L'insuffisance cardiaque (IC) est l'évolution terminale de nombreuses pathologies cardiaques, notamment de l'infarctus du myocarde (IDM). Elle se caractérise par la mort des cellules contractiles du cœur, les cardiomyocytes (CMs), et par l'incapacité des CMs survivants à régénérer le tissu lésé. Les thérapeutiques disponibles ralentissent sans toutefois stopper la progression de l'IC, expliquant l'émergence du concept de régénération tissulaire pour restaurer la fonction du cœur. Dans ce domaine, la thérapie cellulaire par injection de cellules exogènes (souches/progénitrices) dans le myocarde représente l'approche la plus conventionnelle. Malgré des résultats expérimentaux prometteurs, l'efficacité de cette stratégie reste encore mal établie en clinique. Ainsi, identifier de nouvelles voies thérapeutiques s'avère essentiel en médecine régénérative cardiaque. Jusqu'à récemment, les CMs survivant à l'IDM ont été ignorés en régénération cardiaque car considérés comme bloqués dans un état post-mitotique. Or, des études récentes ont montré que ces CMs adultes différenciés résidents sont dotés d'un potentiel naturel faible de prolifération qu'il est possible de stimuler afin de régénérer le tissu lésé. L'enjeu est maintenant d'identifier les mécanismes moléculaires naturels bloquant la prolifération du CM en vue d'une modulation en thérapeutique et a constitué le contexte de mon travail de thèse. Dans ce contexte, l'équipe a récemment identifié Ephrine-B1 comme nouvelle protéine spécifique de la membrane latérale (ML) du CM stabilisant la morphologie en brique du CM adulte et la cohésion du tissu cardiaque. De façon inattendue, au cours de mon travail de doctorat, nous avons montré, grâce à de multiples approches in vitro et in vivo, que la délétion d'Ephrine-B1 (souris KO ou thérapie génique avec interférence à l'ARN) confère au CM adulte un potentiel de prolifération important (~ 15%), mobilisé uniquement en situation de stress cardiaque (vieillissement, apectomie adulte ou IDM) et permettant la régénération cardiaque. Ephrine-B1 apparaît donc comme une cible prometteuse en médecine régénératrice cardiaque. [...] / Heart failure (HF) is the convergent evolution of many cardiac diseases, particularly myocardial infarction (MI). At the tissue level, HF is characterized by the death of cardiac contractile cells, the cardiomyocytes (CMs), and by the incapability of survivor CMs to regenerate the damaged tissue. Various pharmacological therapies have proven to slow HF progression but not to block it. Thus, major efforts have been developed in regenerative cardiac medicine to repair the scar tissue of failing heart to restore the function. In this context, cardiac cell therapy (injection of exogenous stem/progenitor cells) has been one of the more promising approaches. Besides its encouraging results in laboratories, its clinical benefit still remains elusive. Thus, there is an urgent need for identifying new therapeutic strategies for cardiac regenerative medicine. Until now, surviving resident CMs to MI have been ignored in cardiac regeneration since considered in a post-mitotic state, unable to proliferate. However, recent studies demonstrated that adult differentiated CMs can naturally proliferate but at low rate and that it is possible to stimulate this potential to regenerate the damaged tissue. The issue remains now to identify the natural molecular mechanisms involved in the post-mitotic blockage of adult CMs and has constituted my main thesis project. In this context, the team has recently identified Ephrin-B1 as a new specific protein of the CM lateral membrane (LM),stabilizing the adult CM rod-shape and the overall cardiac tissue cohesion. Surprisingly, during my thesis, we demonstrated, based on multiple in vitro and in vivo approaches, that Ephrin-B1 deletion (KO mice or RNA-interference based-gene therapy) confers an important proliferative potential to the adult CM. Interestingly, this potential is only mobilized under cardiac stress (aging, adult apectomy or MI). Thus, Ephrin-B1 deletion in CMs leads to substantial cardiac regeneration through their proliferation. Ephrin-B1 appears as a promising target for cardiac regenerative medicine. [...]

