ABSTRACT Immature skeletal muscle cells, or myoblasts, have been used in cellular cardiomyoplasty... more ABSTRACT Immature skeletal muscle cells, or myoblasts, have been used in cellular cardiomyoplasty, which attempts to regenerate cardiac muscle tissue by injecting cells into damaged,myocardium. In some studies, muscle tissue within myoblast implant sites may be morphologically similar to cardiac muscle. We hypothesized that identifiable aspects of the cardiac milieu may contribute to the growth and development of implanted myoblasts in vivo. To test this hypothesis, we designed a novel in vitro system to mimic some aspects of the electrical and biochemical
Immature skeletal muscle cells, or myoblasts, have been used in cellular cardiomyoplasty, which a... more Immature skeletal muscle cells, or myoblasts, have been used in cellular cardiomyoplasty, which attempts to regenerate cardiac muscle tissue by injecting cells into damaged,myocardium. In some studies, muscle tissue within myoblast implant sites may be morphologically similar to cardiac muscle. We hypothesized that identifiable aspects of the cardiac milieu may contribute to the growth and development of implanted myoblasts in vivo. To test this hypothesis, we designed a novel in vitro system to mimic some aspects of the electrical and biochemical
Transcriptomics is the study of how our genes are regulated and expressed in different biological... more Transcriptomics is the study of how our genes are regulated and expressed in different biological settings. Technical advances now enable quantitative assessment of all expressed genes (ie, the entire "transcriptome") in a given tissue at a given time. These approaches provide a powerful tool for understanding complex biological systems and for developing novel biomarkers. This chapter will introduce basic concepts in transcriptomics and available technologies for developing transcriptomic biomarkers. We will then review current and emerging applications in cardiovascular medicine.
ABSTRACT Spectral imaging as a modality combines two powerful techniques of imaging and fluoresce... more ABSTRACT Spectral imaging as a modality combines two powerful techniques of imaging and fluorescence spectroscopy. By generating a 2-D image of an object with fluorescence information at every pixel, spectral imaging has the potential to provide clinicians with a valuable tool which can not only diagnosis the tissue but also provide an image of the boundary of where the normal and cancerous tissue intersect. The system used in these experiments was a modified spectral imaging system from Applied Spectral Imaging (SD-200, Carlsbad, CA). The system was mounted in an off-microscope configuration so that instead of performing microscopic measurements of tissue, macroscopic measurements on the order of several millimeters in size were collected. Preliminary results indicate that the spectra acquired from human brain tissues in vitro at individual pixels of the spectral image cube appear similar to that acquired using the single pixel system. Based on the findings of this study, spectral imaging has the potential to be a useful tool for tissue diagnostics and is currently limited by the speed of data acquisition and size of the data.
Methods in molecular biology (Clifton, N.J.), 2014
Micropatterning is a powerful technique to control cell shape and position on a culture substrate... more Micropatterning is a powerful technique to control cell shape and position on a culture substrate. In this chapter, we describe the method to reproducibly create large numbers of micropatterned heterotypic cell pairs with defined size, shape, and length of cell-cell contact. These cell pairs can be utilized in patch clamp recordings to quantify electrical interactions between cardiomyocytes and non-cardiomyocytes.
The Journal of Heart and Lung Transplantation, 2009
Purpose: Continuous flow LVADs may impair normal blood clotting and therefore, could predispose t... more Purpose: Continuous flow LVADs may impair normal blood clotting and therefore, could predispose to increased bleeding events. To study this, bleeding and thromboembolic event rates were compared for patients supported with the HeartMate XVE (pulsatile) versus the HeartMate II (continuous-flow) LVAD. Methods and Materials: After IRB approval, we reviewed 99 consecutive patients who were supported with either the HeartMate XVE (nϭ50) or HeartMate II (nϭ49) devices between 2004 and 2007 at a single institution. Isolated periprocedural events (within 7 days) were excluded. A bleeding event was defined as any bleeding requiring hospitalization or transfusion. Thromboembolism was identified by CT scan, angiography, or autopsy. Data were analyzed with a t-test, Fischer's Exact test, or the Kaplan-Meier method, as appropriate. Results: Baseline characteristics for the 2 groups were similar, including age, heart failure etiology and overall survival. More female patients received support with the HeartMate II device. Average INR at the time of the events was 1.65 for the HeartMate II group and 1.85 for the XVE group (p ϭ 0.3). Thromboembolic event rates were very low for both groups where there were no events in the XVE cohort and one event in the HeartMate II cohort. More HeartMate II patients bled (32/49, 65% vs 22/50, 44% pϭ0.03); HeartMate II patients bled sooner than XVE patients (see . Conclusions: Thromboembolic event rates were exceptional low for both the XVE and HeartMate II cohorts. Patients supported with the HeartMate II device experienced earlier and more frequent bleeding events. Anticoagulation does not fully account for the increased bleeding observed with HeartMate II patients as the groups had similar INRs.
