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Simvastatin is one of the statins, which are a class of drugs originally developed to fight cardiovascular disease by lowering cholesterol. However, it is now clear that they have effects independent of cholesterol. For example, statin therapy, like exercise, induces adaptations within the heart that protect it against I/R injury. Patients are frequently advised to undergo a combination treatment of statins and chronic exercise, although little is known about how this combination treatment affects cardioprotective adaptations. Both treatments appear to exert their cardioprotective effects through different mechanisms, therefore it appears plausible that combining the two treatments would provide added cardioprotection than either treatment alone. Purpose: To investigate the effects of a combination treatment of statins and exercise upon parameters of post-ischemic myocardial function and damage. Methods: Female Sprague-Dawley rats (6 months of age) were separated into 4 groups for a period of 4 weeks: Sedentary (S, n=10), sedentary plus 10 mg simvastatin (Zocor®)/kg body wt/ day (SD, n=9), exercise (R, n=9), and exercise plus simvastatin (RD, n=9). R and RD were exercised identically on a treadmill for 5 days/week at an intensity of about 70% VO2max and for a duration that was gradually increased to 60 min/day. Twenty-four hrs following the last exercise bout, isolated perfused working hearts were subjected to 30 min of global ischemia followed by 30 min of normoxic reperfusion. Coronary effluents were used to determine lactate dehydrogenase (LDH) leakage and prostaglandin generation. Results: Cardiac function was similar in all groups prior to ischemia. Post I/R recovery of cardiac function in S was 17.6 [greater than or equal to]6.6% of pre-ischemic cardiac output times systolic pressure. Recovery was significantly higher in SD (37.7[greater than or equal to]7.7%) and R (40.1[greater than or equal to]7.8%) and tended to be highest in RD (49.7[greater than or equal to]7.1%). SD had significantly higher pre-ischemic coronary flow per g heart weight (CF/g) than all other groups. At 10 min post-ischemia, simvastatin treatment significantly increased CF/g compared to S (p<0.05). Exercise had an effect on increasing post-ischemic myocardial efficiency and RD had significantly higher post-ischemic myocardial efficiency vs. S (p<0.05). Compared to S, LDH leakage during reperfusion was dramatically decreased in SD, R and RD by approximately similar amounts. Simvastatin treatment doubled the basal production of protective prostaglandins, whereas exercise did not significantly alter their production and combining both treatments yielded a lower prostaglandin release than simvastatin treatment. SD had s significantly higher basal prostaglandin release than R (p<0.05). Conclusion: Although the combination treatment of simvastatin and exercise did not result in a statistically significant addition in cardioprotection compared to either treatment alone, there was a trend for improved parameters of post-ischemic cardiac function and damage upon combining both treatments compared to each treatment alone. Specifically, the prostaglandin, CF/g and efficiency data suggest that exercise and statininduced cardioprotection against I/R injury appears to occur by different mechanisms and combining the two treatments may provide greater protection than either alone.

