Diverses études épidémiologiques ont mis en évidence une relation étroite entre pollution urbaine au monoxyde de carbone (CO) et mortalité cardiovasculaire. Récemment il a été mis en évidence, chez le rat, qu'une exposition prolongée à ce polluant urbain avait pour conséquence le développement d'un phénotype cellulaire pathologique, pouvant influencer la vulnérabilité du coeur à un stress aigu. L'objectif de nos travaux était donc i) d'évaluer l'impact de la pollution au CO, sur la sensibilité du myocarde de rats au syndrome d'ischémie-reperfusion (IR) ; et ii) d'évaluer les effets potentiellement cardioprotecteurs d'un exercice pratiqué régulièrement à intensité modérée, sur le remodelage phénotypique cellulaire myocardique. Pour cela, 187 rats Wistar ont été séparés en 3 groupes : des rats contrôles, des rats exposés pendant 4 semaines au CO (30-100 ppm), et des rats entraînés en endurance avant d'être exposés au CO. La sensibilité à l'IR était évaluée par ischémie régionale réalisée sur modèle de coeur isolé perfusé de Langendorff. La fonction et les mouvements calciques de cardiomyocytes isolés était évalués en condition basale et consécutivement à un protocole d'anoxie-réoxygénation. Les résultats de ce travail confirment l'apparition d’un phénotype pathologique chez les rats exposés de façon prolongée au CO. Ce phénotype pathologique caractérisé dans notre travail par une altération de l’homéostasie calcique et du statut redox cellulaire ainsi qu'une expression tissulaire de iNOS apparait comme à l'origine de la plus grande vulnérabilité du coeur à un stress d’IR. Un autre résultat majeur de ce travail est qu’une stratégie de cardioprotection par un exercice d'intensité modérée pratiqué de manière régulière, permet de prévenir le remodelage pathologique cardiomyocytaire et ainsi l'augmentation de la sensibilité du myocarde à l'IR / Epidemiological studies suggested that carbon monoxide (CO) urban air pollution is mainly related to cardiovascular mortality. In addition, recent experimental studies have highlighted that CO exposure was responsible for the development of cardiomyocytes’ pathological remodeling, which can render the heart more vulnerable to acute stresses. Therefore, the aim of this experimental work was to i) evaluate the impact of prolonged exposure to simulated CO urban pollution on the sensitivity of the myocardium to IR ; and ii) evaluate potential cardioprotective effects of regular bouts of endurance training in this model. 187 Wistar rats were separated into 3 groups : control rats, CO rats exposed during 4 weeks to CO (30-100 ppm), and CO exercised rats. Myocardial sensibility to IR was evaluated with a regional ischemia performed on a Langendorff model of isolated heart. Moreover, the cardiomyocytes’ function and calcium handling were evaluated at basal conditions, following a protocol of cellular anoxia and reoxygenation. The results of this study confirm that chronic exposure to CO is responsible for cardiac phenotypic changes, which are characterized in this work by an imbalance in the cardiomyocytes’ oxidative status, an impairment of calcium handling and iNOS expression. These phenotypic changes were associated in this work with higher heart vulnerability to IR. Another major result of this study is that regular bouts of endurance training conducted prior to CO exposure prevented the pathological cardiac remodeling, consequently leading to higher heart vulnerability due to IR

Orientadores: Antonio Carlos Boschiero, Helena Coutinho Franco de Oliveira / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-20T07:29:42Z (GMT). No. of bitstreams: 1 Souza_JaneCristinade_D.pdf: 1851485 bytes, checksum: 9bc182bf9f44ff62ddc24d9ea9603f71 (MD5) Previous issue date: 2012 / Resumo: Alterações no conteúdo de colesterol celular podem contribuir para o mau funcionamento das células-beta pancreáticas. Camundongos knockout para o receptor de LDL (LDLR-/-) possuem maior teor de colesterol nas ilhotas pancreáticas e secretam menos insulina em comparação a camundongos selvagens (WT). Neste estudo, investigamos a associação entre o conteúdo