IEEE Engineering in Medicine and Biology Magazine, 2000
ABSTRACT Currently, the largest ongoing effort in cellular and tissue cardiomyoplasty therapies i... more ABSTRACT Currently, the largest ongoing effort in cellular and tissue cardiomyoplasty therapies is the search for a suitable cell source. The following are the desirable attributes of an ideal cell source: 1) autologous (not requiring immune suppression), 2) able to substantially proliferate in vitro, 3) having a similar function when compared to host cardiomyocytes, and 4) able to couple with the host cells electrically and mechanically. A number of cell types have been attempted for cellular and tissue engineering therapies in the heart.
To summarize the available evidence concerning the occurrence and treatment of ventricular arrhyt... more To summarize the available evidence concerning the occurrence and treatment of ventricular arrhythmias in patients supported with long-term ventricular assist devices (VADs). Approximately one-third of left ventricular assist device-supported patients experience significant ventricular arrhythmias, with higher rates in certain patient subsets. Ventricular arrhythmias are associated with both increased mortality and morbidity in VAD-supported patients. Mechanical factors, myocardial fibrosis and alterations in cardiac myocyte physiology because of myocardial unloading are contributors to ventricular arrhythmias in this population. In the absence of definitive trials, current evidence supports implanted cardioverter defibrillators (ICDs) in long-term VAD patients to mitigate the risks associated with ventricular arrhythmias. Though antiarrhythmic therapies have limited efficacy, amelioration of inflow cannula contact with the endocardium and suction events or ablation of specific anatomic origins of ventricular arrhythmias, when present, are also efficacious in VAD-supported hearts. As the application of long-term VAD support continues to grow, it will be increasingly important to clarify and target the mechanisms contributing to ventricular arrhythmias in this population. Prospective trials assessing the benefits of de-novo ICD placement, ablative strategies and other prophylactic and therapeutic interventions will be increasingly important to further improve the survival and quality of life among VAD-supported patients.
The purpose was to summarize the findings of the proangiogenic clinical trials using protein and ... more The purpose was to summarize the findings of the proangiogenic clinical trials using protein and gene therapy, with analysis of the problems and an interpretation of the results. Recent findings include several new large clinical trials, using both gene and protein therapies. There has been development of new basic science concepts, especially with regard to endothelial activation and stabilization of newly formed microvessels. This review provides a critical analysis of the most recent clinical trials, both in efforts to understand the pitfalls of earlier clinical trials, and also to focus on requirements for future studies. This article reviews many of the clinical trials utilizing proangiogenic therapy, assesses the pitfalls seen within the current trials, and discusses the conclusions drawn and the future of angiogenesis therapy.
Aims The pathological proliferation of cardiac fibroblasts (CFs) in response to heart injury resu... more Aims The pathological proliferation of cardiac fibroblasts (CFs) in response to heart injury results in fibrosis, which correlates with arrhythmia generation and heart failure. Here we systematically examined the effect of fibroblast-derived paracrine factors on electrical propagation in cardiomyocytes. Methods and results Neonatal rat cardiac monolayers were exposed for 24 h to media conditioned by CFs. Optical mapping, sharp microelectrode recordings, quantitative RT-PCR, and immunostaining were used to assess the changes in the propagation and shape of the action potential and underlying changes in gene and protein expression. The fibroblast paracrine factors produced a 52% reduction in cardiac conduction velocity, a 217% prolongation of action potential duration, a 64% decrease of maximum capture rate, a 21% increase in membrane resting potential, and an 80% decrease of action potential upstroke velocity. These effects were dose dependent and partially reversible with removal of the conditioned media. No fibroblast proliferation, cardiomyocyte apoptosis, or decreased connexin-43 expression, phosphorylation, and function were found in conditioned cardiac cultures. In contrast, the expression of the fast sodium, inward rectifying potassium, and transient outward potassium channels were, respectively, reduced 3.8-, 6.6-fold, and to undetectable levels. The expression of b-myosin heavy chain increased 17.4-fold. No electrophysiological changes were observed from media conditioned by CFs in the presence of cardiomyocytes. Conclusion Paracrine factors from neonatal CFs alone produced significant electrophysiological changes in neonatal rat cardiomyocytes resembling those found in several cardiac pathologies.
