Objective: Brugada Syndrome is an autosomal dominant congenital heart disease that is responsible for 20% of sudden deaths of patients with structurally normal hearts. The majority of mutations involve the cardiac sodium channel gene SCN5A and give rise to classical symptoms, which include an abnormal electrocardiogram with ST segment elevation and a predisposition to ventricular fibrillation. To date, the implantation of a cardioverter defibrillator is the only proven effective treatment of the disease. The ability to reprogram dermal fibroblasts to induced pluripotent stem (iPS) cells and to differentiate these into cardiomyocytes with the same genetic background provides a novel approach to studying inherited cardiac channelopathies with advantages over existing model systems. Whilst this technique has enormous potential to model inherited channelopathies, such as Brugada Syndrome, the derived cells have not been fully characterised and compared to foetal and adult cardiomyocytes. Methods: Dermal fibroblasts from a patient with Brugada syndrome (SCN5A; c.1100G > A - pARG367HIS) and an age- and sex-matched control were reprogrammed using episomal vectors. All newly derived iPS cell lines were fully characterised using immunocytochemistry, flow cytometry, real-time quantitative reverse transcription PCR and single nucleotide polymorphism analysis and were compared to established human embryonic stem (hES) cell and in-house derived healthy control iPS cell lines. The same control cell lines were used to compare the efficiencies of several cardiac differentiation media. Spontaneously contracting areas, derived from control as well as patient iPS cell lines, were disaggregated and single cardiomyocytes were compared to foetal and adult cardiomyocytes isolated from primary human tissue using immunocytochemistry, transmission electron microscopy, membrane visualisation, calcium imaging and electrophysiology. Results: Comparison of cardiac differentiation protocols using healthy control hES and iPS cell lines found that despite significant inter-line variability with regard to efficiency of cardiac formation guided differentiation protocols could be used to reliably and efficiently generate beating bodies. Spontaneous contraction was observed in stem cell-derived cardiomyocytes and human foetal cardiomyocytes. Pluripotent stem cell-derived cardiomyocytes stained for markers of the cardiac contractile apparatus such as α-actinin, cardiac troponin I and cardiac troponin T. They also expressed functional voltage-activated sodium channels and exhibited action potential triggered calcium-induced calcium release. Stem cell-derived cardiomyocytes showed organisation of myofibrils, ultrastructure and calcium handling more similar to foetal than adult cardiomyocytes. Brugada Syndrome patient-specific cardiomyocytes were structurally indistinguishable from healthy control iPS cell line-derived cardiomyocytes. Electrophysiological analysis of sodium current density confirmed a ~50% reduction in patient-derived compared to healthy control-derived cardiomyocytes. Conclusion: Although iPS cells give rise to a mixture of immature and more mature cardiomyocytes, they all express typical cardiac proteins and have functional cardiac sodium channels. Results illustrate the ability of patient-specific iPS cell technology to model the abnormal functional phenotype of an inherited channelopathy that is independent of structural abnormalities and that the relative immaturity of iPS cell-derived cardiomyocytes does not prevent their use as an accurate model system for channelopathies affecting the cardiac sodium channel Nav1.5. This iPS cell based model system for classical Brugada Syndrome allows for the first time to study the mutation in its native environment and holds promise for further studies to investigate disease mechanisms of known and unknown mutations and to develop new therapies.