de colesterol, a secreção de insulina e a movimentação de cálcio citoplasmático nessas ilhotas. Além disso, analisamos o efeito da dieta rica em gordura (HFD) sobre a homeostasia glicêmica, secreção e ação da insulina nesses camundongos. Os resultados mostraram que a primeira e segunda fase de secreção de insulina assim como a movimentação de Ca2+, estimuladas por glicose, foram reduzidas nos LDLR-/-. Camundongos LDLR-/- também apresentaram menor conteúdo de proteínas envolvidas com a extrusão dos grânulos de insulina tais como: VAMP-2 e SNAP-25 (p<0,05). A remoção do excesso de colesterol pelo uso da metil-beta-ciclodextrina (M?CD) normalizou a secreção de insulina, estimulada por glicose (GSIS) ou tolbutamida, assim como a movimentação de cálcio estimulada por glicose. A remoção do colesterol das ilhotas WT com 0.1 e 1 mmol/L de M?CD reduziu a secreção bem como a movimentação de cálcio. No entanto, ilhotas incubadas com 10 mmol/L de M?CD apresentaram aumento significativo na secreção de insulina, apesar da redução na movimentação de cálcio. A dieta hiperlipídica (H) promoveu maior ganho de peso e acúmulo de gordura visceral nos camundongos LDLR-/-H em relação aos WTH. A dieta aumentou a glicemia tanto no jejum quanto alimentado, porém não houve alterações nas concentrações plasmáticas de insulina nos camundongos LDLR-/-H. Já nos camundongos WTH, a dieta causou aumento nas glicemias de jejum e alimentado bem como na insulinemia. A área sob a curva glicêmica durante o oGTT foi 30% maior nos camundongos LDLR-/-H. A GSIS não foi significativamente alterada pela dieta hiperlipídica em ambos os grupos. Camundongos LDLR-/- em dieta padrão apresentaram maior fosforilação do receptor de insulina (IR?) e da AKT em fígado e músculo. O conteúdo da enzima que degrada a insulina (IDE) se mostrou reduzido nos LDLR-/-. A dieta hiperlipídica reduziu a sinalização da insulina em fígado, músculo e tecido adiposo dos camundongos LDLR-/-H. Nos camundongos WTH essa dieta promoveu apenas uma redução na fosforilação do IR? no músculo. A análise conjunta dos resultados nos permitiu concluir que, tanto o aumento de colesterol verificado em ilhotas LDLR-/- quanto a diminuição excessiva do conteúdo de colesterol em ilhotas WT (tratadas com M?CD) alteram a movimentação de cálcio e conseqüentemente a secreção de insulina. A redução do colesterol nas ilhotas dos camundongos LDLR-/- corrigiu a redução da secreção de insulina, apesar das concentrações reduzidas de VAMP-2 e SNAP-25. Camundongos LDLR-/-, alimentados com dieta padrão, são mais sensíveis à insulina, provavelmente como um mecanismo adaptativo a menor secreção de insulina. No entanto, estas adaptações não são suficientes para manter a homeostase glicêmica visto que estes animais são intolerantes à glicose. Quando alimentados com dieta hiperlipídica, os LDLR-/-H se tornam resistentes à insulina provavelmente devido ao aumento do tecido adiposo visceral / Abstract: Changes in cellular cholesterol levels may contribute to beta cell dysfunction. Islets from LDL receptor knockout (LDLR-/-) possess higher cholesterol content and secrete less insulin than wild type (WT) mice. Here, we investigated the association between cholesterol content, insulin secretion and Ca2+ handling in these islets. In addition, we analyzed the effects of highfat- diet (HFD) on glucose homeostasis, insulin secretion and action in these mice. Both first and second phase of glucose-stimulated insulin secretion (GSIS) were lower in LDLR-/- compared with WT islets. This lower secretion was paralleled by impairment in Ca2+ handling in these islets. The contents of SNAP-25 and VAMP-2 proteins, which participate in the extrusion of the insulin containing granules, were reduced in LDLR-/- compared with WT islets. Removal of the excess of cholesterol from LDLR-/- islets (Methyl-?