Immature skeletal muscle cells, or myoblasts, have been used in cellular cardiomyoplasty in attem... more Immature skeletal muscle cells, or myoblasts, have been used in cellular cardiomyoplasty in attempts to regenerate cardiac muscle tissue by injection of cells into damaged myocardium. In some studies, muscle tissue within myoblast implant sites may be morphologically similar to cardiac muscle. We hypothesized that identifiable aspects of the cardiac milieu may contribute to growth and development of implanted myoblasts in vivo. To test this hypothesis, we designed a novel in vitro system to mimic some aspects of the electrical and biochemical environment of native myocardium. This system enabled us to separate the three-dimensional (3-D) electrical and biochemical signals that may be involved in myoblast proliferation and plasticity. Myoblasts were grown on 3-D polyglycolic acid mesh scaffolds under control conditions, in the presence of cardiac-like electrical current fluxes, or in the presence of culture medium that had been conditioned by mature cardiomyocytes. Cardiac-like electrical current fluxes caused increased myoblast number in 3-D culture, as determined by DNA assay. The increase in cell number was due to increased cellular proliferation and not differences in apoptosis, as determined by proliferating cell nuclear antigen and TdT-mediated dUTP nick-end labeling. Cardiomyocyte-conditioned medium also significantly increased myoblast proliferation. Expression of transcription factors governing differentiation along skeletal or cardiac lineages was evaluated by immunoblotting. Although these assays are qualitative, no changes in differentiation state along skeletal or cardiac lineages were observed in response to electrical current fluxes. Furthermore, from these experiments, conditioned medium did not appear to alter the differentiation state of skeletal myoblasts. Hence, cardiac milieu appears to stimulate proliferation but does not affect differentiation of skeletal myoblasts.
Well-controlled studies of the structural and functional interactions between cardiomyocytes and ... more Well-controlled studies of the structural and functional interactions between cardiomyocytes and other cells are essential for understanding heart pathophysiology and for the further development of safe and efficient cell therapies. We established a novel in vitro assay composed of a large number of individual micropatterned cell pairs with reproducible shape, size, and region of cell-cell contact. This assay was applied to quantify and compare the frequency of expression and distribution of electrical (connexin43) and mechanical (N-cadherin) coupling proteins in 5,000 cell pairs made of cardiomyocytes (CMs), cardiac fibroblasts (CFs), skeletal myoblasts (SKMs), and mesenchymal stem cells (MSCs). We found that for all cell pair types, side-side contacts between two cells formed 4.5-14.3 times more often than end-end contacts. Both connexin43 and N-cadherin were expressed in all homotypic CM pairs but in only 13.4-91.6% of pairs containing noncardiomyocytes, where expression was either junctional (at the site of cell-cell contact) or diffuse (inside the cytoplasm). CM expression was exclusively junctional in homotypic pairs but predominantly diffuse in heterotypic pairs. Noncardiomyocyte homotypic pairs exhibited diffuse expression 1.7-8.7 times more often than junctional expression, which was increased 2.6-4.4 times in heterotypic pairs. Junctional connexin43 and N-cadherin expression, respectively, were found in 38.6 +/- 7.3 and 39.6 +/- 6.2% of CM-MSC pairs, 21.9 +/- 5.0 and 13.6 +/- 1.9% of CM-SKM pairs, and in only 3.8-9.6% of CM-CF pairs. Measured frequencies of protein expression and distribution were stable for at least 4 days. Described studies in micropatterned cell pairs shed new light on cellular interactions relevant for cardiac function and cell therapies.
