Resuscitation Science

Hydrogen Sulfide Improves Survival After Cardiac Arrest

and Cardiopulmonary Resuscitation via a Nitric Oxide
Synthase 3–Dependent Mechanism in Mice
Shizuka Minamishima, MD; Masahiko Bougaki, MD; Patrick Y. Sips, PhD; Jia De Yu, BS;
Yoji Andrew Minamishima, MD, PhD; John W. Elrod, PhD; David J. Lefer, PhD;
Kenneth D. Bloch, MD; Fumito Ichinose, MD, PhD

Background—Sudden cardiac arrest (CA) is one of the leading causes of death worldwide. We sought to evaluate the
impact of hydrogen sulfide (H2S) on the outcome after CA and cardiopulmonary resuscitation (CPR) in mouse.
Methods and Results—Mice were subjected to 8 minutes of normothermic CA and resuscitated with chest compression and
mechanical ventilation. Seven minutes after the onset of CA (1 minute before CPR), mice received sodium sulfide (Na2S) (0.55
mg/kg IV) or vehicle 1 minute before CPR. There was no difference in the rate of return of spontaneous circulation, CPR time to
return of spontaneous circulation, and left ventricular function at return of spontaneous circulation between groups.
Administration of Na2S 1 minute before CPR markedly improved survival rate at 24 hours after CPR (15/15) compared with
vehicle (10/26; P⫽0.0001 versus Na2S). Administration of Na2S prevented CA/CPR-induced oxidative stress and ameliorated
left ventricular and neurological dysfunction 24 hours after CPR. Delayed administration of Na2S at 10 minutes after CPR did
not improve outcomes after CA/CPR. Cardioprotective effects of Na2S were confirmed in isolated-perfused mouse hearts
subjected to global ischemia and reperfusion. Cardiomyocyte-specific overexpression of cystathionine ␥-lyase (an enzyme
that produces H2S) markedly improved outcomes of CA/CPR. Na2S increased phosphorylation of nitric oxide synthase 3 in
left ventricle and brain cortex, increased serum nitrite/nitrate levels, and attenuated CA-induced mitochondrial injury and cell
death. Nitric oxide synthase 3 deficiency abrogated the protective effects of Na2S on the outcome of CA/CPR.
Conclusions—These results suggest that administration of Na2S at the time of CPR improves outcome after CA possibly
via a nitric oxide synthase 3– dependent signaling pathway. (Circulation. 2009;120:888-896.)
Key Words: apoptosis 䡲 cardiopulmonary resuscitation 䡲 heart arrest 䡲 myocardial contraction
䡲 nitric oxide synthase 䡲 physiology

R estoration of spontaneous circulation after prolonged

whole body ischemia is an unnatural pathophysiolog-
ical state created by successful cardiopulmonary resusci-
Clinical Perspective on p 896
Hydrogen sulfide (H2S) is a colorless gas with a characteristic
tation (CPR). According to recent epidemiological data, rotten-egg odor found in various natural and industrial sources.3
the in-hospital mortality rate after cardiac arrest (CA) was In mammalian tissues, H2S is generated through the degradation
55% to 71% in adults and children who regained any of L-cysteine mainly by 2 enzymes, cystathionine ␤-synthase
spontaneous circulation after CPR.1 Post-CA syndrome, (CBS) and cystathionine ␥-lyase (CGL).4 Both CBS and CGL
including neurological dysfunction, myocardial damage, are expressed at high levels in liver, kidney, and pancreas,
and “sepsis-like” systemic inflammation, is likely to con- presumably for cysteine synthesis. CGL is also expressed in
tribute to the multisystem organ dysfunction and the brain, heart, and smooth muscle.5,6 Although deficiency of CBS
ultimate demise of many victims.2 Although hypothermia causes hyperhomocysteinemia, an established risk factor for
has proven effective in clinical studies, no pharmacologi- cardiovascular disorders including sudden cardiac death, CGL
cal agent yet tested has proven successful in improving deficiency causes cystathioninuria, an apparently benign condi-
outcome from CA/CPR. tion albeit with an inconsistent and variable disease association.7

Received November 17, 2008; accepted June 15, 2009.

From the Anesthesia Center for Critical Care Research, Department of Anesthesia and Critical Care, Massachusetts General Hospital (S.M., M.B.,
P.Y.S., J.D.Y., K.D.B., F.I.), and Department of Medical Oncology, Brigham and Women’s Hospital and Dana-Farber Cancer Institute, Harvard Medical
School (Y.A.M.), Boston, Mass; Department of Molecular and Cardiovascular Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio
(J.W.E.); and Department of Surgery, Emory University School of Medicine, Atlanta, Ga (D.J.L.).
The online-only Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.108.833491/DC1.
Correspondence to Fumito Ichinose, MD, PhD, Department of Anesthesia and Critical Care, Massachusetts General Hospital, 149 13th St, 3405,
Charlestown, MA 02129. E-mail [email protected]
© 2009 American Heart Association, Inc.
Circulation is available at http://circ.ahajournals.org DOI: 10.1161/CIRCULATIONAHA.108.833491

888
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 Minamishima et al Hydrogen Sulfide in Cardiac Arrest and CPR 889