-cyclodextrine, M?CD) normalized glucose- and tolbutamide-induced insulin release. Glucose-stimulated Ca2+ handling was also normalized in cholesterol-depleted LDLR-/- islets. Cholesterol removal from WT islets by 0.1 and 1.0 mmol/L M?CD impaired both GSIS and Ca2+ handling. However, 10 mmol/L M?CD markedly increased insulin secretion induced by glucose or tolbutamide in WT islets, despite a significant reduction in Ca2+ handling. The HFD promoted higher body weight gain and visceral fat pad depot in LDLR-/-H than in WTH. LDLR-/-H mice showed fasted and fed glucose levels significantly higher whereas no changes in fasted plasma insulin levels were observed. WTH mice also showed an increase in fasted and fed glucose levels, but a higher fasted plasma insulin level (p<0.05) was noticed. The area under the curve of the oGTT in LDLR-/-H, but not in WTH, was increased (30%) by HFD. GSIS was not significantly altered by HFD in both groups. LDLR-/- mice showed higher IR? and AKT phosphorylation in liver and skeletal muscle. Insulin degrading enzyme (IDE) protein content was lower in liver of LDLR-/-. The HFD reduced insulin signaling in liver, skeletal muscle and adipose tissue in LDLR-/-H. In WTH the HFD reduced only the IR? phosphorylation in muscle. In conclusion, our results indicate that abnormal high (LDLR-/- islets) or low (WT islets treated with M?CD) cholesterol contents alter both GSIS and Ca2+ handling. Normalization of islet cholesterol content improved Ca2+ handling and insulin secretion in LDLR-/- islets, despite the lower expression of SNAP-25 and VAMP-2. LDLR-/- mice, fed a chow diet, are more sensitive to insulin probably due adaptive mechanisms that compensate the low insulin secretion. However, these changes are not sufficient to promote glucose homeostasis since these mice were glucose intolerants. When fed a high-fat diet, they became also insulin resistant probably due to an increase in the mass of the visceral adipose tissue / Doutorado / Fisiologia / Doutor em Biologia Funcional e Molecular

In adult CMs, ryanodine receptor 2 (RYR2) is an indispensable Ca2+ release channel that ensures the integrity of excitation-contraction (E-C) coupling, which is fundamental for every heartbeat. However, the role and importance of RYR2 during human embryonic cardiac development are still poorly understood. In this study, after the knockout of RYR2 gene (RYR2–/–), induced pluripotent stem cells (iPSCs) were able to differentiate into cardiomyocytes (CMs) with an efficiency similar to control iPSCs (Ctrl-iPSCs); however, the survival of iPSC-CMs was markedly affected by the lack of functional RYR2. While Ctrl-iPSC-CMs exhibited regular Ca2+ handling, significantly reduced frequency and intense abnormalities of Ca2+ transients were observed in RYR2–/–-iPSC-CMs. Ctrl-iPSC-CMs displayed sensitivity to extracellular calcium ([Ca2+]o) and caffeine in a concentration-dependent manner, while RYR2–/–-iPSC-CMs showed inconsistent reactions to [Ca2+]o and were insensitive to caffeine, indicating there is no RYR2-mediated Ca2+ release from the sarcoplasmic reticulum (SR). Instead, the compensatory mechanism for Ca2+ handling in RYR2–/–-iPSC-CMs is partially mediated by the Inositol 1,4,5-trisphosphate receptor (IP3R). Similar to Ctrl-iPSC-CMs, SR Ca2+ refilling in RYR2–/–-iPSC-CMs is mediated by sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA). Additionally, RYR2–/–-iPSC-CMs showed a decreased beating rate and a reduced L-type Ca2+ current (ICaL) density. These findings demonstrate that RYR2 is not required for CM lineage commitment but is important for CM survival and contractile function. IP3R-mediated Ca2+ release is one of the major compensatory mechanisms for Ca2+ cycling in human CMs with the RYR2 deficiency. Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a life-threatening inherited arrhythmogenic disorder. RYR2 mutations are the genetic cause of CPVT Type 1. So far, the pathogenic mechanism of how RYR2 mutations remodel cardiac rhythm remains controversial, because all existing hypotheses cannot independently and universally represent the mechanism behind CPVT. Patient-specific iPSCs offer a unique opportunity for CPVT modeling and investigation in vitro. In this study, the effects of four different RYR2 mutations (R420W, A2254C, E4076K, and H4742Y) on cardiac Ca2+ handling were examined individually. The R420W mutation