High mortality rates from cardiovascular disease underscore the need for improved therapies. Thus, it is important to further our understanding of factors and mechanisms promoting cardiac protection and repair. Fibroblast growth factor-2 (FGF-2), administered to the heart before or during injury exerts beneficial effects such as cytoprotection and angiogenesis. However, the effects of a chronic elevation in endogenous FGF-2 on recovery/remodeling after ischemic injury are not known. My hypothesis was that chronic elevation in endogenous FGF-2 expression (in FGF-2 overexpressing transgenic mice) exerts beneficial effects such as improved function after isoproterenol-induced injury in vivo. The first study showed that treatment with the β-adrenergic agonist isoproterenol resulted in exaggerated levels of cellular infiltration and myocardial disarray in transgenic FGF-2 versus non-transgenic mouse myocardium. This was suggestive of increased cardiac injury in transgenic FGF-2 mice. Inhibition of T cells using the immunosuppressants cyclosporine A or antibodies against CD3ε attenuated cellular infiltration in transgenic FGF-2 mice, to levels comparable to those of non-transgenic mice, suggesting a T lymphocyte-mediated effect. Overall morphological data suggested that chronic FGF-2 elevation might have created an adverse outcome after cardiac injury. In a follow-up study the effect of chronic FGF-2 elevation on cardiac function was examined, as measured by tissue Doppler imaging (TDI), after isoproterenol administration. FGF-2 overexpressing mice displayed improved cardiac function compared to controls, after isoproterenol, both acutely (24 h) and in a sustained fashion (2-4 weeks). The FGF-2 associated functional improvement at 2-4 weeks was attenuated following immunosuppression with cyclosporine A, but not treatment with anti-CD3ε antibodies. The FGF-2–associated functional improvement may be partially attributed to a cyclosporine A-sensitive (but anti-CD3-insensitive) infiltrating cell population. Thus cellular infiltration, in response to elevated FGF-2, may have a net beneficial effect. In a third study, non-transgenic mice were put through a brief swimming protocol (exercise) prior to isoproterenol. This acute bout of exercise resulted in significant improvement in TDI function, compared to control groups, measured at 24 hours up to 4 weeks post-isoproterenol. In conclusion, increased endogenous cardiac FGF-2 expression, or an acute bout of exercise, exert sustained beneficial effects against isoproterenol-induced cardiac injury.

Ischemic heart disease is a major cause of death worldwide. Identifying the mechanisms mediating cardiac resistance to injury (‘cardioprotection’) can contribute to therapies for cardiac injury. These studies investigated the role of the membrane channel forming protein connexin43 (Cx43), a downstream effector of PKCε-mediated cardioprotection. In isolated cardiomyocytes FGF-2/PKCε –induced cytoprotection is mediated by Cx43 phosphorylation at PKCε-target site, serine(S)262. Hypothesis (1). PKCε-mediated cardioprotection increases Cx43 phosphorylation at PKCε-target sites and prevents ischemia and/or reperfusion-induced Cx43 remodeling. Rat hearts were subjected to protective treatments (ischemic preconditioning, FGF-2, diazoxide), followed by 30 min global ischemia and/or 60 min reperfusion. All treatments elicited above-physiological levels of phospho(p)S262-Cx43 and pS368-Cx43 (P*Cx43), which were sustained during global ischemia, and reperfusion, and accompanied by attenuation of ischemia-induced Cx43 dephosphorylation, and prevention of Cx43 lateralization. Cx43 is present in cardiac subsarcolemmal (SSM) but not interfibrillar (IFM) mitochondria. Hypothesis (2). FGF-2 exerts protective effects on both mitochondrial populations, but is associated with mitochondrial (mito) P*Cx43 state in SSM, and mediated by mitoCx43 function. FGF-2 treatment increased calcium tolerance in SSM and IFM by 2.9- and 1.7-fold, respectively, compared to controls. In the presence of Gap27, a Cx43 hemi-channel blocker, the salutary effect of FGF-2 were lost in SSM but not IFM, indicating a functional role for Cx43. FGF-2 increased levels of PKCε, pPKCε and Tom-20 translocase in SSM and IFM. In SSM, FGF-2 increased pS262-and pS368-Cx43 by 30-fold and 8-fold, compared to controls. Stimulation of untreated SSM with a PKC activator (phorbol 12-myristate 13-acetate; PMA) also increased pS262-and-S368-Cx43 and calcium tolerance, which was prevented by εV1-2, a PKCε-inhibiting peptide. The effect of FGF-2 on isolated cardiac mitochondria is unknown. Hypothesis (3). FGF-2 exerts a direct protective effects on SSM. Direct stimulation of SSM with FGF-2 increased pS262-and-S368-mitoCx43, and calcium resistance to mPTP, and was dependent on mitoPKCε. FGF receptor inhibitors, SU5402 and FGFR neutralizing antibodies, blocked the effect of FGF-2, suggesting a FGFR1-like protein is responsible for the direct protective effects of FGF-2. This new intracellular mechanism of cytoprotection implies that endogenous intracellular levels of FGF-2 may determine constitutive levels of cardiac mitochondrial resistance to mPTP.