H2S exerts a host of biological effects on various targets, Study drug (Na2S) or vehicle was injected via the femoral venous
resulting in responses that range from cytotoxic to cytopro- line 1 minute before CPR. On the basis of pilot studies, we
administered 0.55 mg/kg (7.0 ␮mol/kg) Na2S. Effects of delayed
tective effects.5 Although breathing high concentrations of
administration of Na2S at 10 minutes after the start of CPR
H2S is toxic,5 breathing low concentrations of H2S reversibly (post-CPR Na2S) were examined in a subgroup of mice.
reduces metabolism in rodents and improves survival after
hemorrhagic shock in rats.8 –10 Although administration of Assessment of Neurological Function
NaHS has been shown to worsen cerebral ischemia11 and Neurological function was assessed at 24 hours after CA/CPR or
lipopolysaccharide-induced systemic inflammation,12 recent sham surgery with the use of a previously reported neurological
studies showed that administration of NaHS or Na2S can function scoring system with minor modifications (see Methods in
the online-only Data Supplement).18,19
attenuate ischemia/reperfusion (IR) injury in heart,13–15
liver,16 and kidney.17 The protective effects of H2S appear to
Effects of Na2S on Myocardial Function in
be mediated via antiapoptotic and/or anti-inflammatory ef-
Isolated-Perfused Mouse Heart
fects. However, the impact of Na2S on the outcome of Mice were heparinized and anesthetized with the use of pentobarbital
CA/CPR, which is complicated by whole body IR injury and (60 mg/kg IP). The heart was removed quickly and mounted on a
systemic inflammation, remains to be elucidated. In the 20-gauge cannula in ice-cold perfusion buffer (a modified Krebs-
present study, we report that administration of Na2S at the Henseleit buffer, containing [in mmol/L] 118.5 NaCl, 24.9 NaHCO3,
time of CPR markedly improves myocardial and neurological 11.2 glucose, 4 KCl, 1.2 MgSO4, 1.2 KH2PO4, 2 sodium pyruvate,
and 2.5 CaCl2). Retrograde perfusion was started with the use of
function and survival after CA/CPR in mice. perfusion buffer continuously gassed with 95% O2/5% CO2 at 37°C.
A constant hydrostatic coronary pressure of 65 mm Hg was main-
Methods tained throughout the experiment. The tip of an SPR-671 transducer
(Millar Instruments, Houston, Tex) was placed in a saline-filled
Mice polyvinyl chloride balloon that was inserted into the left ventricle
After approval by the Massachusetts General Hospital Subcommittee (LV). The balloon was inflated to obtain a diastolic pressure of 5 to
on Research Animal Care, we studied 2- to 3-month-old age- and 10 mm Hg, and LV pressure was monitored via the Millar transducer
weight-matched male C57BL/6J wild-type and nitric oxide synthase and continuously recorded. LV developed pressure, rate-pressure
3 (NOS3)– deficient mice on a C57BL/6J background (NOS3⫺/⫺). product, maximum and minimum rate of LV pressure change
We also studied 2- to 3-month-old male mice with cardiomyocyte- (dP/dtmax and dP/dtmin), and the time constant of isovolumic LV
specific overexpression of CGL (CS-CGLtg)13 and their wild-type relaxation (␶) were calculated. Hearts were electrically paced at 7 Hz
littermates (FVBN/J). with the use of a Grass S88 stimulator (Grass Technologies, West
Warwick, RI). Coronary flow rate was measured with an N1 in-line
Material flowprobe and a T106 flowmeter (Transonic Systems, Ithaca, NY).
Sodium sulfide (Na2S; IK-1001) was a generous gift from Ikaria Inc After a 30-minute stabilization period, hearts were subjected to 20
(Seattle, Wash). Na2S was produced by using H2S gas (Matheson, minutes of ischemia followed by 60 minutes of reperfusion. Na2S (10
Newark, Calif) as a starting material and was formulated to pH ␮mol/L) or vehicle was administered into the perfusate starting
neutrality and iso-osmolarity. Na2S was diluted in normal (0.9%) saline immediately or 40 seconds after the start of reperfusion and
to the desired concentration immediately before administration. continued for 5 minutes. Myocardial function was measured for the
first 60 minutes after reperfusion.
Animal Preparation
Mice were anesthetized with 100 ␮g/g ketamine and 0.25 ␮g/g Measurement of Serum Nitrite/Nitrate
fentanyl delivered by intraperitoneal injection and mechanically Concentrations of nitrite and nitrate were measured in serum samples
ventilated (FIO2⫽0.21; Minivent, Harvard Apparatus). Arterial blood obtained at 15 minutes after CA/CPR or sham surgery with a
pressure was measured via a left femoral arterial line. A saline-filled Nitrate/Nitrite Fluorometric Assay Kit (Cayman Chemical, Ann
microcatheter (PE-10, Becton Dickinson) was inserted into the left Arbor, Mich) according to the manufacturer’s instructions.
femoral vein for drug and fluid administration. Blood pressure and
needle-probe ECG monitoring data were recorded and analyzed with
the use of a PC-based data acquisition system. Mitochondrial Permeability Transition
Mouse heart mitochondria were isolated 15 minutes after CA/CPR or
Murine CPR Model sham operation by differential centrifugation with the use of the
MITO-ISO1 kit (Sigma-Aldrich, St Louis, Mo) following the man-
CA was induced by administration of 0.08 mg/g potassium chloride
ufacturer’s instructions. Mitochondrial permeability transition was
through the femoral catheter and was confirmed by loss of arterial
measured by monitoring the decrease in absorbance at 540 nm
pressure and asystolic rhythm on ECG. After 8 minutes of CA, chest
associated with mitochondrial swelling, as described previously.20
compressions were delivered with a finger at a rate of 340 to 360
Briefly, mitochondrial suspensions were diluted to a final concen-
beats per minute with resumption of mechanical ventilation
(FIO2⫽1.0). Chest compressions were adjusted to provide a uniform tration of 0.5 mg/mL in a buffer containing (in mmol/L) 200
rate seen on femoral artery pressure monitoring and a target femoral mannitol, 70 sucrose, 10 HEPES, and 5 succinate (pH 7.4). After 2
diastolic pressure of ⬎20 mm Hg. Epinephrine was infused at 0.3 minutes of stabilization, 400 ␮mol/L CaCl2 was added to induce
␮g/min starting 30 seconds before CPR and continued until heart rate mitochondrial permeability transition. The decrease in absorbance
became ⬎300 bpm. Core body temperature was maintained at 37°C was monitored for 1000 seconds at room temperature with a
by a warming lamp. Return of spontaneous circulation (ROSC) was spectrophotometer. The maximal rate of the absorbance decrease
defined as the return of sinus rhythm with a mean arterial pressure was obtained by linear regression over a 3-minute period.
⬎40 mm Hg lasting at least 1 minute. Mice were weaned from
mechanical ventilation at 1 hour after and extubated at 2 hours after Measurements of Peroxide Levels in Serum
CPR. In a subgroup of mice, at 24 hours after CPR, cardiac function Concentrations of hydrogen peroxide were measured in serum
was examined with a conductance pressure-volume catheter (SPR- samples obtained at 5 minutes after CA/CPR or sham surgery with a
839, Millar Instruments Inc) under anesthesia. Mice subjected to QuantiChrom Peroxide Assay Kit (BioAssay Systems, Hayward,
sham surgery that were not subjected to CA were used as controls. Calif) according to the manufacturer’s instructions.

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890 Circulation September 8, 2009