in CPVTa-iPSC-CMs showed no effect on the amplitude of paced Ca2+ transients but led to an increased Ca2+ leak and a decreased SR Ca2+ content. Moreover, CPVTa-iPSC-CMs presented an enhanced sensitivity to [Ca2+]o and caffeine but a lower ICaL density. Compared to Ctrl cells, CPVTb-iPSC-CMs carrying the A2254V mutation displayed Ca2+ transients with smaller amplitude and higher frequency. More importantly, CPVTb-iPSC-CMs showed remarkably severe Ca2+ leak and unaltered SR Ca2+ content. The A2254V mutation also enhanced the sensitivity of iPSC-CMs to [Ca2+]o and caffeine. Interestingly, the ICaL density was found higher in CPVTb-iPSC-CMs. As for the E4076K mutation, it caused a reduction in both amplitude and frequency of Ca2+ transients in CPVTc-iPSC-CMs. In addition, the sensitivity to [Ca2+]o was diminished in CPVTc-iPSC-CMs, while the caffeine sensitivity and ICaL density were not changed. Regarding the H4742Y mutation, it led to a reduction of Ca2+ transient amplitude. In addition, CPVTd-iPSC-CMs manifested unique SR Ca2+ leak, which was resistant to tetracaine, suggesting a conformational remodeling of the H4742Y-mutated RYR2. Furthermore, CPVTd-iPSC-CMs also showed enhanced sensitivity to [Ca2+]o and caffeine, although the ICaL density was comparable to Ctrl-iPSC-CMs. In summary, the A2254V variation presented a typical gain-of-function mutation, rendering the RYR2 hyperactive, while the E4076K variation was identified as a loss-of-function mutation, leading to hypoactive RYR2. R420W and H4742Y mutations did not enhance or suppress the activity of RYR2. However, by destabilizing the N-terminal domain (NTD) of RYR2, the R420W mutation caused Ca2+ leak via the mutant channel, which could be blocked by RYR2 inhibitor. As for the H4742Y mutation, it led to a consistent and inhibitor-resistant Ca2+ leak via RYR2, suggesting a structural remodeling of RYR2 that disturbs complete closure of the channel. These results confirmed the importance of RYR2 on the maintenance of Ca2+ handling and gained evidence for the theory that the underlying mechanisms of CPVT caused by mutations in RYR2 should be mutation-specific rather than unified. This study suggests hiPSC-CMs as a suitable platform for modeling cardiac arrhythmogenic disease, interpreting potential molecular and pathophysiological mechanisms, testing new therapeutic compounds, and guiding mechanism-specific therapy.:Abbreviations V List of figures VIII List of tables X 1 Introduction 1 1.1 Human induced pluripotent stem cells 1 1.1.1 Generation and characteristics of human induced pluripotent stem cells 1 1.1.2 Differentiation of hiPSCs into cardiomyocytes 3 1.1.3 Modeling of inherited cardiac disease with hiPSCs 4 1.2 Catecholaminergic polymorphic ventricular tachycardia 7 1.2.1 Clinical characteristics and diagnosis of CPVT 7 1.2.2 Genetic background of CPVT 8 1.2.3 Clinical descriptions of CPVT patients recruited in this study 10 1.2.4 Patient-specific iPSC-CMs recapitulate the phenotypes of CPVT in vitro 10 1.3 Cardiac excitation-contraction coupling 11 1.3.1 Cardiac action potential 12 1.3.2 Ca2+ homeostasis in cardiomyocytes 14 1.3.2.1 Ca2+ influx via L-type Ca2+ channel 14 1.3.2.2 Initiation and termination of SR Ca2+ release 15 1.3.2.3 Ca2+ removal from cytosol 17 1.3.3 Cardiomyocyte contraction 20 1.4 Cardiac ryanodine receptor 21 1.4.1 Distribution and classification of RYRs 22 1.4.2 Regulation of RYR2 23 1.4.2.1 Cytosolic Ca2+ 23 1.4.2.2 Luminal Ca2+ 24 1.4.2.3 Phosphorylation by PKA and CaMKII 25 1.4.2.4 Calmodulin 27 1.4.2.5 Caffeine 27 1.4.3 Pathophysiological mechanisms of CPVT associated with RYR2 mutations 28 2 Aims of this study 33 3 Materials and methods 34 3.1 Materials 34 3.1.1 Cells 34 3.1.2 Laboratory devices and experimental hardware 34 3.1.3 Disposable items 36 3.1.4 Chemicals, solutions, and buffers for physiological and molecular experiment 36 3.1.5 Antibodies 40 3.1.6 Primers 41 3.1.7 Chemicals, media and solutions used for cell culture 42 3.1.8 Software 44 3.2 Methods 44 3.2.1 Cell culture 44 3.2.1.1 Preparation of glass coverslips for cell culture 44 3.2.1.2 Coating of