GLUT4 trafficking in muscle cells has been studied to determine how distinct signalling pathways induce GLUT4 translocation. Two different cell models were adopted for these investigations; cardiomyocytes isolated from a transgenic mouse line expressing HA-GLUT4-GFP in muscle and L6 myotubes retrovirally expressing HA-GLUT4. The GLUT4 constructs were largely excluded from the external membrane under basal conditions in both cell models. GLUT4 was trafficked to the external membrane in to response all stimuli studied in cardiomyocytes (insulin, contraction and hypoxia) and L6 myotubes (insulin, AICAR and A-769662). By comparing the anti-HA and GFP signals at the sarcolemma and transverse tubules in cardiomyocytes, it has also be possible to observe an enhancement of GSV fusion with the sarcolemma following stimulation with insulin and contraction. This effect was specific to these stimuli and to the sarcolemma. Insulin-stimulation of GLUT4 exocytosis was not detected under steady-state conditions in L6 myotubes. Here, the major effect of insulin-stimulation and AMPK-activation was on GLUT4 internalisation. The rate constant for GLUT4 internalisation was very rapid in basal cells and was decreased during the steady-state responses to insulin and the AMPK-activators AICAR and A-769662. In cardiomyocytes, internalising GLUT4 colocalised with clathrin at puncta at the sarcolemma. This indicates that GLUT4 is internalised via a clathrin-mediated route. Investigations into the amount of GLUT4 recycling in L6 cells under steady-state conditions revealed that a large proportion of cellular GLUT4 recycles with the cell surface under basal conditions. Insulin-stimulation and AMPK-activation additively mobilised GLUT4 in L6 cells. This implies a non-convergent mobilisation of GLUT4 in response to activation of the PKB/Akt and AMPK signalling pathways. Data obtained from an in vitro kinase assay confirmed that serine 237 of TBC1D1 is a bone fide AMPK phosphorylation site. Furthermore, phosphorylation of this site in L6 myotubes incubated with AMPK activators has been confirmed using a novel antibody specific to TBC1D1 phosphorylated at serine 237. This thesis discusses the consequences and importance of multiple controls impinging on GLUT4 traffic and highlights the advantages and limitations of kinetic studies of these processes.

"Recent data show that 7.6 million Americans have survived a myocardial infarction (MI), and 5.1 million Americans suffer from severe heart failure. Stem cell therapy has the potential to improve cardiac function after MI. Two promising cells for cardiovascular regeneration therapies include human mesenchymal stem cells (hMSCs) and pluripotent stem cell derived cardiomyocytes (hPS-CM) each with their own unique method for improving cardiac function post-infarct. However, a limiting factor to cell therapies is that the methods currently used to deliver cells to the myocardium, including intramyocardial injection (considered the gold standard), suffer from low retention rates. To promote localization of delivered cells to the infarct and increase retention rates, our lab has developed a fibrin biological suture that can deliver human mesenchymal stem cells (hMSCs) with an efficiency of 64% compared to just 11% with intramyocardial injection in the normal rat heart. In this dissertation we sought to examine the functionality of hMSC and hPS-CM seeded sutures and their impact on cardiovascular regeneration applications. We began by delivering hMSC seeded fibrin sutures to an infarcted rat heart and found that the sutures are an effective method to deliver cells to the infarcted myocardium and demonstrated a trend towards improved regional mechanical function in the infarct region over infarct alone. Next, we transitioned to using hPS-CM and developed methods to seed the sutures, as well as a method to measure hPS-CM contractility with high spatial and temporal resolution, while concurrently capturing calcium transients. This technique allowed us to examine the contractile behavior in terms of contractile strain and conduction velocity of hPS-CM seeded