mice (Figure 2B). The caspase 3 activation was markedly

attenuated by Na2S. Histological studies revealed that the
number of neurons containing cleaved caspase 3 in the CA1
region of the hippocampus was markedly increased at 24
hours after CA/CPR (Figure 2C and 2D). Similarly, the
number of dead neurons identified by morphological crite-
ria21 in the CA1 region was markedly increased at 72 hours
after CA/CPR (Figure 2E and 2F). Administration of Na2S 1
minute before CPR decreased the numbers of cleaved caspase
Figure 1. Survival during the first 24 hours after CA and CPR.
3–positive or dead neurons in the hippocampus. Because
Vehicle indicates mice subjected to CPR treated with vehicle;
Na2S, mice treated with Na2S 1 minute before CPR. *P⫽0.0001 phosphorylation of GSK-3␤ has been suggested to inhibit
vs vehicle. apoptosis, we examined phosphorylation status of GSK-3␤ in
the brain cortex extracts. The neuroprotective effects of Na2S
Detailed description of reagents and protocol for immunoblots and were associated with enhanced GSK-3␤ phosphorylation at
histological studies are provided in Methods in the online-only Data Ser9 (Figure 2G). These results suggest that administration of
Supplement.
Na2S 1 minute before CPR prevents apoptosis in the brain.
Statistical Analysis
All data are expressed as mean⫾SEM. Normally distributed data
Na2S Prevents Myocardial Dysfunction After CA
were analyzed with the use of unpaired t test, 2-way repeated- and CPR
measures ANOVA, or 1-way ANOVA with a Holm-Sidak or There was no difference in heart rate and mean arterial
Bonferroni post hoc test. Neurological function scores of CS-CGLtg pressure between mice treated with vehicle or Na2S at ROSC
versus FVBN/J wild-type mice were compared by Mann-Whitney or 60 minutes after CPR (Supplemental Table I in the
rank sum test because the values are not normally distributed.
Difference in survival rate was analyzed by Gehan-Breslow test.
online-only Data Supplement). Twenty-four hours after CA/
Sigmastat 3.01a (Systat Software Inc, San Jose, Calif) and GraphPad CPR, heart rate, LV end-systolic pressure, dP/dtmax, end-sys-
Prism 5.0 (GraphPad Software Inc, La Jolla, Calif) were used for tolic elastance, and preload-recruitable stroke work were
statistical analyses. depressed in vehicle-treated mice compared with sham but
not in Na2S-treated mice (Table). Myocardial mechanical
Results efficiency (estimated by the ratio of end-systolic elastance to
Na2S Improves Survival Rate After CA and CPR arterial elastance) was impaired (decreased ratio) in vehicle-
The rate of ROSC was ⬎95% in mice treated with either treated mice but not in Na2S-treated mice (Table). These
vehicle or Na2S. There was no difference between treatment results show that administration of Na2S at the time of CPR
groups in the CPR time to ROSC or the total epinephrine dose prevents post-CA myocardial dysfunction 24 hours after
(Table I in the online-only Data Supplement). Although only CA/CPR.
10 of 26 mice treated with vehicle survived 24 hours after
CPR, all 15 mice treated with Na2S 1 minute before CPR Na2S Improves Myocardial Function After In
survived 24 hours after CA and CPR (P⫽0.0001; Figure 1). Vitro IR
In contrast, delayed administration of Na2S at 10 minutes To examine the impact of Na2S on myocardial function after
after CPR did not improve the survival rate at 24 hours after global IR, we studied the effects of Na2S in isolated-perfused
CPR (5 of 11 survived; P⫽0.9 versus vehicle). Although mouse hearts. Myocardial function was depressed after 20
formal long-term survival analysis was not done in the minutes of ischemia in vehicle-treated hearts. Administration
present study, in our pilot studies, ⬎90% of mice treated with of Na2S (10 ␮mol/L) starting at the time of reperfusion after
Na2S recovered completely by 72 hours after CPR and lived 20 minutes of ischemia markedly improved the recovery of
indefinitely. On the other hand, ⬍20% of vehicle-treated myocardial function (Figure 3A through 3F). Recovery of
mice survived 72 hours after CPR. Because delayed admin- both systolic (dP/dtmax, LV developed pressure, and rate-
istration of Na2S did not improve survival compared with pressure product) and diastolic (dP/dtmin and ␶) myocardial
vehicle, neurological and myocardial function and tissue function was markedly augmented by Na2S compared with
injury were compared between groups of mice that received vehicle. Na2S also improved the recovery of coronary flow.
vehicle or Na2S 1 minute before CPR. Of note, delaying the start of administration of Na2S by 40
seconds after the start of reperfusion abolished the protective
Na2S Improved Neurological Function 24 Hours effects of Na2S on myocardial function after IR (Figure I in
After CA and CPR the online-only Data Supplement). These results further
Although neurological function was depressed in surviving support the concept that Na2S can preserve cardiac function
mice 24 hours after CA/CPR, it was better in Na2S-treated after global myocardial IR when it is administered at the time
mice than in vehicle-treated mice (P⬍0.0001; Figure 2A). of reperfusion.

Na2S Prevents Apoptosis in Brain After CA Na2S Attenuates Oxidative Stress and Protects
and CPR Mitochondria in the Heart After CA and CPR
Immunoblot analysis revealed that caspase 3 was activated at CA accelerated the rate of Ca2⫹-induced mitochondrial swell-
15 minutes after CA/CPR in cerebral cortex of vehicle-treated ing, a measure of mitochondrial permeability transition pore
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 Minamishima et al Hydrogen Sulfide in Cardiac Arrest and CPR 891

Figure 2. Neuroprotective effects of

Na2S. A, Neurological function score in
surviving mice at 24 hours after
CA/CPR. Dead mice (indicated by
score⫽0) were excluded from the sta-
tistical analysis. *P⬍0.0001 vs vehicle
by unpaired t test. B, Representative
immunoblots showing cleaved caspase
3 and ␤-tubulin protein expression in
brain cortex of mice 15 minutes after
sham operation or CA/CPR treated with
vehicle or Na2S. Cleaved caspase 3
protein levels were determined by
dividing cleaved caspase 3 immunore-
activity by ␤-tubulin immunoreactivity
and normalized to values of sham-
operated mice. *P⬍0.05 vs sham;
#P⬍0.05 vs vehicle. C, Representative
photomicrographs of brain sections of
mice treated with vehicle or Na2S
showing cleaved caspase 3–immunore-
active neurons (indicated with black
arrowheads) at 24 hours after CPR. D,
Summary of the number of neurons
containing cleaved caspase 3 in the
CA-1 region of the brain. #P⬍0.05 vs
vehicle. n⫽3 for each group. E, Repre-
sentative photomicrographs of brain
sections of mice treated with vehicle or
Na2S showing dead neurons (indicated
with black arrowheads) at 72 hours
after CPR in hippocampal CA-1 region.
F, Summary of the number of dead
neurons in the hippocampal CA-1
region. #P⬍0.05 vs vehicle. n⫽3 for
each group. G, Representative immu-
noblots showing phosphorylated and
total GSK-3␤ at Ser9 in the brain cortex
of mice 15 minutes after sham opera-
tion or CA/CPR treated with vehicle or
Na2S. Relative phospho-GSK-3␤ levels
were quantified by dividing the
phospho-GSK-3␤ immunoreactivity by
total GSK-3␤ immunoreactivity and
normalized to values of sham-operated
mice. n⫽6 to 8 in each group. *P⬍0.05
vs sham; #P⬍0.05 vs vehicle.