plates and dishes 44 3.2.1.3 Cultivation of iPSCs with feeder-free method 45 3.2.1.4 Cryopreservation and thawing of iPSCs 45 3.2.1.5 Spontaneous differentiation of iPSCs in vitro 45 3.2.1.6 Directed differentiation of iPSCs into cardiomyocytes 46 3.2.1.7 First digestion of iPSC-CMs 46 3.2.1.8 Cryopreservation and thawing of iPSC-CMs 46 3.2.1.9 Time-dependent proliferation analysis of iPSC-CMs 47 3.2.1.10 Second digestion of iPSC-CMs 47 3.2.1.11 Collection of cell pellets for molecular experiment 47 3.2.2 Cell viability assay 48 3.2.3 Gene expression analyses 48 3.2.3.1 RNA isolation 48 3.2.3.2 Reverse transcription reaction 48 3.2.3.3 Polymerase chain reaction 49 3.2.3.4 Agarose gel electrophoresis 49 3.2.4 Protein expression analyses 49 3.2.4.1 Western blot 49 3.2.4.1.1 Lysis of cultured cells 49 3.2.4.1.2 SDS-polyacrylamide gel electrophoresis 50 3.2.4.1.3 Transfer and detection of proteins 50 3.2.4.2 Flow cytometry 51 3.2.4.3 Immunofluorescence staining 51 3.2.5 Calcium imaging 51 3.2.5.1 Measurement of spontaneous Ca2+ transients 52 3.2.5.2 Evaluation of diastolic SR Ca2+ leak and SR Ca2+ content 52 3.2.5.3 Assessment of iPSC-CM sensitivity to [Ca2+]o 53 3.2.5.4 Quantification of iPSC-CM response to caffeine 53 3.2.6 Patch-clamp 53 3.2.6.1 Preparation of agar salt bridge 53 3.2.6.2 Assessment of liquid junction 53 3.2.6.3 Measurement of action potential and L-type calcium current 54 3.2.7 Statistical analysis 54 4 Results 55 4.1 IP3R-mediated SR Ca2+ release partially restores the impaired Ca2+ handling in iPSC-CMs with RYR2 deficiency 55 4.1.1 Loss of RYR2 does not alter the pluripotency of RYR2–/–-iPSCs 55 4.1.2 Loss of RYR2 leads to increased death of RYR2–/–-iPSC-CMs 56 4.1.3 Loss of RYR2 does not affect the expression of IP3R in iPSC-CMs 58 4.1.4 RYR2–/–-iPSC-CMs show abnormal Ca2+ transients 60 4.1.5 The sensitivity of RYR2–/–-iPSC-CMs to [Ca2+]o and caffeine is changed 62 4.1.6 IP3R is critical for the generation Ca2+ transients in RYR2–/–-iPSC-CMs 63 4.1.7 SERCA-mediated SR Ca2+ uptake is crucial for the Ca2+ handling in both Ctrl- and RYR2–/–-iPSC-CMs 65 4.1.8 RYR2–/–-iPSC-CMs display abnormal action potentials 66 4.2 Investigation of the impaired function of RYR2 in CPVTa-iPSC-CMs 69 4.2.1 The R420W mutation leads to increased SR Ca2+ leak and decreased SR Ca2+ content 69 4.2.2 The R420W mutation leads to an enhanced sensitivity of iPSC-CMs to [Ca2+]o 70 4.2.3 CPVTa-iPSC-CMs shows increased sensitivity to caffeine 71 4.2.4 CPVTa-iPSC-CMs show reduced ICaL density 72 4.3 Investigation of the impaired function of RYR2 in CPVTb-iPSC-CMs 74 4.3.1 CPVTb-iPSC-CMs show abnormal Ca2+ transients 74 4.3.2 The A2254V mutation intensifies the SR Ca2+ leak in iPSC-CMs 75 4.3.3 The A2254V mutation enhances the sensitivity of iPSC-CMs to [Ca2+]o 76 4.3.4 The A2254V mutation increases the sensitivity of iPSC-CMs to caffeine 78 4.3.5 CPVTb-iPSC-CMs show increased ICaL density 78 4.4 Investigation of the impaired function of RYR2 in CPVTc-iPSC-CMs 79 4.4.1 CPVTc-iPSC-CMs show abnormal Ca2+ transients 79 4.4.2 The E4076K mutation shows no effect on the SR Ca2+ leak and content 80 4.4.3 The E4076K mutation diminishes the sensitivity of iPSC-CMs to [Ca2+]o 81 4.4.4 The E4076K mutation shows almost no effect on the response of iPSC-CMs to caffeine 82 4.4.5 The E4076K mutation does not alter the ICaL density in iPSC-CMs 83 4.5 Investigation of the impaired function of RYR2 in CPVTd-iPSC-CMs 84 4.5.1 CPVTd-iPSC-CMs show abnormal Ca2+ transients 84 4.5.2 The H4742Y mutation leads to a tetracaine-resistant Ca2+ leak in iPSC-CMs 84 4.5.3 The H4742Y mutation improves the sensitivity of iPSC-CMs to [Ca2+]o 86 4.5.4 The H4742Y mutation enhances the response of iPSC-CMs to caffeine 87 4.5.5 The H4742Y mutation alters the gating properties of LTCC in iPSC-CMs 88 5 Discussion 90 5.1 IP3R-mediated compensatory mechanism for Ca2+ handling in iPSC-CMs with RYR2 deficiency 90 5.2 Pathophysiological mechanisms of RYR2 mutation-related CPVT are mutation-specific 93 5.2.1 Dysfunctional Ca2+ handling caused by RYR2-R420W mutation 94 5.2.2 Dysfunctional Ca2+ handling caused by RYR2-A2254V mutation 96 5.2.3 Dysfunctional Ca2+ handling caused by RYR2-E4076K mutation 99 5.2.4 Dysfunctional Ca2+ handling caused by RYR2-H4742Y mutation 101 5.3 Conclusions and future perspectives 104 6 Summary 106 7 Zusammenfassung 108 8 References 111 9 Acknowledgements 131 10 Declaration 132