on fibrin microthreads over 21 days in culture. We found that the fibrin microthread is a suitable scaffold for hPS-CM attachment and contraction and that extended culture promotes cell alignment along the length of the suture as well as improvements in contractile function in terms of increases in contractile strain and conduction velocity. Finally, we delivered the hPS-CM seeded microthreads to an uninjured rat heart and found a delivery efficiency of 67%. Overall, we further demonstrated the technology of the fibrin suture to deliver cells to an infarct as well as the ability to support the attachment, contraction and delivery of hPS-CM to cardiac tissue. "

The role of endogenous molecules in facilitating stem cell differentiation into cardiomyocytes is yet to be fully understood. SPC and S1P, common biolipids, promote cardiac differentiation of mesenchymal stem cells and cardiac progenitor cells, however, the same potential of closely related lysophosphatidic acid (LPA) has only recently become evident. The initial cardio-protection offered by elevated LPA levels in response to acute myocardial infarction and the ability of this biolipid to mediate other cellular fates served as a rationale to investigate the ability of LPA to mediate the cardiac differentiation of the murine P19 teratocarcinoma cell line and further examine the role of signalling molecules critical to lineage commitment. All experiments were carried out using P19 stem cells, cultured in supplemented alpha-minimal essential medium. Cells were aggregated into embryoid bodies in the presence of 5µM LPA in non-tissue grade Petri dishes over the course of 4 days to commence the differentiation process. Inhibitors were added 60 minutes before LPA while control cells were cultured in medium only. Embryoid bodies were transferred to 6-well tissue culture grade plates and cultured for a further 6 days. Cardiac differentiation was assessed by examining the expression of ventricular myosin light chain (MLC1v) by western blot and the role of LPA receptors 1-4, PKC, PI3K, MAPKs, and NF-κB were determined by examining the changes in this expression in the presence of selective inhibitors. The induction and regulation of GATA4, MEF2C, ATF-2, JNK, and YAP was also determined by western blotting. The activity and regulation of transcription factors, AP-1 and NF-κB, and the MAPKs was determined using ELISA kits. LPA induced the differentiation of P19 cells into cardiomyocytes most effectively when used at a concentration of 5µM as evidenced by the expression of MLC1v on day 10 of the differentiation process. Inhibition of LPA receptor 4 (0.1mg/mL Suramin), LPA receptors 1/3 (20µM Ki16425), LPA receptor 2 (7.5nM H2L5186303), PKC (10µM BIM-1), PI3K (20µM LY294002), ERK (20µM PD98059), JNK (10µM SP600125), and NF-κB (0.01nM CAY10470) blocked LPA induced expression of MLC1v. GATA4, MEF2C, pcJun, pJunD, and pATF2 expression increased in a time-dependent manner peaking at day 10 in LPA treated cells. GATA4 and pcJun expression was suppressed by all the inhibitors whereas MEF2C expression was unaffected by CAY10470, pJunD expression was unaffected by H2L5186303, pATF2 and NF-κB expression was unaffected by LY294002, but the latter was enhanced by Suramin. JNK was transiently phosphorylated in all cells whereas YAP was dephosphorylated 24-48 hours after EB formation in LPA treated cells and were both affected by Ki16425 and partially by H2L5186303 treatment. In conclusion, the studies carried out in this thesis have shown that LPA mediates the cardiac differentiation of P19 cells through LPA receptor 2, partially through receptors 1/3, and possibly through receptor 4. Conceivably downstream of these receptors, PKC, PI3K, MAPK, and NF-κB signalling pathways converge on the regulation of cardiac-specific transcription factors GATA4 and MEF2C along with ubiquitous transcription factor AP-1. JNK signalling is initiated through LPA receptors 1/3 and partially through receptor 2 to commence the cardiac program however the role of JNK and YAP in the proliferation of aggregating EBs is yet to be entirely established.