opening, compared with the mitochondria of sham-operated ROSC (1.5⫾0.2 versus 0.9⫾0.1 ␮g; P⫽0.015), and had a
mice. Na2S prevented the acceleration of Ca2⫹-induced mito- higher mean arterial pressure at 60 minutes after CPR (28⫾2
chondrial swelling in cardiac mitochondria after CA/CPR versus 35⫾3 mm Hg; P⫽0.046). Additionally, CS-CGLtg
(Figure 4A and 4B). Na2S also prevented CA-induced incre- mice had markedly improved neurological function (8⫾1
ments in hydrogen peroxide levels in serum 5 minutes after [median⫽7] versus 5⫾1 [median⫽4.5]; P⫽0.048 by Mann-
CPR (Figure 4C). These results suggest that Na2S attenuates Whitney rank sum test) and survival rate (8 of 11 versus 4 of
oxidative stress early after CA/CPR and protects cardiac 13; P⫽0.03; Figure 5) 24 hours after CA/CPR. These results
mitochondria. suggest that augmented cardiac H2S levels facilitated recov-
ery after CA/CPR.
Cardiomyocyte-Specific Overexpression of CGL
Improves Outcomes After CA and CPR Na2S Increases Phosphorylation of Akt and
To examine the impact of endogenously produced H2S in the AMP-Activated Protein Kinase ␣ After CA
heart on the recovery from CA/CPR, we studied CS-CGLtg and CPR
mice, which have been characterized extensively and display Because Akt- and AMP-activated protein kinase (AMPK)–
increased H2S production in the heart.13 Compared with their dependent signal contributes to cell survival in variety of
wild-type littermates, CS-CGLtg mice had a markedly shorter pathological conditions,22,23 we sought to investigate the role
CPR time to ROSC (516⫾62 versus 331⫾24 seconds; of Akt and AMPK in the protective effects of Na2S. CA and
P⫽0.016), required a smaller dose of epinephrine to achieve CPR increased Akt phosphorylation in LV compared with
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892 Circulation September 8, 2009

Table. Myocardial Function 24 Hours After CA and CPR

Sham (n⫽5) Vehicle (n⫽6) Na2S (n⫽8)
Heart rate, bpm 627⫾8 559⫾24* 575⫾19
LVESP, mmHg 118⫾8 78⫾11* 105⫾6†
LVEDP, mmHg 4⫾1 3⫾0 5⫾1
dP/dtmax, mmHg/s 17 969⫾2290 10 437⫾2275* 15 328⫾1417
dP/dtmin, mmHg/s ⫺13 404⫾1752 ⫺11 919⫾2627 ⫺14 559⫾734
Cardiac output, 11⫾1 10⫾1 13⫾1
mL/min
dP/dtmax/IP, s⫺1 203⫾9 191⫾10 197⫾9
Ea, mmHg/␮L 7⫾0 4⫾1* 5⫾1*
Ees, mmHg/␮L 27⫾7 3⫾0* 11⫾2†
Ees/Ea 4.0⫾1.1 0.8⫾0.1* 2.4⫾0.5†
PRSW, mmHg 158⫾22 41⫾8* 118⫾13†
␶, ms 5.2⫾0.3 5.2⫾0.7 4.9⫾0.3
Values are mean⫾SEM. Sham indicates sham-operated mice; Vehicle, mice
subjected to CPR treated with vehicle; Na2S, mice treated with Na2S 1 minute
before CPR; LVESP, LV end-systolic pressure; LVEDP, LV end-diastolic pres-
sure; dP/dtmax/IP, dP/dtmax divided by instantaneous pressure; Ea, arterial
elastance; Ees, LV end-systolic ventricular elastance; and PRSW, preload-
recruitable stroke work.
*P⬍0.05 vs sham-operated mice. Figure 3. Effects of Na2S in global IR in isolated-perfused
†P⬍0.05 vs vehicle. mouse hearts. Percentages of baseline values are shown for
maximal LV developed pressure (LVDevP) (A), rate-pressure
product (RPP) (B), maximal rate of LV pressure rise (dP/dtmax)
sham-operated mice (Figure 6A). Administration of Na2S 1 (C), maximal rate of LV pressure reduction (dP/dtmin) (D), the
minute before CPR increased Akt phosphorylation at Ser473 time constant of isovolumic LV relaxation (␶) (E), and coronary
compared with vehicle both in the LV and brain after flow rate (flow rate) (F). ###P⬍0.001, ##P⬍0.01, and #P⬍0.05
for comparisons of vehicle vs Na2S, as calculated by repeated-
CA/CPR (Figure 6A and 6B). CA and CPR decreased measures 2-way ANOVA; *P⬍0.01 vs vehicle at the same time
phosphorylation of NOS3 at Ser1179 in the LV and brain point, as calculated by Bonferroni post hoc tests.
(Figure 6C and 6D). Administration of Na2S prevented the
dephosphorylation of NOS3 at Ser1179 both in LV and brain 1 minute before CPR did not improve the survival rate in
after CA/CPR. Similarly, CA/CPR-induced dephosphoryla- NOS3⫺/⫺ mice (1 of 7 survived; Figure 6F). These results
tion of AMPK␣ at Thr172 was prevented by Na2S in the brain suggest that the protective effects of Na2S on the outcome of
(Figure 6E). Of note, delayed administration of Na2S at 10 CA/CPR require NOS3.
minutes after CPR (post-CPR Na2S) failed to increase the
phosphorylation of Akt, NOS3, and AMPK␣ compared with Discussion
vehicle after CA/CPR. These observations suggest that pro- The present study revealed that administration of Na2S at the
tective effects of Na2S on organ function are associated with time of CPR markedly improves myocardial and neurological
activation of Akt- and/or AMPK␣-dependent prosurvival function and survival after CA/CPR in mice. The neuropro-
signals. tective effects of Na2S were associated with attenuated
CA/CPR-induced caspase 3 activation in the brain. Global
Na2S Prevents Reduction of Nitrite/Nitrate Levels myocardial IR in isolated-perfused heart model confirmed the
After CA and CPR protective effects of Na2S. Administration of Na2S also
To examine whether the protective effects of Na2S were prevented systemic oxidative stress and acceleration of mito-
associated with augmented NO production, we measured chondrial permeability transition in the heart. Enhanced
serum nitrite and nitrate levels at 15 minutes after CA/CPR. endogenous H2S production by cardiomyocyte-specific over-
Compared with sham-operated mice (4.7⫾0.8 ␮mol/L), ni- expression of CGL improved outcomes after CA/CPR. The
trite/nitrate levels were decreased in vehicle-treated mice salutary impact of Na2S treatment on the outcome of CA/CPR
(3.2⫾0.8 ␮mol/L; P⬍0.01 versus sham) but not in Na2S- was associated with preserved serum nitrite and nitrate levels
treated mice (4.3⫾0.2 ␮mol/L; P⫽0.35 versus sham, P⬍0.05 and enhanced phosphorylation of Akt and NOS3 in the brain
versus vehicle-treated mice) 15 minutes after CA/CPR. and heart and AMPK␣ and GSK-3␤ in the brain. Finally,
NOS3 deficiency abrogated the protective effects of Na2S on
Na2S Does Not Improve Outcome of CA and CPR the outcome of CA/CPR. Taken together, these observations
in NOS3-Deficient Mice suggest that H2S confers organ protection and improves
To elucidate the role of NOS3/NO in the protective effects of survival after CA/CPR at least in part via an NOS3-dependent
Na2S, we examined whether Na2S improves outcomes of mechanism.
CA/CPR in NOS3⫺/⫺ mice. Only 1 of 7 NOS3⫺/⫺ mice The majority of research on CA over the past half century
survived 24 hours after CA/CPR, and administration of Na2S has focused on improving the rate of ROSC, and significant
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 Minamishima et al Hydrogen Sulfide in Cardiac Arrest and CPR 893