Cardiac diseases such as myocardial infarction (MI) can lead to adverse remodeling and impaired contractility of the heart due to widespread cardiomyocyte death in the damaged area. Current therapies focus on improving heart contractility and minimizing fibrosis with modest cardiac regeneration, but MI patients can still progress to heart failure (HF). There is a dire need for clinical therapies that can replace the lost myocardium, specifically by the induction of new myocyte formation from pre-existing cardiomyocytes. Many studies have shown terminally differentiated myocytes can re-enter the cell cycle and divide through manipulations of the cardiomyocyte cell cycle, signaling pathways, endogenous genes, and environmental factors. However, these approaches result in minimal myocyte renewal or cardiomegaly due to hyperactivation of cardiomyocyte proliferation. Finding the optimal treatment that will replenish cardiomyocyte numbers without causing tumorigenesis is a major challenge in the field. Another controversy is the inability to clearly define cardiomyocyte division versus myocyte DNA synthesis due to limited methods. A recent study suggests that systemic hypoxemia in adult male mice can induce cardiac myocytes to proliferate. The goal of the present experiments was to confirm these results, provide new insights on the mechanisms that induce cardiomyocyte cell cycle re-entry, and to determine if hypoxemia also induces cardiomyocyte proliferation and division in female mice. EdU mini pumps were implanted in 3-month-old, male and female C57BL/6 mice. Mice were then placed in a hypoxia chamber and the oxygen was lowered by 1% every day for 14 days to reach 7% oxygen. The animals remained in 7% inspired oxygen for 2 weeks before terminal studies. Myocyte cell cycle re-entry and division was also studied with a mosaic analysis with double markers (MADM) mouse model. MADM mice were exposed to hypoxia at 7% Oxygen as described above. Hypoxia induced cardiac hypertrophy in both left ventricular (LV) and right ventricular (RV) myocytes, with LV myocytes lengthening and RV myocytes widening and lengthening. Hypoxia induced a small increase in cardiomyocytes undergoing DNA synthesis (EdU+) in male and female C57BL/6 mice. Hypoxia induced a significant increase in myocyte cell cycle re-entry in MADM mice, but few myocytes synthesized new DNA (EdU+) and completed cytokinesis. RNA-sequencing showed upregulation in mitotic cell cycle processes but a downregulation of promoter genes for G1 to S phase transition in hypoxic mice when compared to control mice. There was also proliferation of non-myocyte cells and mild cardiac remodeling in hypoxic mice that did not disrupt cardiac function. Male and female mice exhibited similar gene expression profiles following hypoxia. Thus, systemic hypoxia induces adult cardiac myocyte cell cycle re-entry, but very few adult myocytes progress through the cell cycle to synthesize new DNA and divide into two daughter cells. / Biomedical Sciences

Cardiovascular disease remains the number one cause or mortally in the western world. Heart failure is the most rapidly growing cardiovascular disease (Hobbs, 2004; Levy, et al., 2002). Heart failure, by definition, is progressive deteriorating function of the heart due to progressive cardiac myocytes loss. Though after decades of endeavor of searching the pathophysiology and treatments for heart failure, it remains highly lethal. Therefore, it is vital to find novel therapies to help treat such chronic disease. Replace the lost cardiomyocyte with new ones could restore cardiac function and reduce mortality. The purpose of this study is to investigate on how TTCCs (T-type calcium channels) affect cardiomyocyte proliferation. In mice after birth, the major TTCC expressed in the heart is Cav3.1/α1G, and therefore we used Cav3.1/α1G transgenic (TG), knockout (-/-) and wild type mice respectively to define the role of TTCC in cardiomyocyte proliferation. In neonatal mouse ventricular myocyte (NMVMs) right after birth, there is almost no TTCC after birth in α1G-/- NMVMs, whereas there are around 35% NMVMs in wild type (WT) show TTCC. On day 7 after birth, there are no T-type calcium currents in both α1G-/- NMVMs and WT NMVMs. Using BrdU, a DNA synthesis marker, we identified plenty of BrdU positive cardiomyocyte during the first seven days after birth. Cardiomyocyte is special due to its double nucleation property. Our cell cycle studies showed that there is significant difference in cell cycle distribution between α1G-/- and WT NMVMs on day seven after birth. Significantly more NMVMs are arrested in G1 phase in α1G-/-, compared to WT NMVMs. Even until 2 month old, there are still significantly more mono-nucleated cardiomyocyte in α1G-/- than in WT. In conclusion, all these evidence showed that blocking T-type calcium channel could partially prevent binucleation from happening and stop cardiomyocytes withdrawal from cell cycle. Mononucleated cardiomyocyte is still able to proliferate. Hence, mononucleated cardiomyocytes in adult still have potential to proliferation because these cardiomyoctes are arrested in their cell-cycle before their terminal differentiation, which could offer a novel approach for cardiac repair. / Physiology

