Emilie Dubois-Deruy

Le remodelage ventriculaire gauche (RVG) est un processus complexe qui intervient après un infarctus du myocarde chez 30% des patients en dépit des meilleurs traitements connus actuellement. Le but de mon travail de thèse consistait à identifier les déterminants moléculaires du RVG dans le but de mieux en comprendre les mécanismes physiopathologiques. Pour cela, nous avons étudié les modifications post-traductionnelles des protéines contractiles du VG et en particulier, la phosphorylation et la O-N-acétylglucosaminylation (O-GlcNAc). Nous nous sommes ensuite particulièrement intéressés à la troponine T (TnT) pour laquelle nous avons ainsi pu mettre en évidence une diminution de la phosphorylation au niveau de la sérine 208 au niveau du VG et du plasma chez le rat, suggérant que cela pourrait être un marqueur du RVG post-infarctus. Pour cette étude, nous avons travaillé en collaboration avec l’unité INSERM U644 de Rouen sur un modèle expérimental d’insuffisance cardiaque. L’infarctus du myocarde est induit chez le rat par ligature de la branche descendante de l’artère coronaire gauche, les rats témoins subissant l’intervention mais sans ligature. Dans un premier temps, nous avons réalisé une étude globale du phosphoprotéome du VG en phase tardive du RVG (2 mois post-ligature). Pour cela, les protéines extraites du VG ont été séparées par électrophorèse bidimensionnelle puis colorées au Pro-Q®Diamond (spécifique des protéines phosphorylées) puis au Sypro®Ruby (spécifique des protéines totales). Par analyse bioinformatique, nous avons mis en évidences 69 spots polypeptidiques présentant des modulations de phosphorylation. Nous avons donc analysé ces spots par spectrométrie de masse et avons identifié 30 protéines correspondant à 53 spots polypeptidiques présentant des modulations de phosphorylation. Parmi ces protéines, nous avons choisi de nous concentrer et d’étudier 6 protéines contractiles : la TnT, l’alpha-tropomyosine 1 (α-Tm 1), la desmine, l’αB-crystalline et les chaînes légères de myosine 1 et 2 (MLC). Pour chacune de ces protéines, nous avons identifié le type d’acide aminé responsable de la phosphorylation et quantifié les modulations de phosphorylation dans le VG des rats insuffisants cardiaques (IC). De manière intéressante, nous avons observé que le VG des animaux IC présentait une diminution significative de la phosphorylation sur les résidus sérine pour l’α-Tm 1, la TnT et la MLC-2 et sur les résidus de tyrosine pour l’αB-crystalline ainsi qu’une augmentation significative de la phosphorylation sur les résidus tyrosine pour la MLC-1 et sur les résidus de sérines pour la desmine, confirmant ainsi les résultats obtenus en électrophorèse bidimensionnelle. Afin de compléter l’analyse des modifications post-traductionnelles, nous avons étudié les modifications de O-GlcNAc pour chacune de ces protéines. Nous avons ainsi observé une diminution significative de la O-GlcNAcylation de l’α-Tm 1, de la desmine et l’αB crystalline ainsi qu’une augmentation de la O-GlcNAcylation de la MLC-3 et de la TnT. Par ailleurs, nous avons pu corréler ces modulations de phosphorylation et de O-GlcNAcylation avec des modulations de l’activité des enzymes impliquées dans ces modulations. En effet, par analyse bioinformatique de la séquence de la TnT et par recherche bibliographique nous avons mis en évidence que la protéine kinase C et la protéine phosphatase 2A pourrait être impliquées dans ces modulations de phosphorylation. Nous avons alors mis en évidence une diminution de l’activité de la protéine kinase C epsilon dans le VG des rats IC mais sans variation de l’activité de la protéine phosphatase 2A. Par ailleurs, nous avons mis en évidence une augmentation de l’activité de la O-GlcNAc transférase et une diminution de l’activité de la O-GlcNAcase dans le VG des rats IC.

Lysine acetylation is a highly conserved mechanism that affects several biological processes such as cell growth, metabolism, enzymatic activity, subcellular localization of proteins, gene transcription or chromatin structure. This post-translational modification, mainly regulated by lysine acetyltransferase (KAT) and lysine deacetylase (KDAC) enzymes, can occur on histone or non-histone proteins. Several studies have demonstrated that dysregulated acetylation is involved in cardiac dysfunction, associated with metabolic disorder or heart failure. Since the prevalence of obesity, type 2 diabetes or heart failure rises and represents a major cause of cardiovascular morbidity and mortality worldwide, cardiac acetylation may constitute a crucial pathway that could contribute to disease development. In this review, we summarize the mechanisms involved in the regulation of cardiac acetylation and its roles in physiological conditions. In addition, we highlight the effects of cardiac acetylation in physiopathology, with a focus on obesity, type 2 diabetes and heart failure. This review sheds light on the major role of acetylation in cardiovascular diseases and emphasizes KATs and KDACs as potential therapeutic targets for heart failure.