Figure 4. Effects of Na2S on mitochondrial

permeability transition and oxidative stress.
A, Representative tracings of calcium-
induced mitochondrial swelling measured by
spectrophotometry. B, Quantification of the
rate of Ca2⫹-induced mitochondrial swelling
as a measure of mitochondrial permeability
transition. *P⬍0.05 vs sham; #P⬍0.05 vs
vehicle. n⫽7 in each group. C, Hydrogen
peroxide levels in serum obtained at 5 min-
utes after CPR. n⫽3 to 6 in each group.
*P⬍0.05 vs sham; #P⬍0.05 vs vehicle.

progress has been made. However, many patients succumb to the present study (7 ␮mol/kg of Na2S) may explain the
the post-CA syndrome after successful resuscitation. The conflicting results of these studies.16 Proapoptotic effects of
present study was designed to investigate whether H2S could relatively high concentrations of H2S have also been reported
improve the outcome after “successful” resuscitation. Al- in pancreas.25 Nonetheless, our results suggest that physio-
though there was no difference in the rate of ROSC or CPR logical level of H2S can have important protective effects on
time to ROSC, mice that received Na2S immediately before ischemic brain injury.
CPR showed a markedly higher survival rate at 24 hours than To examine the molecular mechanisms responsible for the
did mice that received vehicle before CPR or Na2S 10 antiapoptotic effects of Na2S, we tested whether Na2S pro-
minutes after CPR. These observations suggest that adminis- motes Akt- or AMPK-dependent prosurvival signals in the
tration of Na2S at the time of CPR improved outcomes at 24 brain. We found that Na2S increased phosphorylation of Akt
hours after CA by preventing the development of post-CA and GSK-3␤ in the brain cortex. Akt-dependent phosphory-
organ injury and dysfunction after ROSC. lation of GSK-3␤ inhibits GSK-3␤ activity and thereby
The present results suggest that Na2S can protect against decreases apoptosis.26,27 Similarly, administration of Na2S 1
the development of post-CA neurological dysfunction and minute before CPR prevented the CA/CPR-induced dephos-
injury when administered at the time of CPR (Figure 2A). CA phorylation of AMPK␣ and NOS3. It has been reported that
and CPR activated caspase 3 and induced cell death in the activation of cGMP-dependent protein kinase may confer
brain of vehicle-treated mice. In contrast, the improved cardioprotection via GSK-3␤.27 It is conceivable that Akt-
neurological function at 24 hours after CPR in mice treated and/or AMPK␣-dependent NOS3 activation activates the
with Na2S was associated with marked attenuation of caspase cGMP-dependent protein kinase– dependent neuroprotective
3 activation and cell death in the brain after CPR. Previously, mechanism via GSK-3␤ phosphorylation in our model.
Qu and colleagues11 showed that administration of NaHS Similar to neurological function, myocardial function at 24
increased brain infarct volume after permanent middle cere- hours after CA/CPR was also improved by administration of
bral artery occlusion. Differences in the models and the Na2S (Table). These results are reinforced by our findings
higher dose of NaHS (180 ␮mol/kg of NaHS, equivalent to that administration of Na2S starting immediately after
⬇90 ␮mol/kg of Na2S24) used in the Qu study compared with reperfusion, but not when administered 40 seconds after

Figure 5. Impact of endogenous H2S on the

outcome of CA/CPR. A, Survival during the first
24 hours after CA and CPR in mice with car-
diomyocyte-specific overexpression of CGL
(CS-CGLtg, n⫽11) and their wild-type litter-
mates (FVBN/J, n⫽13). *P⫽0.03 vs FVBN/J by
Gehan-Breslow test. B, Neurological function
score in surviving mice at 24 hours after
CA/CPR. Dead mice (indicated by score⫽0)
were excluded from the statistical analysis.
Horizontal lines show median value of each
group. *P⫽0.048 vs FVBN/J by Mann-Whitney
rank sum test.

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894 Circulation September 8, 2009

Figure 6. Phosphorylated protein expression in brain cortex and LV 15 minutes after sham operation or CA/CPR in mice treated with
vehicle or Na2S 1 minute before CPR (Veh and Na2S, respectively) or Na2S administered at 10 minutes after CPR (post-CPR Na2S). Rel-
ative phospho-Akt Ser473 levels were quantified by dividing the phospho-Akt Ser473 immunoreactivity by total Akt immunoreactivity and
normalized to values of sham-operated mice in the heart (A) and brain (B). Relative phospho-NOS3 Ser1179 levels were quantified by
dividing the phospho-NOS3 Ser1179 immunoreactivity by total NOS3 immunoreactivity and normalized to values of sham-operated mice
in the heart (C) and brain (D). Relative phospho-AMPK␣ Thr172 levels were quantified by dividing the phospho-AMPK␣ Thr172 immunore-
activity by total AMPK␣ immunoreactivity and normalized to values of sham-operated mice in the brain (E). Two representative blots are
shown of 6 to 8 blots in each group for all proteins. Loading controls are shown to demonstrate equal sample loading. *P⬍0.05 vs
sham; #P⬍0.05 vs vehicle; †P⬍0.05 vs Na2S. F, Survival during the first 24 hours after CA and CPR in NOS3⫺/⫺ mice treated with vehi-
cle (NOS3⫺/⫺⫹Vehicle; n⫽7) and NOS3⫺/⫺ mice treated with Na2S (NOS3⫺/⫺⫹Na2S; n⫽7).

reperfusion, markedly improved myocardial function in Mitochondrial permeability transition is a central mechanism of
the isolated-perfused mouse heart subjected to global IR. IR-induced death.20 Na2S prevented CA/CPR-induced acceleration
These results are consistent with a previous report that of Ca2⫹-induced mitochondrial swelling, a measure of mitochon-
NaHS was cardioprotective in isolated rat hearts.15 The drial permeability transition, in LV mitochondria. The beneficial
relatively narrow temporal window of opportunity for effect of Na2S was associated with attenuated oxidative stress
Na2S to improve the outcome of CA/CPR is consistent with indicated by decreased serum hydrogen peroxide levels (Figure
previous studies of ischemic postconditioning. For exam- 4C). Oxidative stress has been shown to trigger mitochondrial
ple, Cohen and colleagues28 recently reported that delay of permeability transition.29 These observations suggest that the
the onset of postconditioning by only 1 minute aborts cardioprotective effects of Na2S may be mediated by inhibition
protection in rabbit hearts. of mitochondrial permeability transition.
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 Minamishima et al Hydrogen Sulfide in Cardiac Arrest and CPR 895