As metaloproteases da matriz (MMPs) são enzimas proteolíticas que participam da degradação da matriz extracelular no organismo de vertebrados. Estudos mostram a grande importância dessas enzimas no processo de remodelação do tecido cardíaco, além de sugerirem a participação da MMP-2 em doenças cardiovasculares. Em estudo recente foi demonstrado que as MMPs clivam o receptor ?2-adrenérgico, contribuindo para o aumento do tônus arteriolar de ratos espontaneamente hipertensos (SHR). Acredita-se que processo semelhante possa ser verificado em relação ao receptor ?-1 adrenérgico e proteínas das junções, que são fundamentais para o funcionamento do coração. As análises in sílico realizadas mostraram regiões prováveis de clivagem pela metaloprotease da matriz 2 humana recombinante (rhMMP-2) na porção extracelular, especificamente na região Nterminal deste receptor, no entanto, as análises de comparação de similaridade de substratos não apresentaram resultados significativos, embora os resultados preliminares obtidos no teste in vitro mostraram que houve hidrolise logo no início do peptídeo sintético ASPPASLLPPAS, entre os resíduos alanina e serina, entre as duas prolinas e por fim entre o resíduo de prolina e alanina, regiões com grandes chances de ocorrer a hidrólise, pois o substrato nativo desta enzima é o colágeno que é composto por uma cadeia polipeptídica com uma sequência de repetições onde geralmente temos glicina-X-Y, onde X normalmente é uma prolina e Y frequentemente uma hidroxiprolina, e raramente lisina e hidroxilisina, no entanto a replicação deste experimento não apresentou o mesmo resultado. Já os resultados obtidos no western blotting mostraram que a expressão do receptor é diminuída quando os cardiomiócitos são previamente tratados com 40mM e 120mM de rhMMP- 2 e esse efeito tem uma reversão significativa quando as células são previamente tratadas com inibidores doxiciclina ou ONO-4817, corroborando com os trabalhos apresentados na literatura em que a rhMMP-2 atua no receptor ?1adrenérgico. / Matrix metalloproteinases (MMPs) are proteolytic enzymes that participate in the degradation of the extracellular matrix in the vertebrate organism. Studies show the great importance of these enzymes in the remodeling process of cardiac tissue, besides suggesting the participation of MMP-2 in cardiovascular diseases. In a recent study, MMPs were shown to cleave the ?2-adrenergic receptor, contributing to the increase in arteriolar tone of spontaneously hypertensive rats (SHR). It is believed that a similar process can be verified also in the ?-1 adrenergic receptor and junction proteins, which are fundamental to the heart function. The in situ analyzes performed revealed sections prone to be cleaved by matrix metalloproteinase 2 recombinant human (rhMMP-2) in the extracellular portion, specifically in the Nterminal region of this receptor, however, the comparative analyzes of the similarity of substrates did not present significant results, but those obtained in the in vitro test showed that there was hydrolysis right at the beginning of the synthetic peptide ASPPASLLPPAS, between alanine and serine residues, between the two proline and finally between the proline residue and alanine. Hydrolysis among proline residues was expected, even though it was not predicted for in silica cleavage, since the native substrate of this enzyme is collagen, which is composed of a polypeptide chain with a sequence of repetitions in which it usually has glycine-XY, where X usually is a proline and Y often a hydroxyproline, and rarely lysine and hydroxylysine, however the replication of this experiment di not present the same result. The obtained results in western blotting have presented that receptor expression is decreased when cardiomyocytes are pretreated with 40mM and 120mM rhMMP-2 and this effect has a significant reversion when cells are pretreated with doxycycline or ONO-4817 inhibitors, supporting previous studies which demonstrate that rhMMP- 2 acts on the ? 1 adrenergic receptor.

