in mRNA extracted from PBS perfused mouse hearts possibly
indicating presence of resident macrophages. Cardiomyocytes stimulated with fMLP showed no signicant decrease in viability compared to
control.
Conclusion: Mitochondrial debris reduced the viability of the
cardiomyocytes, and gave an inammatory response in cardiac broblasts. This response appears not to be mediated through Fpr.

TH-096
Alpha-MHC MitoTimer mouse: in vivo mitochondrial turnover
model reveals remarkable mitochondrial heterogeneity in the heart.
Aleksandr Stotland, Roberta Gottlieb
Cedars-Sinai Heart Institute, Los Angeles, CA, USA
In order to maintain an efcient, energy-producing network in
the heart, dysfunctional mitochondria are cleared through the
mechanism of autophagy, which is closely linked with mitochondrial biogenesis; these, together with fusion and ssion comprise
a crucial process known as mitochondrial turnover. Until recently,
the lack of molecular tools and methods available to researchers
has impeded in vivo investigations of turnover. To investigate the
process at the level of a single mitochondrion, our laboratory has
developed the MitoTimer protein. Timer is a mutant of DsRed uorescent protein characterized by transition from green uorescence
to a more stable red conformation over 48 hrs, and its rate of maturation is stable under physiological conditions. We fused the
Timer cDNA with the inner mitochondrial membrane signal sequence and placed it under the control of a cardiac-restricted promoter. This construct was used to create the alpha-MHCMitoTimer mice. Surprisingly, initial analysis of the hearts from
these mice demonstrated a high degree of heterogeneity in the
ratio of red-to-green uorescence of MitoTimer in cardiac tissue.
Further, scattered solitary mitochondria within cardiomyocytes display a much higher red-to-green uorescence (red-shifted) relative
to other mitochondria in the cell, implying a block in import of
newly synthesized MitoTimer likely due to lower membrane potential. These red-shifted mitochondria may represent older, senescent
mitochondria. Concurrently, the cardiomyocytes also contain a subpopulation of mitochondria that display a lower red-to-green uorescence (green-shifted) relative to other mitochondria, indicative
of germinal mitochondria that are actively engaged in import of
newly-synthesized mito-targeted proteins. These mitochondria
can be isolated and sorted from the heart by ow cytometry for
further analysis. Initial studies suggest that these mice represent
an elegant tool for the investigation of mitochondrial turnover in
the heart.

TH-097
Oncotic and apoptotic mechanisms of toxic cardiomyocyte injury:
role of mitochondria and gene expression
L. Maximilian Buja, Priya Weerasinghe, David Loose, Robert Brown
The University of Texas Health Science Center at Houston, Houston, Texas,
USA
Mechanism of chemotherapy-induced cardiotoxicity was studied in
primary cultures of cardiomyocytes (CMC) derived from mouse

Abstracts

embryonic stem cells (ES) (Reach Bio LLC, Seattle, WA) exposed to
sanguinarine (Sang) and doxorubicin (Dox). CMC exposed to Sang, 4
uM and 33 uM, for 2 hours, or Dox, 2uM and 20uM, for up to 24 hours
displayed the morphologies of typical apoptosis (shrinkage) at the
lower doses and oncosis (swelling) at the higher doses, in most CMC, respectively. In CMC loaded with the cationic green uorochrome rhodamine 123 (rh 123), mitochondrial membrane potential at 2 hours was
maintained in apoptotic CMC but was markedly reduced in oncotic
CMC. To identify genes altered in oncosis vs. apoptosis, high density microarray analysis on RNAs prepared from CMC was performed using
Illumina Beadchips. Sang altered the expression of 2514 probes at the
higher oncosis-inducing dose and 1643 probes at the lower apoptosisinducing dose (p b 0.001), indicating the differential involvement of
multiple biochemical and signaling pathways. With high dose Sang,
perforin, a cytolytic protein found in CD8 T cells and NK cells, was induced more than 11-fold. Silencing of perforin gene by RNA interference
demonstrated salvage of CMC conrming the involvement of perforin in
Sang-induced oncosis. Compared to low dose Sang (n = 4), high dose
Sang (n =8) changed expression of 286 genes of canonical pathways
after 1 hour (p b 0.01, with a false discovery rate of 0.05), particularly
mitochondrial genes: citrate cycle (p= 5.0 x 10-4)(6/30 genes), mitochondrial function (p = 5.1x10-4)(12/130 genes), and oxidative phosphorylation (p = 3.55 x 10-3) (11/150 genes). Thus, CMC exhibit a
biphasic injury response to low and high dose Sang and Dox characterized by apoptosis and oncosis with differential gene expression and rate
of mitochondrial impairment.