A key cardiovascular signaling molecule involved in both physiologic and pathologic regulation of cardiomyocytes is the small molecular weight G-protein, Ras. Differential effects of Ras are mediated by multiple effector molecules, including the RalGEFs which activate Ral. Studies performed in cardiomyocytes have indicated a role for Ral in cardiac hypertrophic signaling and the RalGEF family member, Rgl2, was shown to specifically interact with Ras in the heart. Therefore, I hypothesized that Rgl2 was an important Ras effector that would regulate cardiomyocyte signaling. To elucidate the potential importance of Rgl2 in regulating cardiomyocyte signaling, a gain-of-function approach was utilized in which NRVMs were infected with an adenovirus to increase Rgl2 expression. Using this approach, I found that Rgl2 increased Ral-GTP levels, Ras-GTP levels, and PI3-kinase-Akt signaling, but decreased ERK phosphorylation. Overall, my results suggest a model in which Rgl2 disrupts Ras-Raf and Ras-RasGAP interaction to decrease ERK phosphorylation and increase Ras-GTP, respectively. Furthermore, Rgl2-induced Ral activation promotes the enhanced PI3- kinase-Akt signaling. The physiologic consequence of Rgl2 signaling is difficult to predict, but the increase in PI3-kinase-Akt signaling would be expected to promote cardiomyocyte survival and enhance cardiac function, both of which are characteristic of physiologic hypertrophy.

Background: Ethanol is a cardiotoxic substance that damages the heart by increasing apoptosis, free radical formation and calcium overloading. Consequently, there is an increase in cell death leaving fewer functioning myocytes leading to heart failure. Sildenafil is a phosphodiesterase type-5 (PDE-5) inhibitor approved for treatment of erectile dysfunction. Studies from our lab have demonstrated that PDE-5 inhibition reduces myocardial infarct size and attenuates post-ischemic cardiac dysfunction in both ischemia-reperfusion and permanent coronary artery ligation models. Therefore, in the present study, we hypothesized that treatment with sildenafil will prevent cardiotoxicity associated with acute alcohol exposure by reducing myocyte apoptosis and preserving cardiac function through PKG signaling. Methods and Results: Adult cardiomyocytes were isolated and treated with 100 mM of 100% ethanol ± 10 µM sildenafil. At 24 hours necrosis was assessed via trypan blue exclusion assay, JC-1 staining assessed mitochondrial membrane potential and ROS production was measured by DCF fluorescence. At 48 hours apoptosis was assessed by TUNEL assay. Ethanol increased the rate of necrotic and apoptotic cell death. This was attenuated by co-treatment with sildenafil. Ethanol disrupted the mitochondrial membrane potential and increased ROS production. Sildenafil preserved mitochondrial membrane potential and attenuated ROS production. Treatment of myocytes with 5-HD, a mitochondrial K+atp channel antagonist, blocked the protective effect of sildenafil. Knockdown of PKG using adenoviral siRNA blocked the protective effect of sildenafil, while overexpression of PKG1α conferred protection against ethanol cytotoxicity. To further demonstrate the effect of sildenafil ethanol-cardiotoxicity in vivo, mice were treated with ethanol (3 g/kg/day) with or without sildenafil (0.7 mg/kg) by i.p. injection for three consecutive days. After treatment, the animals were sacrificed and the hearts removed and perfused on a Langendorff system to measure function. After functional analysis, apoptosis and PKG activity was measured in the heart samples. Ethanol decreased the rate-force product and increased myocardial apoptosis. Sildenafil preserved cardiac function and significantly reduced apoptosis. Sildenafil treated myocardium also showed an increase in PKG activity. Conclusion: Sildenafil attenuates the toxic effect of ethanol by reducing apoptosis and maintaining the mitochondrial integrity in cardiomyocytes. Sildenafil also preserved cardiac function in ethanol-treated mice. Protein kinase G-dependent signaling plays a critical role in attenuating cardiotoxic effect of ethanol.

Multi electrode array (MEA) systems have evolved from custom-made experimental tools, exploited for neural research, into commercially available systems that are used throughout non-invasive electrophysiological study. MEA systems are used in conjunction with cells and tissues from a number of differing organisms (e.g. mice, monkeys, chickens, plants). The development of MEA systems has been incremental over the past 30 years due to constantly changing specific bioscientific requirements in research. As the application of MEA systems continues to diversify contemporary commercial systems are requiring increased levels of sophistication and greater throughput capabilities.