Heart failure, mostly associated with cardiac hypertrophy, is a major cause of illness and death. Oxidative stress causes accumulation of reactive oxygen species (ROS), leading to mitochondrial dysfunction, suggesting that mitochondria-targeted therapies could be effective in this context. The purpose of this work was to determine whether mitochondria-targeted therapies could improve cardiac hypertrophy induced by mitochondrial ROS. We used neonatal (NCMs) and adult (ACMs) rat cardiomyocytes hypertrophied by isoproterenol (Iso) to induce mitochondrial ROS. A decreased interaction between sirtuin 3 and superoxide dismutase 2 (SOD2) induced SOD2 acetylation on lysine 68 and inactivation, leading to mitochondrial oxidative stress and dysfunction and hypertrophy after 24 h of Iso treatment. To counteract these mechanisms, we evaluated the impact of the mitochondria-targeted antioxidant mitoquinone (MitoQ). MitoQ decreased mitochondrial ROS and hypertrophy in Iso-treated NCMs and ACMs but altered mitochondrial structure and function by decreasing mitochondrial respiration and mitophagy. The same decrease in mitophagy was found in human cardiomyocytes but not in fibroblasts, suggesting a cardiomyocyte-specific deleterious effect of MitoQ. Our data showed the importance of mitochondrial oxidative stress in the development of cardiomyocyte hypertrophy. We observed that targeting mitochondria by MitoQ in cardiomyocytes impaired the metabolism through defective mitophagy, leading to accumulation of deficient mitochondria.

Mitophagy, which mediates the selective elimination of dysfunctional mitochondria, is essential for cardiac homeostasis. Mitophagy is regulated mainly by PTEN-induced putative kinase protein-1 (PINK1)/parkin pathway but also by FUN14 domain-containing 1 (FUNDC1) or Bcl2 interacting protein 3 (BNIP3) and BNIP3-like (BNIP3L/NIX) pathways. Several studies have shown that dysregulated mitophagy is involved in cardiac dysfunction induced by aging, aortic stenosis, myocardial infarction or diabetes. The cardioprotective role of mitophagy is well described, whereas excessive mitophagy could contribute to cell death and cardiac dysfunction. In this review, we summarize the mechanisms involved in the regulation of cardiac mitophagy and its role in physiological condition. We focused on cardiac mitophagy during and following myocardial infarction by highlighting the role and the regulation of PI NK1/parkin-; FUNDC1-; BNIP3- and BNIP3L/NIX-induced mitophagy during ischemia and reperfusion.

Mitochondrial dysfunctions are implicated in several pathologies, such as metabolic, cardiovascular, respiratory, and neurological diseases, as well as in cancer and aging. These metabolic alterations are usually assessed in human or murine samples by mitochondrial respiratory chain enzymatic assays, by measuring the oxygen consumption of intact mitochondria isolated from tissues, or from cells obtained after physical or enzymatic disruption of the tissues. However, these methodologies do not maintain tissue multicellular organization and cell-cell interactions, known to influence mitochondrial metabolism. Here, we develop an optimal model to measure mitochondrial oxygen consumption in heart and lung tissue samples using the XF24 Extracellular Flux Analyzer (Seahorse) and discuss the advantages and limitations of this technological approach. Our results demonstrate that tissue organization, as well as mitochondrial ultrastructure and respiratory function, are preserved in heart and lung tissues freshly processed or after overnight conservation at 4 °C. Using this method, we confirmed the repeatedly reported obesity-associated mitochondrial dysfunction in the heart and extended it to the lungs. We set up and validated a new strategy to optimally assess mitochondrial function in murine tissues. As such, this method is of great potential interest for monitoring mitochondrial function in cohort samples.