Introduction: Cardiac cell-cell junctions play important roles in maintaining cardiac integrity linking single cardiomyocytes into a single functioning syncytium. There are three main types of cell junctions in the heart: gap junctions (GJ), desmosomes (D) and adherens junctions (AJ). Mutations in the proteins which make-up these junctions are known to cause arrhythmogenic right ventricular cardiomyopathy (ARVC). Pathological features include progressive replacement of right ventricular cardiac muscle with fibrofatty tissue. This can lead to heart failure and life threatening arrhythmias. During normal development of the mammalian heart, protein components of AJ and D gradually fuse to form composite junctions at the intercalated discs, also called areae compositae (singular, area composita, AC). In contrast, the adult heart of lower vertebrates, including the zebrafish, may have few or no AC type junctions. The detailed structure of cardiomyocyte cell-cell junctions in the adult zebrafish heart remain poorly defined and their role in normal development, growth and response to injury have yet to be studied. This thesis will examine the hypothesis that localisation and distribution of myocardial cell-cell junction proteins are crucial in normal myocardial development and in endogenous cardiac regeneration and repair following injury. This will be achieved by understanding the normal development of cell-cell junction proteins in zebrafish from embryonic to adulthood. These findings will then be analysed in comparison to cell-cell junction proteins localisation and distribution in early and late mammalian (mouse and human) myocardium. Once a normal pattern of cell-cell junction proteins will be established, the localisation of cell-cell junction proteins in plakologbin mutant zebrafish model for cardiomyopathy will be studied to understand the distribution and localisation of these proteins in disease manifestation. This model will then be used to test if localisation of cell-cell junction proteins plays an important in cardiac repair following injury by using embryonic laser injury model, this will be further tested by drug intervention study to investigate underlying pathways such as Wnt signalling pathway. Methods: Myocardial cell-cell junctions were assessed using immunohistochemistry in embryonic, juvenile and adult zebrafish hearts and in foetal and adult human hearts. The Plakoglobin mutant zebrafish line (UAS:Gal-4:Plakoglobin Naxos; named as PGNx) was characterised using various functional and morphological assessments including histology, echocardiography and MRI scanning. Similar studies were undertaken in PGNx mutants at different developmental stages. A pharmacological intervention study, using a GSK-3 inhibitor, was carried out in PGNx mutants followed by cardiac structural and functional assessments. Laser-induced cardiac trauma was used to assess the response to injury and repair in normal and PGNx embryos following treatment with the GSK3 inhibitor drug. Results: Cell-cell junction patterning in the embryonic, juvenile and adult zebrafish heart shows a characteristic pearl string appearance of desmoplakin and β-catenin labelled distinct disc shaped AJ. Human foetal heart showed small distinct D and AJ, while the adult human heart had features consistent with AC type junctions. PGNx fish showed reduced ventricle ejection fraction, dilatation of the atrium, reduced amplitude of wall motion and ventricle relaxation velocity compared to age-matched controls. Echocardiography and MRI imaging confirmed severe atrial dilatation and restrictive ventricle physiology in adult fish. The cell-cell junction proteins were over-expressed in the zebrafish PG mutant (PGNx) hearts compared to age-matched controls. Drug studies using a GSK-3β inhibitor showed complete recovery of cardiac function and partial recovery of heart structure. Cardiac injury studies, using laser, showed failure of repair in PGNx embryos compared to age-matched controls. The GSK3 inhibitor failed to improve the functional response following heart laser injury. Conclusions: Cell-cell junctions are distributed abundantly around cardiomyocytes in the zebrafish heart during early development and into adulthood. In contrast to previous studies in adult mammalian heart, there was no evidence of AC type junctions in adult zebrafish cardiomyocytes. The mutant zebrafish line showed restrictive cardiac physiology and abnormal cardiac structure confirming the key role played by plakoglobin in the normal heart development. This is further supported by evidence showing failure of repair in PGNx mutant embryos after injury. Drug treatment with a GSK-3 inhibitor highlights a potentially novel therapeutic pathway for treatment of ARVC involving Wnt signalling.

Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro / Indivíduos obesos apresentam maior risco de morbidade e mortalidade atribuída às doenças cardiovasculares. A composição da dieta é um fator que prediz o fenótipo cardíaco em resposta a obesidade e, o tipo de ácido graxo pode afetar de forma diferencial a estrutura e a função do miocárdio. Estudos têm mostrado que a disfunção mitocondrial exerce um papel chave na patogênese da insuficiência e hipertrofia cardíaca, e as alterações mitocondriais observadas em falhas cardíacas apontam para defeitos em sítios específicos da cadeia transportadora de elétrons. Desta forma, o objetivo deste estudo foi avaliar a função contrátil ventricular em camundongos, alimentados com dieta hiperlipídica, rica em ácidos graxos poliinsaturados, buscando elucidações através da bioenergética mitocondrial. Após desmame, camundongos machos C57Bl/6 passaram a receber dieta manipulada contendo 7% (C) ou 19% (HF) de óleo de soja, até os 135 dias de idade. A ingestão alimentar e a massa corporal foram monitoradas e foi realizado teste de tolerância à glicose. No final do período experimental, os animais foram anestesiados e submetidos à avaliação da composição corporal por Absortimetria de Raios X de Dupla Energia (DXA), e em seguida, sacrificados por exsanguinação. No plasma foram determinados o perfil lipídico e a insulina. O coração, o tecido adiposo intra-abdominal e o subcutâneo foram coletados, pesados, processados para análise histomorfológica. Fibras cardíacas do ventrículo esquerdo foram utilizadas para análise da respiração mitocondrial através de oxígrafo. O coração também foi utilizado para a técnica de perfusão de coração isolado de Langendorff, e para análise da expressão de proteínas relacionadas à bioenergética de cardiomiócitos, através de Western Blotting. O índice de HOMA e de adiposidade foram calculados. O grupo HF apresentou maior adiposidade, sem alteração na ingestão alimentar. Foi observada intolerância a glicose, hiperinsulinemia e resistência à insulina, além de alterações desfavoráveis no perfil lipídico. Foi observado alteração na morfologia cardíaca e quadro de cardiomiopatia hipertrófica, refletindo em alteração hemodinâmica, determinando maior contratilidade, maior pressão ventricular e função diastólica prejudicada. Em relação à atividade mitocondrial dos cardiomiócitos foi observada menor oxidação de carboidratos (-47%) e de ácidos graxos (-60%). Porém, sem alteração na expressão de proteínas relacionadas à bioenergética de cardiomiócitos, CPT1, UCP2, GLUT1, GLUT4, AMPK e pAMPK. A partir desses resultados, concluímos que o tipo e a quantidade de ácidos graxos predizem o fenótipo cardíaco na obesidade, promovendo alteração na capacidade oxidativa mitocondrial, na morfologia e na hemodinâmica cardíaca / Obese individuals have a higher risk of morbidity and mortality attributed to cardiovascular disease. The diet composition is one factor that predicts the cardiac phenotype in response to obesity and the type of fatty acid differentially influences the myocardial structure and function. Studies have showed that mitochondrial dysfunction is considered to play a key role in the pathogenesis of cardiac hypertrophy and failure, also the mitochondrial alterations present in heart failure indicate to defects at specific sites in electron transport chain. Thus, the aim of the study was evaluated the ventricular contractile function in mice fed high fat diet, rich in polyunsaturated fatty acids, looking through mitochondrial bioenergetics. After weaning, mouse C57Bl/6 received manipulated diet containing 7% (C) or 19% (HF) of soybean oil, until 135 days of age. The food intake and the body mass were monitored, and the glucose tolerance test was realized. At the end of the experimental period, the animals had their body composition evaluated by Dual-energy X-ray Absorptiometry (DXA), after, were sacrificed by exsanguination. In plasma was determined the lipid profile and insulin. The heart, intraabdominal and subcutaneous adipose tissue were collected, weighted and processed to morphological analysis. The left ventricular myocardial fibers were used to analyze mitochondrial respiration by technique of high resolution respirometry. The heart was used to the Langendorff technique of isolated heart perfusion, and to analyze the expression of proteins related to the cardiomyocytes bioenergetics, through of Western Blotting. The HOMA-IR and the adiposity index were calculated. The group HF showed higher adiposity, but did not differ about food intake. Was observed glucose intolerance, hiperinsulinemia, insulin resistance and also unfavorable alterations in lipid profile. Was observed alterations in cardiac morphology and hypertrophic cardiomyopathy, reflecting in hemodinamic alterations with increase of contractility, higher ventricular pressure and impaired diastolic function. About the mitochondrial activity of cardiomyocytes was observed lower oxidation of carbohydrates (-47%) and fatty acids (-60%). However, the expression of proteins related to the cardiomyocytes bioenergetics, CPT1, UCP2, GLUT1, GLUT4, AMPK e pAMPK, did not differ between the groups. From these results, we conclude that the type and amount of fatty acids predict the cardiac phenotype in obesity, promoting the impairment of mitochondrial oxidative capacity, alterations in cardiac morphology and hemodynamics