Cardioprotective effects of endogenous H2S after IR have Cavaillon JM. Successful cardiopulmonary resuscitation after cardiac
been suggested by studies showing deleterious effects of arrest as a “sepsis-like” syndrome. Circulation. 2002;106:562–568.
3. Kashiba M, Kajimura M, Goda N, Suematsu M. From O2 to H2S: a
CGL inhibitors30 and protective effects of CS-CGLtg13 in landscape view of gas biology. Keio J Med. 2002;51:1–10.
myocardial IR. Our observation that CS-CGLtg mice had a 4. Stipanuk MH, Beck PW. Characterization of the enzymic capacity for
markedly shorter CPR time to ROSC suggests that endoge- cysteine desulphhydration in liver and kidney of the rat. Biochem J.
1982;15;206:267–277.
nous H2S promotes the recovery of myocardial function after
5. Szabo C. Hydrogen sulphide and its therapeutic potential. Nat Rev Drug
CA/CPR. Although CS-CGLtg mice also had improved Discov. 2007;6:917–935.
neurological function and survival rate 24 hours after CA/ 6. Fiorucci S, Distrutti E, Cirino G, Wallace JL. The emerging roles of
CPR, these findings may be due to the shortened CPR time to hydrogen sulfide in the gastrointestinal tract and liver. Gastroenterology.
2006;131:259 –271.
ROSC, as well as possible neuroprotective effects of endog- 7. Wang J, Huff AM, Spence JD, Hegele RA. Single nucleotide poly-
enously produced H2S. Nonetheless, these observations sup- morphism in CTH associated with variation in plasma homocysteine
port an important salutary effect of endogenous H2S produc- concentration. Clin Genet. 2004;65:483– 486.
tion on the outcomes of CA/CPR. 8. Blackstone E, Morrison M, Roth MB. H2S induces a suspended
animation-like state in mice. Science. 2005;308:518.
Improved myocardial function in Na2S-treated mice was 9. Volpato GP, Searles R, Yu B, Scherrer-Crosbie M, Bloch KD, Ichinose F,
associated with increased phosphorylation of Akt and Zapol WM. Inhaled hydrogen sulfide: a rapidly reversible inhibitor of cardiac
NOS3 in the LV and elevated serum levels of nitrite plus and metabolic function in the mouse. Anesthesiology. 2008;108:659–668.
nitrate combined compared with vehicle-treated mice 15 10. Morrison ML, Blackwood JE, Lockett SL, Iwata A, Winn RK, Roth MB.
Surviving blood loss using hydrogen sulfide. J Trauma. 2008;65:183–188.
minutes after CA/CPR. In contrast, delayed administration 11. Qu K, Chen CPLH, Halliwell B, Moore PK, Wong PTH. Hydrogen sulfide
of Na2S at 10 minutes after CPR did not increase the is a mediator of cerebral ischemic damage. Stroke. 2006;37:889–893.
phosphorylation of Akt and NOS3 in the LV and failed to 12. Li L, Bhatia M, Zhu YZ, Zhu YC, Ramnath RD, Wang ZJ, Anuar FB,
Whiteman M, Salto-Tellez M, Moore PK. Hydrogen sulfide is a novel
improve the outcome of CA/CPR. Furthermore, NOS3
mediator of lipopolysaccharide-induced inflammation in the mouse.
deficiency abrogated the protective effects of Na2S on the FASEB J. 2005;19:1196 –1198.
outcome of CA/CPR. In a previous study, we found that 13. Elrod JW, Calvert JW, Morrison J, Doeller JE, Kraus DW, Tao L, Jiao X,
NOS3 deficiency markedly worsened outcomes of CA/ Scalia R, Kiss L, Szabo C, Kimura H, Chow CW, Lefer DJ. Hydrogen sulfide
attenuates myocardial ischemia-reperfusion injury by preservation of mito-
CPR, suggesting a salutary role of NOS3 on the outcome chondrial function. Proc Natl Acad Sci U S A. 2007;104:15560–15565.
of CA/CPR.19 Taken together, these results suggest a 14. Sodha NR, Clements RT, Feng J, Liu Y, Bianchi C, Horvath EM, Szabo
critical role of NOS3/NO in the mechanisms responsible C, Sellke FW. The effects of therapeutic sulfide on myocardial apoptosis
for the protective effects of Na2S after CA/CPR. in response to ischemia-reperfusion injury. Eur J Cardiothorac Surg.
2008;33:906 –913.
In summary, the present study revealed robust protective 15. Yong QC, Lee SW, Foo CS, Neo KL, Chen X, Bian JS. Endogenous
effects of Na2S on the outcome of CA/CPR in mouse. hydrogen sulphide mediates the cardioprotection induced by ischemic
Administration of Na2S 1 minute before the start of CPR postconditioning. Am J Physiol. 2008;295:H1330 –H1340.
16. Jha S, Calvert JW, Duranski MR, Ramachandran A, Lefer DJ. Hydrogen
markedly improved myocardial and neurological function
sulfide attenuates hepatic ischemia-reperfusion injury: role of antioxidant
and survival 24 hours after normothermic CA/CPR. These and antiapoptotic signaling. Am J Physiol. 2008;295:H801–H806.
observations, if extrapolated to human beings, may be highly 17. Tripatara P, SA Patel N, Collino M, Gallicchio M, Kieswich J, Castiglia
clinically relevant because they suggest that Na2S adminis- S, Benetti E, Stewart KN, Brown PA, Yaqoob MM, Fantozzi R, Thi-
emermann C. Generation of endogenous hydrogen sulfide by cysta-
tration can improve the outcome of CA at the time of CPR thionine ␥-lyase limits renal ischemia/reperfusion injury and dysfunction.
when intravenous access is obtained. Further studies are Lab Invest. 2008;88:1038 –1048.
warranted to elucidate the impact of exogenous and endoge- 18. Abella BS, Zhao D, Alvarado J, Hamann K, Vanden Hoek TL, Becker
nous H2S on the longer-term outcome of CA/CPR compli- LB. Intra-arrest cooling improves outcomes in a murine cardiac arrest
model. Circulation. 2004;109:2786 –2791.
cated by post-CA syndrome. 19. Nishida T, Yu JD, Minamishima S, Sips PY, Searles RJ, Buys ES,
Janssens S, Brouckaert P, Bloch KD, Ichinose F. Protective effects of
Acknowledgments nitric oxide synthase 3 and soluble guanylate cyclase on the outcome of
The authors thank Ikaria Inc for generously providing IK-1001 and cardiac arrest and cardiopulmonary resuscitation in mice. Crit Care Med.
Dr Christophe Adrie for valuable advice. 2009;37:256 –262.
20. Baines CP, Kaiser RA, Purcell NH, Blair NS, Osinska H, Hambleton MA,
Brunskill EW, Sayen MR, Gottlieb RA, Dorn GW, Robbins J, Molkentin
Sources of Funding JD. Loss of cyclophilin D reveals a critical role for mitochondrial per-
This work was supported by National Institutes of Health grants meability transition in cell death. Nature. 2005;434:658 – 662.
HL92141 (Drs Lefer and Elrod), HL92737 (Dr Elrod), HL70896 (Dr 21. Kofler J, Hattori K, Sawada M, DeVries AC, Martin LJ, Hurn PD,
Bloch), HL71987 (Dr Ichinose), and GM79360 (Dr Ichinose) and the Traystman RJ. Histopathological and behavioral characterization of a
Gas-Enabled Medical Innovation Fund (Dr Ichinose). novel model of cardiac arrest and cardiopulmonary resuscitation in mice.
J Neurosci Methods. 2004;136:33– 44.
Disclosures 22. Hausenloy DJ, Yellon DM. New directions for protecting the heart
against ischaemia-reperfusion injury: targeting the reperfusion injury
None. salvage kinase (RISK)-pathway. Cardiovasc Res. 2004;61:448 – 460.
23. Young LH, Li J, Baron SJ, Russell RR. AMP-activated protein kinase: a
References key stress signaling pathway in the heart. Trends Cardiovasc Med. 2005;
1. Nadkarni VM, Larkin GL, Peberdy MA, Carey SM, Kaye W, Mancini 15:110 –118.
ME, Nichol G, Lane-Truitt T, Potts J, Ornato JP, Berg RA, for the 24. Doeller JE, Isbell TS, Benavides G, Koenitzer J, Patel H, Patel RP,
National Registry of Cardiopulmonary Resuscitation Investigators. First Lancaster JR Jr, Darley-Usmar VM, Kraus DW. Polarographic mea-
documented rhythm and clinical outcome from in-hospital cardiac arrest surement of hydrogen sulfide production and consumption by mammalian
among children and adults. JAMA. 2006;295:50 –57. tissues. Anal Biochem. 2005;341:40 –51.
2. Adrie C, Adib-Conquy M, Laurent I, Monchi M, Vinsonneau C, Fitting 25. Yang G, Yang W, Wu L, Wang R. H2S, endoplasmic reticulum stress, and
C, Fraisse F, Dinh-Xuan AT, Carli P, Spaulding C, Dhainaut JF, apoptosis of insulin-secreting beta cells. J Biol Chem. 2007;282:16567–16576.