Le système cardio-respiratoire joue un rôle prépondérant au sein d’un organisme, en permettant notamment l’apport et la distribution du dioxygène et des nutriments à l’ensemble des cellules. Mieux comprendre les modifications du système cardio-respiratoire au cours de la vie est donc un élément central dans l’évaluation de la capacité de l’organisme à répondre à ses différents besoins énergétiques, ainsi que dans la compréhension du phénomène de mortalité naturelle liée à la sénescence. Cette étude a été réalisée sur un vertébré à durée de vie extrêmement courte (~6 mois), le poisson Nothobranchius furzeri. La température, en plus d’être considérée comme l’un des principaux facteurs environnementaux susceptibles d’influencer la physiologie des téléostéens, est connue pour son effet régulateur sur la longévité. Ainsi, les individus étaient acclimatés à deux températures, 26 °C considérée comme la température optimale chez cette espèce, et une température réduite de 22 °C. Au niveau individuel, les réponses biologiques ont été explorées au travers des performances physiologiques comme mesure indirecte de la fitness : la capacité métabolique aérobie (AS), la croissance, les performances de reproduction, de locomotion et de digestion. A l’échelle sub-individuelle, les mécanismes de régulation de la fonction cardiaque ont été examinés via une étude morpho-fonctionnelle des cardiomyocytes. Les résultats mettent en évidence la présence au cours de la vie de deux phases bien distinctes délimitées par un âge optimal : (1) une première phase d’élévation de l’AS des stades juvénile à adulte ; suivie (2) d’une seconde phase, synonyme de déclin de l’AS, des indicateurs cardiaques et des niveaux d’activités ciblés, associés probablement à l’entrée en sénescence. Une réduction de température accroit la longévité, et retarde les effets néfastes du vieillissement sur l’AS, et les performances cardiaques. Cette étude contribuera à établir une vision globale des effets du vieillissement sur la fonction cardio-respiratoire et à mieux comprendre les mécanismes mis en jeu lors d’un allongement de la longévité par une réduction de la température. / Cardio-respiratory system plays a key role in an organism by delivering oxygen and nutrients towards the cells. Exploring its age-dependant changes is therefore a corner stone for assessing the organism capacity to meet its energetic needs along its life cycle, and for the understanding of mechanisms involved in the mortality due to aging process. This study was realized in a vertebrate with an extremely short lifespan (~6 months), the fish Nothobranchius furzeri. Temperature is an external factor that regulates longevity. Here, fish were acclimatized at two temperatures, 26 °C considered as the optimal temperature for this species, and 22 °C. Biological responses of fish were evaluated at individual level through the assessment of aerobic metabolic scope (AS), growth, reproduction, locomotion and digestion, which are considered to be relevant indirect measurement of fitness. Furthermore, mechanisms involved in the cardiac function regulation were explored through a morpho-functional study of cardiomyocytes. Along the individual life cycle, two phases were emphasized: 1) the first corresponds to AS elevation from the juvenile to the adult stages, following by 2) a second phase reflecting the entrance in the senescent stage, characterized by the decline in AS, cardiac performances and in activity level. A temperature reduction increases the individual longevity and slows-down deleterious effect of aging on both AS and cardiac function. This study will contribute to provide a global vision of senescent effects on cardio-respiratory system, and a better understanding of the mechanisms involved in temperature-dependent increase in longevity.