Epigenetic modifications are alterations in the cell nucleus that affect gene expression and can occur in chromatin at the level of DNA methylation or histone modifications. Such ‘epigenetic marks’ can be heritable through cell division but leave the DNA sequence unchanged. Post-­translational modifications can be found on the histone proteins associated with DNA; the majority of histone modifications are found on the lysine-­rich N-‐terminal amino acid “tails”. Histone acetylation and methylation influence the chromatin structure by loosening or tightening the packaging of DNA, respectively, in association with other chromatin modifiers. Condensed chromatin is linked to transcriptional silencing and genetic imprinting and also occurs at chromosomal centromeres, where it is linked to kinetochore binding. Heart development is well studied, but the epigenetic processes involved are not yet completely understood. While active chromatin mechanisms such as histone acetylation and chromatin remodelling have been described in the heart, the role of gene repressive epigenetic mechanisms has been poorly investigated. Cardiomyocytes are post-­mitotic cells that do not divide to regenerate a damaged heart. The regeneration of cardiomyocytes after myocardial infarction is an important topic of interest in cardiovascular science. There are various approaches to heart repair after infarction, including activating cardiomyocytes so they become mitotic once again, or growing cardiomyocytes in vitro to attach to a lesion site. An important factor in these approaches is understanding the epigenetic mechanisms controlling cell division. In this thesis, we aim to advance the current knowledge of the epigenetic repressive mechanisms involved in cardiomyocyte formation and heart development to explain their lack of regenerative capacities. We studied the epigenetic changes that occur during cardiac development leading to a non-­‐regenerative state to pinpoint the moment at which these changes arise. We found that the epigenetic process is independent of whether cardiac lineage differentiation occurs during embryogenesis or during differentiation in vitro. We discovered that cardiac heterochromatin displays a singular epigenetic signature during development as compared to brain, another post-­mitotic tissue, or liver, an actively regenerative tissue. We observed an epigenetic change in the repressive histone modification histone H3 lysine 9 trimethylation that was specific to heart development. This change involved a nuclear reorganisation of heterochromatin and a reduction of the levels of this mark in E13.5 and E14.5 embryos, as compared to E10.5 embryos. This was consistent with our observations of the histone lysine methyltransferase SUV39H1, the levels of which were lower after stage E10.5 of development. However, contradictorily, in differentiated cardiomyocytes in vitro, SUV39H1 was increased but showed low levels of H3K9me3, compared to ES cells, which had low levels of SUV39H1 and high levels of H3K9me3. We detected extremely low levels of the H3K9me3 in adult heart tissue. We observed that in adult hearts, the myocardium had maintained these major changes in H3K9me3, while this effect was not observed in the epicardium. Genomic studies were carried out to determine changes at a genomic level between the two key epigenetic stages in heart development we identified at E10.5 and E13.5. Methylated DNA immunoprecipitation sequencing and chromatin immunoprecipitation sequencing for H3K9me3 analyses were carried out to find overall changes in methylation patterns. No global changes in DNA methylation were detected between these developmental stages. These results imply that the differences observed in H3K9me3 are due to remodelling of the heterochromatin during heart development and cardiomyocyte formation, rather than quantitative changes.

Electrical potentials exist across the membranes of nearly every cell type in the body. In addition, excitable cells, such as neurons, myocytes and even some endocrine cells elicit electrochemical fluctuations, action potentials (AP), in the cell membrane to initiate cell-to-cell communication or intracellular processes. The basis for the electrical potential is rooted within an array of complex interactions between monovalent ions and their associated membrane channels and transporters that regulate the flux of these charged species across the hydrophobic bilayer. Here, an expansion of our recently published work [1] will serve to explore the modern concepts regarding the origin of the AP as well as to examine the mechanisms by which intracellular calcium ([Ca2+]i) is regulated within the HL-1 mouse cardiac myocyte.