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Post-translational modifications of cardiac proteins could participate to left contractile dysfunction resulting in heart failure. Using a rat model of ischemic heart failure, we showed an accumulation of phosphorylated desmin leading to toxic aggregates in cardiomyocytes, but the cellular mechanisms are unknown. The same rat model was used to decipher the kinases involved in desmin phosphorylation and the proteolytic systems present in rat and human failing hearts. We used primary cultures of neonate rat cardiomyocytes for testing specific inhibitors of kinases and for characterizing the autophagic processes able to clear desmin aggregates. We found a significant increase of active PKCζ, no modulation of ubitiquitin-proteasome system, a defect in macroautophagy, and an activation of chaperone-mediated autophagy in heart failure rats. We validated in vitro that PKCζ inhibition induced a significant decrease of GSK3β and of soluble desmin. In vitro activation of ubiquitination of proteins and of chaperone-mediated autophagy is able to decrease soluble and insoluble forms of desmin in cardiomyocytes. These data demonstrate a novel signaling pathway implicating activation of PKCζ in desmin phosphorylation associated with a defect of proteolytic systems in ischemic heart failure, leading to desmin aggrephagy. Our in vitro data demonstrated that ubiquitination of proteins and chaperone-mediated autophagy are required for eliminating desmin aggregates with the contribution of its chaperone protein, α-crystallin Β-chain. Modulation of the kinases involved under pathological conditions may help preserving desmin intermediate filaments structure and thus protect the structural integrity of contractile apparatus of cardiomyocytes by limiting desmin aggregates formation.

Heart failure, mostly associated with cardiac hypertrophy, is still a major cause of illness and death. Oxidative stress causes contractile failure and the accumulation of reactive oxygen species leads to mitochondrial dysfunction, associated with aging and heart failure, suggesting that mitochondria-targeted therapies could be effective in this context. The purpose of this work was to characterize how mitochondrial oxidative stress is involved in cardiac hypertrophy development and to determine if mitochondria-targeted therapies could improve cardiac phenotypes. We used neonatal and adult rat cardiomyocytes (NCMs and ACMs) hypertrophied by isoproterenol (Iso) to induce an increase of mitochondrial superoxide anion. Superoxide dismutase 2 activity and mitochondrial biogenesis were significantly decreased after 24h of Iso treatment. To counteract the mitochondrial oxidative stress induced by hypertrophy, we evaluated the impact of two different anti-oxidants, mitoquinone (MitoQ) and EUK 134. Both significantly decreased mitochondrial superoxide anion and hypertrophy in hypertrophied NCMs and ACMs. Conversely to EUK 134 which preserved cell functions, MitoQ impaired mitochondrial function by decreasing maximal mitochondrial respiration, mitochondrial membrane potential and mitophagy (particularly Parkin expression) and altering mitochondrial structure. The same decrease of Parkin was found in human cardiomyocytes but not in fibroblasts suggesting a cell specificity deleterious effect of MitoQ. Our data showed the importance of mitochondrial oxidative stress in the development of cardiomyocyte hypertrophy. Interestingly, we observed that targeting mitochondria by an anti-oxidant (MitoQ) impaired metabolism specifically in cardiomyocytes. Conversely, the SOD mimic (EUK 134) decreased both oxidative stress and cardiomyocyte hypertrophy and restored impaired cardiomyocyte metabolism and mitochondrial biogenesis.

Reactive oxygen species (ROS) are subcellular messengers in signal transductions pathways with both beneficial and deleterious roles. ROS are generated as a by-product of mitochondrial respiration or metabolism or by specific enzymes such as superoxide dismutases, glutathione peroxidase, catalase, peroxiredoxins, and myeloperoxidases. Under physiological conditions, the low levels of ROS production are equivalent to their detoxification, playing a major role in cellular signaling and function. In pathological situations, particularly atherosclerosis or hypertension, the release of ROS exceeds endogenous antioxidant capacity, leading to cell death. At cardiovascular levels, oxidative stress is highly implicated in myocardial infarction, ischemia/reperfusion, or heart failure. Here, we will first detail the physiological role of low ROS production in the heart and the vessels. Indeed, ROS are able to regulate multiple cardiovascular functions, such as cell proliferation, migration, and death. Second, we will investigate the implication of oxidative stress in cardiovascular diseases. Then, we will focus on ROS produced by NAPDH oxidase or during endothelial or mitochondrial dysfunction. Given the importance of oxidative stress at the cardiovascular level, antioxidant therapies could be a real benefit. In the last part of this review, we will detail the new therapeutic strategies potentially involved in cardiovascular protection and currently under study.

Unhealthy lifestyle choices, such as bad eating behaviors and cigarette smoking, have major detrimental impacts on health. However, the inter-relations between obesity and smoking are still not fully understood. We thus developed an experimental model of high-fat diet-fed obese C57BL/6 male mice chronically exposed to cigarette smoke. Our study evaluated for the first time the resulting effects of the combined exposure to unhealthy diet and cigarette smoke on several metabolic, pulmonary, intestinal, and cardiac parameters. We showed that the chronic exposure to cigarette smoke modified the pattern of body fat distribution in favor of the visceral depots in obese mice, impaired the respiratory function, triggered pulmonary inflammation and emphysema, and was associated with gut microbiota dysbiosis, cardiac hypertrophy and myocardial fibrosis.