ABSTRACT Immature skeletal muscle cells, or myoblasts, have been used in cellular cardiomyoplasty... more ABSTRACT Immature skeletal muscle cells, or myoblasts, have been used in cellular cardiomyoplasty, which attempts to regenerate cardiac muscle tissue by injecting cells into damaged,myocardium. In some studies, muscle tissue within myoblast implant sites may be morphologically similar to cardiac muscle. We hypothesized that identifiable aspects of the cardiac milieu may contribute to the growth and development of implanted myoblasts in vivo. To test this hypothesis, we designed a novel in vitro system to mimic some aspects of the electrical and biochemical
Immature skeletal muscle cells, or myoblasts, have been used in cellular cardiomyoplasty, which a... more Immature skeletal muscle cells, or myoblasts, have been used in cellular cardiomyoplasty, which attempts to regenerate cardiac muscle tissue by injecting cells into damaged,myocardium. In some studies, muscle tissue within myoblast implant sites may be morphologically similar to cardiac muscle. We hypothesized that identifiable aspects of the cardiac milieu may contribute to the growth and development of implanted myoblasts in vivo. To test this hypothesis, we designed a novel in vitro system to mimic some aspects of the electrical and biochemical
Transcriptomics is the study of how our genes are regulated and expressed in different biological... more Transcriptomics is the study of how our genes are regulated and expressed in different biological settings. Technical advances now enable quantitative assessment of all expressed genes (ie, the entire "transcriptome") in a given tissue at a given time. These approaches provide a powerful tool for understanding complex biological systems and for developing novel biomarkers. This chapter will introduce basic concepts in transcriptomics and available technologies for developing transcriptomic biomarkers. We will then review current and emerging applications in cardiovascular medicine.
ABSTRACT Spectral imaging as a modality combines two powerful techniques of imaging and fluoresce... more ABSTRACT Spectral imaging as a modality combines two powerful techniques of imaging and fluorescence spectroscopy. By generating a 2-D image of an object with fluorescence information at every pixel, spectral imaging has the potential to provide clinicians with a valuable tool which can not only diagnosis the tissue but also provide an image of the boundary of where the normal and cancerous tissue intersect. The system used in these experiments was a modified spectral imaging system from Applied Spectral Imaging (SD-200, Carlsbad, CA). The system was mounted in an off-microscope configuration so that instead of performing microscopic measurements of tissue, macroscopic measurements on the order of several millimeters in size were collected. Preliminary results indicate that the spectra acquired from human brain tissues in vitro at individual pixels of the spectral image cube appear similar to that acquired using the single pixel system. Based on the findings of this study, spectral imaging has the potential to be a useful tool for tissue diagnostics and is currently limited by the speed of data acquisition and size of the data.
Methods in molecular biology (Clifton, N.J.), 2014
Micropatterning is a powerful technique to control cell shape and position on a culture substrate... more Micropatterning is a powerful technique to control cell shape and position on a culture substrate. In this chapter, we describe the method to reproducibly create large numbers of micropatterned heterotypic cell pairs with defined size, shape, and length of cell-cell contact. These cell pairs can be utilized in patch clamp recordings to quantify electrical interactions between cardiomyocytes and non-cardiomyocytes.
The Journal of Heart and Lung Transplantation, 2009
Purpose: Continuous flow LVADs may impair normal blood clotting and therefore, could predispose t... more Purpose: Continuous flow LVADs may impair normal blood clotting and therefore, could predispose to increased bleeding events. To study this, bleeding and thromboembolic event rates were compared for patients supported with the HeartMate XVE (pulsatile) versus the HeartMate II (continuous-flow) LVAD. Methods and Materials: After IRB approval, we reviewed 99 consecutive patients who were supported with either the HeartMate XVE (nϭ50) or HeartMate II (nϭ49) devices between 2004 and 2007 at a single institution. Isolated periprocedural events (within 7 days) were excluded. A bleeding event was defined as any bleeding requiring hospitalization or transfusion. Thromboembolism was identified by CT scan, angiography, or autopsy. Data were analyzed with a t-test, Fischer's Exact test, or the Kaplan-Meier method, as appropriate. Results: Baseline characteristics for the 2 groups were similar, including age, heart failure etiology and overall survival. More female patients received support with the HeartMate II device. Average INR at the time of the events was 1.65 for the HeartMate II group and 1.85 for the XVE group (p ϭ 0.3). Thromboembolic event rates were very low for both groups where there were no events in the XVE cohort and one event in the HeartMate II cohort. More HeartMate II patients bled (32/49, 65% vs 22/50, 44% pϭ0.03); HeartMate II patients bled sooner than XVE patients (see . Conclusions: Thromboembolic event rates were exceptional low for both the XVE and HeartMate II cohorts. Patients supported with the HeartMate II device experienced earlier and more frequent bleeding events. Anticoagulation does not fully account for the increased bleeding observed with HeartMate II patients as the groups had similar INRs.