Le vieillissement de l'organisme prédispose à de nombreuses pathologies chroniques telles que l'insuffisance cardiaque (IC). Des études récentes ont montré que l'accumulation de cellules sénescentes dans les organes au cours du vieillissement est associée à l'apparition de ces pathologies. La sénescence cellulaire a initialement été décrite comme un arrêt stable du cycle cellulaire permettant de limiter la prolifération des cellules dont l'ADN est endommagé. Ce processus s'accompagne de profondes modifications de la fonction cellulaire, avec notamment l'acquisition d'un phénotype sécrétoire associé à la sénescence. La sénescence peut être induite par un raccourcissement des télomères ou par l'exposition à des signaux de stress, tels que le stress oxydant ou l'irradiation, qui entrainent l'activation de la réponse cellulaire aux dommages de l'ADN et l'expression des gènes suppresseurs de tumeurs (p16INK4a, p21CIP1, p53). Ces inhibiteurs du cycle cellulaire sont classiquement utilisés comme marqueur de sénescence car leur expression augmente de manière ubiquitaire au cours du vieillissement. Toutefois, ces marqueurs ne sont pas spécifiques du tissu concerné et un des objectifs de ma thèse a été d'identifier de nouveaux marqueurs de sénescence tissu-spécifiques qui pourraient caractériser un vieillissement cardiaque pathologique. Le vieillissement cardiaque se caractérise par une hypertrophie des cardiomyocytes, une sensibilité accrue au stress et une prédisposition à l'IC. Les cardiomyocytes étant des cellules post-mitotiques, les mécanismes de sénescence mis en jeu, les marqueurs associés et leur rôle potentiel dans l'IC demeurent à l'heure actuelle peu caractérisés. Au cours de ce travail de thèse nous avons donc entrepris : 1) d'étudier le rôle des télomères et des dysfonctions mitochondriales dans l'induction de la sénescence du cardiomyocyte et 2) d'identifier des marqueurs spécifiques. Nous avons tout d'abord montré que les cardiomyocytes de souris âgées expriment les marqueurs classiques de la sénescence comme p16INK4a, p53 et p21CIP1. Concernant les mécanismes inducteurs, nous avons étudié l'implication des dommages télomériques (telomere associated foci, TAF). Au cours du vieillissement, nous avons observé une augmentation du nombre de TAFs par cardiomyocytes en association avec l'hypertrophie. De plus, l'induction de TAFs in vitro est suffisante à l'activation de la voie de sénescence p53/p21CIP1 et l'hypertrophie dans une lignée de cardiomyoblastes H9c2. La formation des TAFs est augmentée chez des souris avec une dysfonction mitochondriale et est associée à l'activation des voies p53/p21CIP1. Par ailleurs, les cardiomyocytes âgés présentent une dérégulation des gènes impliqués dans la biologie mitochondriale pouvant rendre compte de l'augmentation des TAFs. Par l'analyse haut débit du transcriptome (RNAseq) nous avons identifié six nouveaux gènes qui sont surexprimés dans les cardiomyocytes sénescents (Prom2, Kcnk1, Pah, Edn3, Gdf15, Tgfb2). La comparaison d'expression de ces gènes dans le cœur avec d'autres tissus et avec le stroma cardiaque lors vieillissement a permis de confirmer la spécificité d'expression de ces marqueurs au niveau des cardiomyocytes. Nous avons validé cette signature dans deux modèles in vitro de sénescence induite par le stress et démontré que l'expression de certains de ces marqueurs est dépendante de la voie p53. De plus, l'expression de Prom2 est associée à l'hypertrophie des cardiomyocytes. En conclusion, nous avons démontré, qu'avec le vieillissement, les cardiomyocytes présentent un programme de sénescence associé à une dysfonction mitochondriale et une augmentation des TAFs. Cette sénescence se caractérise par l'activation des voies classiques de sénescence (p16INK4, p53/p21CIP1), une hypertrophie et l'acquisition d'une signature spécifique. Ces marqueurs offrent de nouvelles perspectives dans la compréhension de la sénescence cardiaque et dans son implication potentielle dans l'IC. / Ageing of the organism is associated with several chronic pathologies such as heart failure (HF). Recent studies have demonstrated the link between the accumulation of senescent cells during ageing and age-associated diseases. Cellular senescence, originally defined as a stable cell cycle arrest, acts as a tumorigenic repressor by limiting the proliferation of DNA damaged cells. Despite this protective effect, senescence is characterized by deep remodeling of cell biology which drives functional disorders, such as the acquisition of a senescence-associated secretory phenotype (SASP). Senescence can be induced by telomeric attrition and by exposition to cellular stress signals such as oxidative stress or irradiation, which induce telomeric damage, activation of the DNA Damage Response (DDR) and increased expression of antitumoral genes (p16INK4a, p21CIP1, p53). These genes are classically used as markers of senescence