High mortality rates from cardiovascular disease underscore the need for improved therapies. Thus, it is important to further our understanding of factors and mechanisms promoting cardiac protection and repair. Fibroblast growth factor-2 (FGF-2), administered to the heart before or during injury exerts beneficial effects such as cytoprotection and angiogenesis. However, the effects of a chronic elevation in endogenous FGF-2 on recovery/remodeling after ischemic injury are not known. My hypothesis was that chronic elevation in endogenous FGF-2 expression (in FGF-2 overexpressing transgenic mice) exerts beneficial effects such as improved function after isoproterenol-induced injury in vivo. The first study showed that treatment with the β-adrenergic agonist isoproterenol resulted in exaggerated levels of cellular infiltration and myocardial disarray in transgenic FGF-2 versus non-transgenic mouse myocardium. This was suggestive of increased cardiac injury in transgenic FGF-2 mice. Inhibition of T cells using the immunosuppressants cyclosporine A or antibodies against CD3ε attenuated cellular infiltration in transgenic FGF-2 mice, to levels comparable to those of non-transgenic mice, suggesting a T lymphocyte-mediated effect. Overall morphological data suggested that chronic FGF-2 elevation might have created an adverse outcome after cardiac injury. In a follow-up study the effect of chronic FGF-2 elevation on cardiac function was examined, as measured by tissue Doppler imaging (TDI), after isoproterenol administration. FGF-2 overexpressing mice displayed improved cardiac function compared to controls, after isoproterenol, both acutely (24 h) and in a sustained fashion (2-4 weeks). The FGF-2 associated functional improvement at 2-4 weeks was attenuated following immunosuppression with cyclosporine A, but not treatment with anti-CD3ε antibodies. The FGF-2–associated functional improvement may be partially attributed to a cyclosporine A-sensitive (but anti-CD3-insensitive) infiltrating cell population. Thus cellular infiltration, in response to elevated FGF-2, may have a net beneficial effect. In a third study, non-transgenic mice were put through a brief swimming protocol (exercise) prior to isoproterenol. This acute bout of exercise resulted in significant improvement in TDI function, compared to control groups, measured at 24 hours up to 4 weeks post-isoproterenol. In conclusion, increased endogenous cardiac FGF-2 expression, or an acute bout of exercise, exert sustained beneficial effects against isoproterenol-induced cardiac injury.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

L'infarctus du myocarde est la première cause de mortalité cardiovasculaire dans les pays occidentaux. La reperfusion la plus précoce possible est actuellement le seul traitement validé pour réduire la taille d'infarctus, facteur pronostique fondamental de morbi-mortalité. Cependant, la reperfusion engendre des lésions d'ischémie-reperfusion (IR) irréversibles qui précipitent la mort par apoptose des cardiomyocytes. Une approche transcriptomique nous a permis d'identifier les gènes spécifiques du postconditionnement ischémique (PostC) dans le cœur de souris. Parmi ceux-ci, l'expression du gène codant pour le récepteur métabotrope de type 1 du glutamate (mGluR1) est augmentée par le PostC. L'objectif de mon travail de thèse a été d'étudier le rôle de mGluR1 au cours de l'IR myocardique. Notre stratégie, basée sur l'utilisation de souris knock-out, a permis de confirmer l'implication de mGluR1 dans la cardioprotection. L'injection de glutamate au moment de la reperfusion myocardique permet de diminuer significativement la taille de l'infarctus par inhibition de l'apoptose. Cet effet cardioprotecteur est diminué en présence de l'antagoniste spécifique YM 298198 ou en présence de wortmannin, inhibiteur de la PI3-kinase, activée dans la cascade de signalisation du récepteur. La réduction de la taille d'infarctus par le glutamate semble associée à une amélioration de la fonction contractile du ventricule gauche en échocardiographie (par speckle tracking, méthode de quantification de la déformation myocardique) dans un modèle murin d'IR myocardique. Ces résultats, bien que préliminaires, semblent prometteurs et nous permettent d'envisager une application clinique chez le patient coronarien. / Myocardial infarction is the major cause of cardiovascular mortality in western countries. Reperfusion as early as possible is the only treatment recognized to reduce