IEEE Engineering in Medicine and Biology Magazine, 2000
ABSTRACT Currently, the largest ongoing effort in cellular and tissue cardiomyoplasty therapies i... more ABSTRACT Currently, the largest ongoing effort in cellular and tissue cardiomyoplasty therapies is the search for a suitable cell source. The following are the desirable attributes of an ideal cell source: 1) autologous (not requiring immune suppression), 2) able to substantially proliferate in vitro, 3) having a similar function when compared to host cardiomyocytes, and 4) able to couple with the host cells electrically and mechanically. A number of cell types have been attempted for cellular and tissue engineering therapies in the heart.
To summarize the available evidence concerning the occurrence and treatment of ventricular arrhyt... more To summarize the available evidence concerning the occurrence and treatment of ventricular arrhythmias in patients supported with long-term ventricular assist devices (VADs). Approximately one-third of left ventricular assist device-supported patients experience significant ventricular arrhythmias, with higher rates in certain patient subsets. Ventricular arrhythmias are associated with both increased mortality and morbidity in VAD-supported patients. Mechanical factors, myocardial fibrosis and alterations in cardiac myocyte physiology because of myocardial unloading are contributors to ventricular arrhythmias in this population. In the absence of definitive trials, current evidence supports implanted cardioverter defibrillators (ICDs) in long-term VAD patients to mitigate the risks associated with ventricular arrhythmias. Though antiarrhythmic therapies have limited efficacy, amelioration of inflow cannula contact with the endocardium and suction events or ablation of specific anatomic origins of ventricular arrhythmias, when present, are also efficacious in VAD-supported hearts. As the application of long-term VAD support continues to grow, it will be increasingly important to clarify and target the mechanisms contributing to ventricular arrhythmias in this population. Prospective trials assessing the benefits of de-novo ICD placement, ablative strategies and other prophylactic and therapeutic interventions will be increasingly important to further improve the survival and quality of life among VAD-supported patients.
The purpose was to summarize the findings of the proangiogenic clinical trials using protein and ... more The purpose was to summarize the findings of the proangiogenic clinical trials using protein and gene therapy, with analysis of the problems and an interpretation of the results. Recent findings include several new large clinical trials, using both gene and protein therapies. There has been development of new basic science concepts, especially with regard to endothelial activation and stabilization of newly formed microvessels. This review provides a critical analysis of the most recent clinical trials, both in efforts to understand the pitfalls of earlier clinical trials, and also to focus on requirements for future studies. This article reviews many of the clinical trials utilizing proangiogenic therapy, assesses the pitfalls seen within the current trials, and discusses the conclusions drawn and the future of angiogenesis therapy.
Aims The pathological proliferation of cardiac fibroblasts (CFs) in response to heart injury resu... more Aims The pathological proliferation of cardiac fibroblasts (CFs) in response to heart injury results in fibrosis, which correlates with arrhythmia generation and heart failure. Here we systematically examined the effect of fibroblast-derived paracrine factors on electrical propagation in cardiomyocytes. Methods and results Neonatal rat cardiac monolayers were exposed for 24 h to media conditioned by CFs. Optical mapping, sharp microelectrode recordings, quantitative RT-PCR, and immunostaining were used to assess the changes in the propagation and shape of the action potential and underlying changes in gene and protein expression. The fibroblast paracrine factors produced a 52% reduction in cardiac conduction velocity, a 217% prolongation of action potential duration, a 64% decrease of maximum capture rate, a 21% increase in membrane resting potential, and an 80% decrease of action potential upstroke velocity. These effects were dose dependent and partially reversible with removal of the conditioned media. No fibroblast proliferation, cardiomyocyte apoptosis, or decreased connexin-43 expression, phosphorylation, and function were found in conditioned cardiac cultures. In contrast, the expression of the fast sodium, inward rectifying potassium, and transient outward potassium channels were, respectively, reduced 3.8-, 6.6-fold, and to undetectable levels. The expression of b-myosin heavy chain increased 17.4-fold. No electrophysiological changes were observed from media conditioned by CFs in the presence of cardiomyocytes. Conclusion Paracrine factors from neonatal CFs alone produced significant electrophysiological changes in neonatal rat cardiomyocytes resembling those found in several cardiac pathologies.