Downloaded from http://circ.ahajournals.org/ by guest on April 15, 2015

896 Circulation September 8, 2009

26. Tong H, Imahashi K, Steenbergen C, Murphy E. Phosphorylation of 28. Cohen MV, Yang XM, Downey JM. The pH hypothesis of postcon-
glycogen synthase kinase-3 ␤ during preconditioning through a ditioning: staccato reperfusion reintroduces oxygen and perpetuates
phosphatidylinositol-3-kinase-dependent pathway is cardioprotective. myocardial acidosis. Circulation. 2007;115:1895–1903.
Circ Res. 2002;90:377–379. 29. Tsujimoto Y, Shimizu S. Role of the mitochondrial membrane perme-
27. Das A, Xi L, Kukreja RC. Protein kinase G-dependent cardiopro- ability transition in cell death. Apoptosis. 2007;12:835– 840.
tective mechanism of phosphodiesterase-5 inhibition involves phos- 30. Pan TT, Feng ZN, Lee SW, Moore PK, Bian JS. Endogenous hydrogen sulfide
phorylation of ERK and GSK3 ␤ . J Biol Chem.. 2008;283: contributes to the cardioprotection by metabolic inhibition preconditioning in the
29572–29585. rat ventricular myocytes. J Mol Cell Cardiol. 2006;40:119–130.

CLINICAL PERSPECTIVE
Sudden cardiac arrest is one of the leading causes of death worldwide. Despite advances in cardiopulmonary resuscitation
(CPR) methods, including the introduction of the automatic electric defibrillator and therapeutic hypothermia, 60% to 80%
of these arrests result in immediate death, and of the remaining, only ⬇5% are successfully resuscitated to the extent that
they are returned to productive lives. Poor outcome from cardiac arrest is at least partly due to the lack of therapeutic
possibilities. In this study, we examined effects of hydrogen sulfide, a gaseous molecule with multifaceted protective
properties, on the outcome of cardiac arrest and CPR in mice. We found that administration of sodium sulfide, a hydrogen
sulfide donor, 1 minute before CPR markedly improved neurological and myocardial function and survival after 8 minutes
of cardiac arrest in mice. These observations, if extrapolated to human beings, may be highly clinically relevant because
they suggest that sodium sulfide administration can improve the outcome of sudden cardiac arrest at the time of CPR when
intravenous access is obtained. Further studies are warranted to elucidate the impact of exogenous and endogenous
hydrogen sulfide on the longer-term outcome of cardiac arrest and CPR complicated by post– cardiac arrest syndrome.

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 Supplemental Material MS ID#: CIRCULATIONAHA/2008/833491 Page 1

SAPPLEMENTAL MATERIAL for “Hydrogen sulfide improves survival after cardiac

arrest and cardiopulmonary resuscitation via a nitric oxide synthase 3 dependent
mechanism in mice. (MS ID#: CIRCULATIONAHA/2008/833491)”

Supplemental Methods

Assessment of neurological function—Neurological function was assessed at 24h after

CA/CPR or sham surgery using a previously-reported neurological function scoring

system with minor modifications 1. Briefly, five parameters were assessed and scored:

level of consciousness (no reaction to pinching of tail = 0, poor response to tail pinch =

1, normal response to tail pinch = 2), corneal reflex (no blinking = 0, sluggish blinking =

1, normal blinking = 2), respirations (irregular breathing pattern = 0, decreased

breathing frequency with normal pattern = 1, normal breathing frequency and pattern =

2), coordination (no movement = 0, moderate ataxia = 1, normal coordination = 2), and

movement/activity (no spontaneous movement = 0, sluggish movement = 1, normal

movement = 2). Total score was reported as the neurological function score (total

possible score = 10).

Histological studies—Twenty-four or 72h after CA/CPR or sham surgery, mice were

sacrificed, and brains were harvested, fixed in 4% formalin in PBS, and embedded in

paraffin. Brains were cut with a microtome in coronal planes including the hippocampus

(6 µm thickness). Activation of caspase 3 was assessed by immunohistochemistry in

paraffin-embedded brain sections obtained 24h after CA/CPR using a rabbit monoclonal
 Supplemental Material MS ID#: CIRCULATIONAHA/2008/833491 Page 2

antibody against cleaved caspase 3 (1:80, Cell Signaling) according to the protocol

recommended by the manufacturer. Non-viable neurons were counted in brain sections

obtained 72h after CA/CPR stained with hematoxylin and eosin based on cellular

morphology as previously reported.2 Cleaved caspase 3-positive or non-viable neurons

in the CA1 sector of the hippocampus were manually counted by an investigator blinded

to the treatment group, and the number of these neurons per square millimeters of

examined area was reported.