<p>The role of cardiac tissue structure in both normal and abnormal impulse conduction has been extensively studied by researchers in cardiac electrophysiology. However, much is left unknown on how specific micro- and macroscopic structural features affect conduction and conduction block. Progress in this field is constrained by the inability to simultaneously assess intramural cardiac structure and function, as well as the intrinsic complexity and variability of intact tissue preparations. Cultured monolayers of cardiac cells, on the other hand, present a well-controlled in vitro model system that provides the necessary structural and functional simplifications to enable well-defined studies of electrical phenomena. In this thesis, I developed a novel, reproducible cell culture system that accurately replicates the realistic microstructure of cardiac tissues. This system was then applied to systematically explore the influence of natural structure (e.g. tissue boundaries, expansions, local fiber directions) on normal and arrhythmogenic electrical conduction.</p><p>Specifically, soft lithography techniques were used to design cell cultures based on microscopic DTMRI (diffusion tensor magnetic resonance imaging) measurements of fiber directions in murine ventricles. Protein micropatterns comprised of mosaics of square pixels with angled lines that followed in-plane cardiac fiber directions were created to control the adhesion and alignment of cardiac cells on a two-dimensional substrate. The high accuracy of cell alignment in the resulting micropatterned monolayers relative to the original DTMRI-measured fiber directions was validated using immunofluorescence and image processing techniques.</p><p>Using this novel model system, I first examined how specific structural features of murine ventricles influence basic electrical conduction. (1) Realistic ventricular tissue boundaries, either alone or with (2) microscopic fiber directions were micropatterned to distinguish their individual functional roles in action potential propagation. By optically mapping membrane potentials and applying low-rate pacing from multiple sites in culture, I found that ventricular tissue boundaries and fiber directions each shaped unique spatial patterns of impulse propagation and additively increased the spatial dispersion of conduction velocity.</p><p>To elucidate the roles that natural tissue structure play in arrhythmogenesis, I applied rapid-rate pacing from multiple sites in culture in an attempt to induce unidirectional conduction block remote from the pacing site--a precursor to reentry. The incidence of remote block was found to be highly dependent on the direction of wave propagation relative to the underlying tissue structure, and with a susceptibility that was synergistically increased by both realistic tissue boundaries and fiber directions. Furthermore, all instances of remote block in these micropatterned cultures occurred at the anterior and posterior junctions of the septum and right ventricular free wall. At these sites, rapid excitation yielded more abrupt conduction slowing and promoted wavefront-waveback interactions that ultimately evolved into transmural lines of conduction block. The location and shape of these lines of block was found to strongly correlate with the spatial distribution of the electrotonic source-load mismatches introduced by ventricular structures, such as tissue expansions and sharp turns in fiber direction.</p><p>In summary, the overall objective of the work described in this thesis was to reveal the distinct influences of realistic cardiac tissue structure on action potential conduction and conduction block by engineering neonatal rat cardiomyocyte monolayers that reproducibly replicated the anatomical details of murine ventricular cross-sections. In the future, this novel model system is expected to further our understanding of structure-function relationships in normal and structurally diseased hearts, and possibly enable the development of novel gene, cell, and ablation therapies for cardiac arrhythmias.</p> / Dissertation

The ability to generate cardiomyocytes from either embryonic stem cells or induced pluripotent stem cells provides an unprecedented opportunity to establish human in vitro models of cardiovascular disease as well as to develop platforms for the testing of novel cardiac therapeutics. We designed two different platforms, a biowire platform and post deflection platform, to generate engineered heart tissues (EHTs) to study a fundamental process in cardiomyocytes: hypertrophy. Both pathological and physiological hypertrophy was studied in order to garner a better understanding of each process. Physiological hypertrophy characteristics were observed using the biowire platform seen in improved myofibril alignment and downregulation of fetal genes. When electrical stimulation was added, a rate dependent effect on sarcomere maturation was observed by the increased frequency of I-bands and H-zones. Certain hallmark features of pathological hypertrophy, such as upregulation of brain natriuretic peptide and sarcomere structure breakdown, were recapitulated when EHTs were treated with isoproterenol.

The ability to generate cardiomyocytes from either embryonic stem cells or induced pluripotent stem cells provides an unprecedented opportunity to establish human in vitro models of cardiovascular disease as well as to develop platforms for the testing of novel cardiac therapeutics. We designed two different platforms, a biowire platform and post deflection platform, to generate engineered heart tissues (EHTs) to study a fundamental process in cardiomyocytes: hypertrophy. Both pathological and physiological hypertrophy was studied in order to garner a better understanding of each process. Physiological hypertrophy characteristics were observed using the biowire platform seen in improved myofibril alignment and downregulation of fetal genes. When electrical stimulation was added, a rate dependent effect on sarcomere maturation was observed by the increased frequency of I-bands and H-zones. Certain hallmark features of pathological hypertrophy, such as upregulation of brain natriuretic peptide and sarcomere structure breakdown, were recapitulated when EHTs were treated with isoproterenol.

Cardiac hypertrophy is clinically defined as a relative increase in heart size associated with a thickening of the ventricular wall. It is a common feature of individuals suffering from different cardio-vascular or metabolic conditions and leads to heart failure. The structural, functional and molecular mechanisms which induce hypertrophy independent of hemodynamic alterations are poorly characterized. In this study, questions about whether cardiac-specific neuro-endocrine activation or metabolic imbalance are sufficient to induce hypertrophic structural and functional remodeling are addressed using genetically manipulated mouse models of primary cardiac hypertrophy. (For complete abstract open document)