infarct size, crucial prognostic factor of morbidity and mortality. However, reperfusion leads to ischemia-reperfusion (IR) injury leading to irreversible apoptotic death of cardiomyocytes. A transcriptomic approach has allowed us to identify genes specifically regulated upon ischemic postconditioning (PostC) in the mouse heart. Among them, the expression of the metabotropic glutamate receptor type 1 (mGluR1) gene is up-regulated by PostC. The aim of my thesis work was to study the role of mGluR1 during myocardial IR. Our strategy, based on the use of knockout mice, confirmed the involvement of mGluR1 in cardioprotection. Injection of glutamate at the time of reperfusion significantly reduced infarct size via apoptosis inhibition. This cardioprotective effect was reduced in presence of the specific antagonist YM 298198 or in presence of wortmannin, an inhibitor of PI3-kinase, which is activated downstream mGluR1. In our mouse model of myocardial IR injury, decrease in infarct size after glutamate treatment seems to be associated to an improved left ventricular contractile function assessed by echocardiography (speckle tracking method quantifying myocardial strain). These preliminary results are promising and allow us to consider a clinical trial for coronary patients.

Dans ce travail, nous avons montré qu'un nouveau ligand du TSPO, le TRO40303, possède des propriétés cardioprotectrices, confirmant que le TSPO joue un rôle important dans les effets délétères engendrés par l'ischémie-reperfusion myocardique. Des effets similaires ont déjà été observés avec d'autres ligands du TSPO, notamment le 4'-chlorodiazépam, mais le mécanisme d'action par lequel ces molécules exercent leur effet cardioprotecteur reste pour une large part encore mal connu. Nous avons montré chez le rat que la reperfusion d'un myocarde ischémié s'accompagne d'une augmentation du cholestérol mitochondrial, de la formation d'oxystérols et d'un stress oxydant majeur. Le 4'-chlorodiazépam inhibe ces effets et améliore les fonctions mitochondriales post-ischémiques, révélant que le TSPO est responsable du transport du cholestérol dans la mitochondrie cardiaque et que ce dernier, sous des formes oxydées ou non, pourrait participer aux effets délétères de la reperfusion. La limitation de l'entrée du cholestérol dans la mitochondrie lors de la reperfusion d'un myocarde ischémié est un mécanisme original qui pourrait donc contribuer à l'effet cardioprotecteur des ligands du TSPO. Enfin, nous avons montré que chez des rats génétiquement modifiés et associant hypercholestérolémie, obésité et diabète de type II, le cholestérol mitochondrial est d'une part très élevé, avec ou sans ischémie-reperfusion myocardique et d'autre part que le 4'-chlorodiazépam n'a pas d'effet sur le cholestérol mitochondrial après ischémie-reperfusion. Ces résultats suggèrent que les mécanismes d'action des ligands du TSPO sont probablement différents et que les traitements doivent être adaptés en présence de facteurs de co-morbidité. / In the present work we showed that a new TSPO ligand, TRO40303, has cardioprotective properties confirming that TSPO plays a key role in the deleterious effects of myocardial ischemia-reperfusion. Similar effects have been observed with other TSPO ligands, such as 4'-chlorodiazepam, but the mechanism of action of these molecules is not known. We showed that ischemia-reperfusion in rats increased mitochondrial concentration of cholesterol, oxysterol formation and oxidative stress. 4'-Chlorodiazepam inhibited these effects and improved post-ischemic mitochondrial functions, revealing that TSPO is responsible for cholesterol transport in cardiac mitochondria and that cholesterol, in oxidized or non-oxidized forms, could participate in the deleterious effects of reperfusion. The limitation of the increase in cholesterol in mitochondria during ischemia-reperfusion is an original mechanism which could contribute to the cardioprotective effect of TSPO ligands. We then showed, in genetically modified rats with hypercholesterolemia, obesity and type II diabetes, that the concentration of mitochondrial cholesterol is very high with or without ischemia-reperfusion. We further demonstrated that 4'-chlorodiazepam has no effect on mitochondrial cholesterol after ischemia-reperfusion. These results suggest that the mechanisms of action of TSPO ligands probably differ in these conditions and that treatment must be adapted in accordance with the presence of factors of co-morbidity.