TH-098
Monoamine oxidases are major contributors to mitochondrial ROS
formation and dysfunction, and cardiac damage in diabetic
cardiomyopathy
Soni Deshwal1, Chou-Hui Hu2, Guido Buonincontri2, Marleen Forkink2,
Salvatore Antonucci1, Mike Murphy3, Thomas Krieg2, Nina Kaludercic4,
Fabio Di Lisa1,4
1

University of Padova, Padova, Italy

University of Cambridge, Cambridge, UK
3
Mitochondrial Biology Unit, MRC, Cambridge, UK
4
CNR Neuroscience Institute, Padova, Italy
2

Recent studies highlight the important role of monoamine oxidases (MAOs) in the oxidative stress and cardiovascular damage. Reactive oxygen species (ROS) and inammation play a major role in
the pathogenesis of diabetes, but so far the involvement of MAO in
these processes has been overlooked. Thus, we investigated whether
MAOs contribute to high glucose (HG) and inammation induced oxidative stress as well as mitochondrial dysfunction in vitro and cardiac
damage in type 1 diabetes (T1D) in vivo. Neonatal rat ventricular
myocytes (NRVMs) displayed a signicant increase in mitochondrial
ROS formation and loss of mitochondrial membrane potential when
exposed to HG. Moreover, co-treatment with HG and interleukin-1
(IL-1), a pro-inammatory cytokine found to be elevated in diabetes,
further increased mitochondrial ROS levels. MAO inhibitor pargyline
reduced ROS formation in both conditions, suggesting that HG and
IL-1 induce oxidative stress in a MAO-dependent manner. Interestingly, mitochondrial ROS formation was accompanied by upregulated
endoplasmic reticulum (ER) stress markers in IL-1 treated

S85

cardiomyocytes and pargyline treatment prevented it, suggesting

that mitochondrial ROS generated by MAO is responsible for triggering ER stress in these conditions. Furthermore, in an in vivo model
of streptozotocin-induced T1D, oxidative stress, brosis and ER stress
markers were upregulated in the heart and diastolic stiffness, a marker of diastolic dysfunction, was increased. Pargyline administration to
these mice prevented these events, indicating that MAO contributes
to cardiac damage in diabetes. In conclusion, we demonstrated that
pharmacological inhibition of MAO is able to prevent HG and IL-1
induced mitochondrial ROS formation and dysfunction in vitro, as
well as diastolic dysfunction, oxidative stress and brosis in an
in vivo model of T1D. Furthermore, we show that ER stress occurring
in these conditions is MAO-dependent, suggesting an important role
of these avoenzymes in coordinating the interplay between mitochondrial dysfunction and ER stress in diabetes.
TH-099
Factors controlling MAO-dependent oxidative stress in myocytes
and non-myocytes of the heart
Veronica Costiniti1, Alessandra Castegna2, Roberta Menab1,3, Erika
Mariana Palmieri2, Marcella Canton1, Fabio Di Lisa1,3
1

University of Padova, Padova, Italy

University of Bari, Bari, Italy, 3Institute of Neuroscience CNR, Padova, Italy

Background: Monoamine oxidases (MAOs) are mitochondrial enzymes producing H2O2. As MAO inhibitors (iMAO) protect the heart in
experimental models of cardiac injury, the molecular mechanisms underlying MAO activation was evaluated by (i) the availability of MAO
substrates under stress conditions and (ii) their main cellular sources
in the whole heart.
Methods and results: Mass spectrometry (MS) was used to identify
and quantitate potential MAO substrates. We exploited two protocols of
oxidative stress by means of (i) H2O2 perfusion or (ii) post-ischemic reperfusion in the absence and the presence of iMAO in mouse
Langendorff model. The iMAO pargyline caused a relevant increase in
the heart content of N1-methyl-histamine (NMH) in both protocols.
Histidine-decarboxylase and histamine-N1-methyltransferase that are
involved in NMH production are found in heart. The basal MAO substrate content was measured by MS in isolated cardiomyocytes and
NMH was found to be the most abundant. Furthermore, upon histamine
addition to cardiomyocytes, we measured an increase in ROS level that
was inhibited both in the presence of a histamine-2-receptor (H2R) specic inhibitor, and pargyline, suggesting that H2R stimulation increase
histamine effect without excluding MAO activity although the signaling
pathway remains to be claried. To investigate non-cardiac sources for
MAO substrates under oxidative stress conditions we focused our attention on the synaptic terminals that innervate heart and commonly represent a pivotal source of neurotransmitters. Mice were denervated by
6-hydroxydopamine injection, hearts were subjected to the I/R protocol
and the MS analysis showed no relevant differences upon these treatments suggesting that synaptic terminals did not represent a major
sources of MAO substrate.
Conclusion: Histamine appears to promote MAO activity through
both receptor and non-receptor pathway. In fact besides the intracellular generation of NMH, MAO-induced ROS formation results from H2R
activation.