because their expression increases in several tissues during ageing but they are not tissue-specific. Therefore, At the cardiac level, ageing is characterized by cardiomyocytes hypertrophy, increased sensitivity to stress and highest risk of developing HF. Cardiomyocytes are post- mitotic cells and the senescence inductor mechanisms, specifics markers and their role in HF remains poorly understood. This thesis project is articulated around two aims, 1/ studying the role of telomeric damages and mitochondrial dysfunction in triggering cardiomyocyte senescence and 2/ identification of specifics markers. Fisrtly, we shown that aged cardiomyocytes overexpress classic markers of senescence such as p16INK4a, p53 et p21CIP1. Concerning the inductors mechanisms, we studied the implication of telomeric damages (telomere associated foci, TAF). During ageing, we found an increased number of TAFs per cardiomyocytes and their association with hypertrophy. Moreover, TAF- induction in cardiac H9c2 in vitro activated the p53/p21 pathway and induced senescence. These data confirmed the role of TAFs in cardiomyocyte senescence induction. Furthermore, aged cardiomyocytes exhibit a global alteration of genes involved in mitochondrial biology, oxidative stress and metabolism in aged cardiomyocytes that could play a prominent role in TAF accumulation with ageing. In a second part of the study, by using a next generation sequencing method (RNA-seq) we identified 6 new genes highly expressed in senescent cardiomyocytes (Prom2, Kcnk1, Pah, Edn3, Gdf15 and Tgfb2). Expression comparison with other senescent organs and cardiac stromal cells confirmed these new genes as cardiomyocyte specific. Thanks to an in vitro approach, we validate this signature by using different models of stress-induced senescence in cardiac H9c2 cells and demonstrated the implication of the p53 in the regulation of some of these genes. Moreover, Prom2 expression is associated with cardiomyocytes hypertrophy. In conclusion, we demonstrated that, with ageing, cardiomyocytes display a senescence phenotype associated with mitochondrial dysfunction and TAFs. This process is characterized by classic markers (p16INK4, p53/p21CIP1), hypertrophy and new identified signature. These new markers offer innovative perspectives in the understanding and the identification of the cardiac senescence and their potential deleterious role in heart failure.

The proper orchestration of voltage-gated ion channel gating is vital to maintaining normal heart rhythms throughout an animal's lifespan. Voltage-gated sodium channels, Nav, are responsible for the initiation of the cardiac action potential, which leads to cardiac systole. Comparison of neonatal ventricular and atrial myocyte Nav gating with adult indicated that the neonatal ventricular Nav gated following a ~10 mV greater depolarization than did atrial or adult ventricular Nav. In this study I questioned whether development- and/or chamber-dependent changes in Nav-associated functional sialic acids could account for these differences. When desialylated with neuraminidase, all gating characteristics for the lower voltage activated atrial and adult ventricular Nav shifted significantly to more depolarized potentials. However, desialylation of the higher voltage activated neonatal ventricular Nav had no effect on channel gating. Furthermore, channels were stripped of their N-glycosylation via PNGase-F in an attempt to separate the potential effects of the remaining glycosylation structure on Nav gating. Following treatment, neonatal ventricular Nav gating remained unchanged while atrial and adult ventricular Nav gating again shifted to depolarized potentials nearly identical to those of the neonatal ventricular channel. Immunoblot analyses indicated that atrial and adult ventricular Nav α subunits are more heavily sialylated than the neonatal ventricular a subunit, with approximately 15 more sialic acid residues. The data indicate that differential sialylation of myocyte Nav α subunits is responsible for much of the developmental and chamber-specific remodeling of Nav gating observed here. In addition, the Nav1.5 α subunit can associate with β subunits, also believed to be sialylated. The potential for functional β1 trans sialic acids to further modulate Nav1.5 gating was tested via co-transfection of β1 with the Nav1.5 α subunit into the Pro5/Lec2 mammalian expression system. Co-transfection revealed that the additional b1 trans sialic acids caused a hyperpolarizing shift in all tested gating parameters. When transfected into neonatal ventricular myocytes, β1 expression revealed no effect, implying that β1 expression alone is not responsible. Together, the myocyte and expression system studies describe a novel mechanism by which Nav gating, and subsequently cardiac excitability, are modulated by the regulated change in channel-associated functional sialic acids.