L'infarctus du myocarde (IDM) est l'une des principales causes de morbi-mortalité dans les pays développés et ce malgré l'amélioration enregistrée ces dernières années dans sa prise en charge thérapeutique. L'obésité est classée comme étant un facteur de risque majeur pour les maladies coronaires par l'American Heart Association, et concerne 14,5 % de la population française (enquête ObEpi-Roche/INSERM, 2009). En utilisant un modèle expérimental d'obésité, la souris ob/ob génétiquement dépourvue en leptine, l'objectif du présent travail de thèse a été d'étudier l'efficacité de stratégies cardioprotectrices comme le postconditionnement (PCD) ischémique ou l'exercice physique chronique contre l'IDM. La première partie de ce travail de thèse a mis en évidence une perte de la cardioprotection par PCD ischémique au cours de l'obésité. L'étude des voies de signalisation aura permis de mettre en évidence l'implication des protéines phosphatases PTEN, MKP3 et PP2C dans l'inefficacité du PCD. La seconde partie de ce travail de thèse a montré un effet cardioprotecteur de l'exercice physique chronique contre l'IDM dans un contexte expérimental d'obésité. Cet effet est associé à une augmentation des défenses enzymatiques antioxydantes, à une amélioration des fonctions mitochondriales, à une activation des voies de signalisation cardioprotectrices RISK et SAFE et enfin à une diminution des protéines phosphatases impliquées dans la régulation négative des acteurs des voies de signalisation cardioprotectrices. La preuve scientifique des bienfaits de l'exercice est aujourd'hui un argument de poids pour poursuivre les efforts entrepris par les pouvoirs publics ces dernières années à travers le Programme National Nutrition Santé pour favoriser la pratique d'une activité physique et sportive et en particulier chez les obèses. / Myocardial infarction (MI) remains the leading cause of morbidity and mortality in the developing countries despite significant therapeutic advances over these last years. Obesity is a major risk factor for coronary heart disease according to the American Heart Association and concern 14.5% of the French population (ObEpi-Roche/INSERM survey, 2009). Using the leptin-deficient ob/ob mice, an animal model of obesity, the aim of the present thesis was to investigate the efficacy of cardioprotective strategies such as ischemic postconditioning (PCD) or chronic physical exercise against MI. In the first part of this thesis, we have found that the cardioprotective effects of PCD vanish with obesity. The investigation of the cardioprotective pathways has revealed that protein phosphatases such as PTEN, MKP3 and PP2C are involved in the inability of PCD to protect the heart. The second part of this thesis has demonstrated for the first time a cardioprotective effect of chronic physical exercise against MI in an experimental model of obesity. This effect was associated with increased antioxidant enzymes, improved mitochondrial function, activation of the cardioprotective RISK and SAFE pathways and finally a decrease in the related protein phosphatases levels. The scientific proofs given by this work underlines the “Programme National Nutrition Santé” developed by the French government to encourage all people and especially obese people to observe physical and sport activities.