Measurement of protein levels and phosphorylation—LV and cerebral cortex tissue

were obtained 15 min after CA/CPR or sham operation, and tissue homogenates were

centrifuged for 20 min at 20,000 g. Supernatant proteins (15-60 µg) were fractionated

on 7 or 12% SDS-PAGE gels and transferred to PVDF membranes. Membranes were

blocked for 1 h in 5% BSA or 2% ECL-Advance reagent (GE Healthcare) and incubated

overnight with primary antibodies (Cell Signaling Technology Inc. unless otherwise

noted) against total Akt (1:1,000), phospho-Akt (Ser473,1:1,000), total NOS3 (1:5,000;

BD Biosciences), phospho-NOS3 (Ser1179, 1:5,000), total AMP-dependent kinase α

(AMPKα, 1:10,000), phospho-AMPKα (Thr172, 1:10,000), total glycogen synthase kinase

3β (GSK3β, 1:10,000), phospho-GSK3β (Ser9, 1:10,000), cleaved caspase-3 (1:5,000),

vinculin (1:2000 Sigma), and β-tubulin (1:2,000). Bound antibody was detected with a

horseradish peroxidase-linked antibody directed against rabbit IgG (1:5000; Cell

Signaling Technology Inc.) or mouse IgG (1:20,000 Sigma) and was visualized using

chemiluminescence with ECL Plus or ECL Advanced kit.

 Supplemental Material MS ID#: CIRCULATIONAHA/2008/833491 Page 3

Supplemental Table 1. Group characteristics before cardiac arrest and in the first hour

after cardiac arrest and CPR

Vehicle Na2S Post-CPR Na2S

(n=21) (n=13) (n=11)

Weight, g 25.3±0.3 25.2±0.4 25.4±0.4

HR before CA, bpm 517±16 567±17 510±16

MAP before CA, mmHg 127±3 125±3 129±3

Total dose of Epinephrine, µg 0.7±0.0 0.7±0.0 0.7±0.1

CPR time to ROSC, s 221±10 204±8 228±19

HR at ROSC, bpm 521±4 531±4 506±25

MAP at ROSC, mmHg 54±3 69±5 56±4

HR at 60 min after CPR, bpm 333±13 345±14 341±12

MAP at 60min after CPR, mmHg 48±4 50±3 46±4

Values are mean±SEM. Vehicle, mice subjected to CPR treated with vehicle; Na2S,

mice treated with Na2S 1 min before CPR; post-CPR Na2S, mice treated with Na2S 10

min after CPR; HR, heart rate; MAP, mean arterial pressure; CPR, cardiopulmonary

resuscitation; ROSC, return of spontaneous circulation. No differences were found

statistically significant.
 Supplemental Material MS ID#: CIRCULATIONAHA/2008/833491 Page 4

Supplemental Figure Legend

Supplemental Figure 1

Effects of delayed administration of Na2S in simulated ischemia-reperfusion in isolated-

perfused mouse hearts. Percentage of baseline values are shown for maximal LV

developed pressure (LVDevP), rate pressure product (RPP), maximal rate of LV

pressure rise (dP/dtmax), maximal rate of LV pressure reduction (dP/dtmin), the time

constant of isovolumic LV relaxation (Tau), and coronary flow rate (Flow Rate). No

difference was found between vehicle and delayed administration of Na2S on all

parameters. N=4 each.

Supplemental Figure 2

Additional immunoblots images are shown for phosphorylated (Ser473) and total Akt

(heart and brain), phosphorylated (Ser1179) and total NOS3 (heart and brain),

phosphorylated (Ser9) and total GSK3β (brain), and phosphorylated (Thr172) and total

AMPKα (brain). Expressoin level of vinculin or β-tubulin is shown as loading control for

some of the blots. Sham, sham-operated mice not subjected to cardiac arrest; Veh,

mice treated with vehicle 1 min before CPR; Na2S, mice treated with Na2S 1 min before

CPR; post-CPR Na2S, mice treated with Na2S 10 min after CPR.
 Supplemental Material MS ID#: CIRCULATIONAHA/2008/833491 Page 5

LVDevP RPP
100 100
Vehicle
80 Na2S 80
% of baseline

% of baseline
60 60

40 40

20 20

0 0
0 10 20 30 40 50 60 0 10 20 30 40 50 60
Time after reperfusion (min) Time after reperfusion (min)

dP/dtmax dP/dtmin
100 100

80 80
% of baseline

% of baseline
60 60

40 40

20 20

0 0
0 10 20 30 40 50 60 0 10 20 30 40 50 60
Time after reperfusion (min) Time after reperfusion (min)

Flow Rate Tau

100 500

80 400
% of baseline

% of baseline

60 300

40 200

20 100

0 0
0 10 20 30 40 50 60 0 10 20 30 40 50 60
Time after reperfusion (min) Time after reperfusion (min)

Supplemental Figure 1
 Supplemental Material MS ID#: CIRCULATIONAHA/2008/833491 Page 6

post-CPR
Sham Veh Na2S Na2S

Heart p-Akt Ser473

total Akt

post-CPR
Sham Veh Na2S Na2S

Heart p-NOS3 Ser1179

total NOS3

Vinculin

Sham Vehicle Na2S

Brain p-GSK3β Ser9

total-GSK3β

β-tubulin

post-CPR
Sham Veh Na2S Na2S

Brain p-Akt Ser473

total Akt

post-CPR
Sham Veh Na2S Na2S

Brain p-NOS3 Ser1179

total NOS3
Vinculin

post-CPR
Sham Veh Na2S Na2S

Brain p-AMPKα Thr172

total AMPKα
Vinculin

Supplemental Figure 2
 Supplemental Material MS ID#: CIRCULATIONAHA/2008/833491 Page 7

Supplemental Reference List

(1) Abella BS, Zhao D, Alvarado J, Hamann K, Vanden Hoek TL, Becker LB. Intra-
arrest cooling improves outcomes in a murine cardiac arrest model. Circulation
2004 June 8;109(22):2786-91.

(2) Kofler J, Hattori K, Sawada M, DeVries AC, Martin LJ, Hurn PD, Traystman RJ.
Histopathological and behavioral characterization of a novel model of cardiac
arrest and cardiopulmonary resuscitation in mice. J Neurosci Methods 2004 June
15;136(1):33-44.
 Hydrogen Sulfide Improves Survival After Cardiac Arrest and Cardiopulmonary
Resuscitation via a Nitric Oxide Synthase 3−Dependent Mechanism in Mice
Shizuka Minamishima, Masahiko Bougaki, Patrick Y. Sips, Jia De Yu, Yoji Andrew
Minamishima, John W. Elrod, David J. Lefer, Kenneth D. Bloch and Fumito Ichinose

Circulation. 2009;120:888-896; originally published online August 24, 2009;

doi: 10.1161/CIRCULATIONAHA.108.833491
Circulation is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2009 American Heart Association, Inc. All rights reserved.
Print ISSN: 0009-7322. Online ISSN: 1524-4539

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