Immature skeletal muscle cells, or myoblasts, have been used in cellular cardiomyoplasty in attem... more Immature skeletal muscle cells, or myoblasts, have been used in cellular cardiomyoplasty in attempts to regenerate cardiac muscle tissue by injection of cells into damaged myocardium. In some studies, muscle tissue within myoblast implant sites may be morphologically similar to cardiac muscle. We hypothesized that identifiable aspects of the cardiac milieu may contribute to growth and development of implanted myoblasts in vivo. To test this hypothesis, we designed a novel in vitro system to mimic some aspects of the electrical and biochemical environment of native myocardium. This system enabled us to separate the three-dimensional (3-D) electrical and biochemical signals that may be involved in myoblast proliferation and plasticity. Myoblasts were grown on 3-D polyglycolic acid mesh scaffolds under control conditions, in the presence of cardiac-like electrical current fluxes, or in the presence of culture medium that had been conditioned by mature cardiomyocytes. Cardiac-like electrical current fluxes caused increased myoblast number in 3-D culture, as determined by DNA assay. The increase in cell number was due to increased cellular proliferation and not differences in apoptosis, as determined by proliferating cell nuclear antigen and TdT-mediated dUTP nick-end labeling. Cardiomyocyte-conditioned medium also significantly increased myoblast proliferation. Expression of transcription factors governing differentiation along skeletal or cardiac lineages was evaluated by immunoblotting. Although these assays are qualitative, no changes in differentiation state along skeletal or cardiac lineages were observed in response to electrical current fluxes. Furthermore, from these experiments, conditioned medium did not appear to alter the differentiation state of skeletal myoblasts. Hence, cardiac milieu appears to stimulate proliferation but does not affect differentiation of skeletal myoblasts.
Well-controlled studies of the structural and functional interactions between cardiomyocytes and ... more Well-controlled studies of the structural and functional interactions between cardiomyocytes and other cells are essential for understanding heart pathophysiology and for the further development of safe and efficient cell therapies. We established a novel in vitro assay composed of a large number of individual micropatterned cell pairs with reproducible shape, size, and region of cell-cell contact. This assay was applied to quantify and compare the frequency of expression and distribution of electrical (connexin43) and mechanical (N-cadherin) coupling proteins in 5,000 cell pairs made of cardiomyocytes (CMs), cardiac fibroblasts (CFs), skeletal myoblasts (SKMs), and mesenchymal stem cells (MSCs). We found that for all cell pair types, side-side contacts between two cells formed 4.5-14.3 times more often than end-end contacts. Both connexin43 and N-cadherin were expressed in all homotypic CM pairs but in only 13.4-91.6% of pairs containing noncardiomyocytes, where expression was either junctional (at the site of cell-cell contact) or diffuse (inside the cytoplasm). CM expression was exclusively junctional in homotypic pairs but predominantly diffuse in heterotypic pairs. Noncardiomyocyte homotypic pairs exhibited diffuse expression 1.7-8.7 times more often than junctional expression, which was increased 2.6-4.4 times in heterotypic pairs. Junctional connexin43 and N-cadherin expression, respectively, were found in 38.6 +/- 7.3 and 39.6 +/- 6.2% of CM-MSC pairs, 21.9 +/- 5.0 and 13.6 +/- 1.9% of CM-SKM pairs, and in only 3.8-9.6% of CM-CF pairs. Measured frequencies of protein expression and distribution were stable for at least 4 days. Described studies in micropatterned cell pairs shed new light on cellular interactions relevant for cardiac function and cell therapies.
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Papers by Dawn Pedrotty