L'infarctus du myocarde est une des première cause de mortalité mondiale et l'amélioration de la prise en charge et des techniques de reperfusion ont permis une amélioration de la survie des patients. Les méthodes de cardioprotection se sont développées afin de limiter la taille d'infarctus après l'épisode d'ischémie reperfusion dans le but de limiter les effets délétères de l'infarctus du myocarde, tels que le remodelage ventriculaire et l'insuffisance cardiaque.Dans notre étude, nous nous sommes particulièrement focalisés sur le rôle majeur de la mitochondrie dans les phénomènes de mort cellulaire associés à l'ischémie reperfusion. Au sein de la mitochondrie, notre cible principale était la sirtuine 3 (SIRT3), dont les modifications post-traductionnelles associées ont été montrées comme ayant un rôle potentiel dans la modulation de la mort cellulaire liée à l'ischémie reperfusion. Ainsi, dans la première partie de notre travail, nous avons pu mettre en évidence (1) la nécessité de la SIRT3 dans les mécanismes de cardioprotection chez les souris jeunes et (2) l'importance de la déacétylation par la SIRT3 de la cyclophiline D (CypD) dans la réussite de la cardioprotection.Récemment, de nombreuses études précliniques ont mis en avant de nouvelles techniques de cardioprotection efficace chez différents modèles animaux, qui n'ont pourtant pas fait leur preuve lors de la transposition en étude clinique. Dans une seconde partie de notre travail, nous nous sommes attachés à comprendre une des raisons de ces différents échec cliniques, en nous appuyant sur le simple constat que les études précliniques sont réalisées sur des animaux jeunes, alors que l'infarctus du myocarde est une pathologie des personnes sénescentes. Nous avons ainsi étudié l'impact du vieillissement sur différentes méthodes de cardioprotection utilisées au laboratoire, et avons mis en évidence (3) des modifications mitochondriales impactant la déacétylation de la CypD en réponse aux techniques de cardioprotection chez les souris âgées. Cette étude nous a permis de soulever un problème majeur des études précliniques, à savoir l'âge des modèles animaux utilisés par rapport à la pathologie étudiée.Ce travail a donc permis de déterminer le rôle crucial de la déacétylation de la CypD et de la SIRT3 dans la cardioprotection, mais aussi de soulever la question de l'âge des modèles animaux avant de possibles transferts des avancées thérapeutiques en clinique humaine / Myocardial infarction is one of the leading causes of global mortality and improved management and reperfusion techniques have improved patient survival. Cardioprotection methods have been developed to limit the infarct size after ischemia reperfusion episode in order to limit the deleterious effects of myocardial infarction, such as ventricular remodeling and heart failure.In our study, we focused particularly on the major role of mitochondria in cell death phenomena associated with ischemia reperfusion. Within the mitochondria, our main target was sirtuin 3 (SIRT3), whose associated post-translational modifications have been shown to have a potential role in modulating ischemia-related reperfusion cell death. Thus, in the first part of our work, we were able to highlight (1) the need for SIRT3 in cardioprotection mechanisms in young mice and (2) the importance of SIRT3 deacetylation of cyclophilin D (CypD) in the success of cardioprotection.Recently, numerous preclinical studies have highlighted new techniques of effective cardioprotection in different animal models, which have not yet proved their worth after transposition in clinical study. In a second part of our work, we have endeavored to understand one of the reasons for these different clinical failures, based on the simple observation that preclinical studies are carried out on young animals, while myocardial infarction is a pathology of senescent people. We studied the impact of aging on various cardioprotection methods used in the laboratory, and demonstrated (3) mitochondrial changes impacting the deacetylation of CypD in response to cardioprotection techniques in aged mice. This study allowed us to raise a major problem of preclinical studies, namely the age of animal models used in relation to the pathology studied.This work has thus made it possible to determine the crucial role of deacetylation of CypD and SIRT3 in cardioprotection, but also to raise the question of the age of animal models before possible transfer of therapeutic advances in human clinical practice