La fibrillation auriculaire (FA) est l’arythmie cardiaque la plus fréquente et elle peut entraîner des complications médicales sévères, notamment des accidents vasculaires cérébraux. On observe des différences sexuelles importantes dans la présentation clinique de la FA. Son incidence est 1,5 à 2 fois plus élevée chez les hommes, tandis que les femmes tendent à développer de la FA plus tardivement et plus sévèrement. Malheureusement, on ignore toujours les causes de ces différences sexuelles. La FA est une pathologie multifactorielle généralement causée par un débalancement des propriétés électrophysiologiques et/ou structurelles des oreillettes favorisant l’initiation et/ou le maintien de cette arythmie. Le but de ce projet est de déterminer s’il existe des différences sexuelles parmi les mécanismes impliqués dans la pathogenèse de la FA chez la souris. Sachant que les hormones sexuelles peuvent avoir un impact considérable sur l’électrophysiologie cardiaque, un objectif complémentaire de ce projet est de déterminer la contribution des hormones sexuelles dans les différences observées. Au cours de ce projet, nous avons découvert que la prédisposition masculine à la FA est également retrouvée chez la souris mâle. Nous avons identifié des différences sexuelles dans la régulation du calcium intracellulaire favorisant l’initiation de la FA chez les mâles. Celles-ci sont reliées à une expression et une activité plus élevée de l’échangeur Na+-Ca2+ chez les mâles. Nous avons également observé que le maintien de la FA était favorisé par des oreillettes de plus grande taille et par une latéralisation plus prononcée des connexines chez les mâles. L’orchiectomie réduit la susceptibilité des mâles à la FA en diminuant la latéralisation des connexines ainsi que la taille des cardiomyocytes auriculaires, suggérant un rôle des androgènes. À terme, ce projet permettra de mieux comprendre les mécanismes impliqués dans les différences sexuelles dans la pathogenèse de la FA. Une meilleure compréhension de ces mécanismes pourrait mener à une approche thérapeutique mieux adaptée au sexe des patients, pour une meilleure prise en charge de ceux-ci. / Atrial fibrillation (AF) is the most common type of cardiac arrhythmia, and it can lead to severe medical complications, including stroke. There are significant sex differences in the clinical presentation of AF. Its incidence is 1.5- to 2-fold higher in men, whereas women tend to develop AF later and more severely. Unfortunately, the mechanisms underlying these sex differences remain unknown. AF is a multifactorial pathology generally caused by an imbalance in electrophysiological and/or structural properties of the atria that promote AF initiation and/or maintenance. The aim of this project is to determine whether there are sex differences among the mechanisms involved in the pathogenesis of AF in mice.. Knowing that sex hormones can have a considerable impact on cardiac electrophysiology, a complementary objective of this project was to explore the contribution of sex hormones in the sex differences we observed. We first discovered that the male predisposition to AF is also found in mice. We have identified major sex differences in the regulation of intracellular calcium that promote AF initiation in males. These differences are related to a higher Na+-Ca2+ exchanger expression and function in males. We have also observed that AF maintenance was favoured by larger atria and more pronounced lateralization of connexins in males. Orchiectomy reduced AF susceptibility of males by reducing connexin lateralization and the dimensions of atrial myocytes, suggesting a role for androgens. Ultimately, this project will provide a better understanding of the mechanisms underlying sex differences in the pathogenesis of AF. This information could lead to a therapeutic approach better adapted to the sex of the patients, for a better management of AF.