The limited treatment options available for heart disease patients has lead to increased interest in the development of embryonic stem cell (ESC) therapies to replace heart muscle. The challenges of developing usable ESC therapeutic strategies are associated with the limited ability to obtain a pure, defined population of differentiated cardiomyocytes, and the design of in vivo cell delivery platforms to minimize cardiomyocyte loss. These challenges were addressed in Chapter 2 by designing a cardiomyocyte selectable progenitor cell line that permitted evaluation of a collagen-based scaffold for its ability to sustain stem cell-derived cardiomyocyte function (“A P19 Cardiac Cell Line as a Model for Evaluating Cardiac Tissue Engineering Biomaterials”). P19 cells enriched for cardiomyocytes were viable on a transglutaminase cross-linked collagen scaffold, and maintained their cardiomyocyte contractile phenotype in vitro while growing on the scaffold. The potential for a novel cell-surface marker to purify cardiomyocytes within ESC cultures was evaluated in Chapter 3, “Dihydropyridine Receptor (DHP-R) Surface Marker Enrichment of ES-derived Cardiomyocytes”. DHP-R is demonstrated to be upregulated at the protein and RNA transcript level during cardiomyogenesis. DHP-R positive mouse ES cells were fluorescent activated cell sorted, and the DHP-R positive cultured cells were enriched for cardiomyocytes compared to the DHP-R negative population. Finally, in Chapter 4, mouse ESCs were characterized while growing on a clinically approved collagen I/III-based scaffold modified with the RGD integrin-binding motif, (“Collagen (+RGD and –RGD) scaffolds support cardiomyogenesis after aggregation of mouse embryonic stem cells”). The collagen I/III RGD+ and RGD- scaffolds sustained ESC-derived cardiomyocyte growth and function. Notably, no significant differences in cell survival, cardiac phenotype, and cardiomyocyte function were detected with the addition of the RGD domain to the collagen scaffold. Thus, in summary, these three studies have resulted in the identification of a potential cell surface marker for ESC-derived cardiomyocyte purification, and prove that collagen-based scaffolds can sustain ES-cardiomyocyte growth and function. This has set the framework for further studies that will move the field closer to obtaining a safe and effective delivery strategy for transplanting ESCs onto human hearts.

Myocardial ischemia and ischemia/reperfusion activate several protein kinase pathways. Protein kinase activation potentially regulates the onset of myocardial cell injury and the reduction of this injury by ischemic and pharmacologic preconditioning. The primary protein kinase pathways that are potentially activated by myocardial ischemia/reperfusion include: the MAP kinases, ERK 1/2, JNK 1/2, p38 MAPKα/β; the cell survival kinase, Akt; and the sodium-hydrogen exchanger (NHE) kinase, p90RSK. The literature does not support a role for ischemia/reperfusion in the activation of the tyrosine kinases, Src and Lck, or the translocation and activation of PKC. This review will detail the role of these protein kinases in the onset of myocardial cell death by necrosis and apoptosis and the reduction of this injury by preconditioning.

Sarcolemmal blebbing and rupture are prominent features of irreversible ischemic myocardial injury. Dystrophin and spectrin are sarcolemmal structural proteins. Dystrophin finks the transmembrane dystroglycan complex and extracellular laminin receptors to intracellular F-actin. Spectrin forms the backbone of the membrane skeleton confering an elastic modulus to the sarcolemmal membrane. An ischemic loss of membrane dystrophin and spectrin, in ischemically pelleted rabbit cardiomyocytes or in vivo 30-45 rain permanently ischemic. LAD-ligated hearts, was detected by immunofluorescence with monoclonal antibodies. Western blots of light and heavy microsomal vesicles and Triton-extracted membrane fractions from ischemic myocytes demonstrated a rapid loss of dystrophin coincident with sub-sarcolemmal bleb formation, subsequent to a hypotonic challenge. The loss of spectrin from purified sarcolemma of autolysed rabbit heart, and both isolated membrane vesicles and Triton solubilized membrane fractions of ischemic cardiomyocytes correlated linearly with the onset of osmotic fragility as assessed by membrane rupture, subsequent to a hypotonic challenge. In contrast to the ischemic loss of dystrophin and spectrin from the membrane, the dystrophin-associated proteins. α-sarcoglycan and β-dystroglycan and the integral membrane protein, sodium-calcium exchanger, were maintained in the membrane fraction of ischemic cells as compared to oxygenated cells. Preconditioning protected cells, but did not significantly alter ischemic dystrophin or spectrin translocation. This previously unrecognized loss of sarcolemmal dystrophin and spectrin may be the molecular basis for sub-sarcolemmal bleb formation and membrane fragility during the transition from reversible to irreversible ischemic myocardial injury.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18893号 / 医博第4004号 / 新制||医||1009(附属図書館) / 31844 / 京都大学大学院医学研究科医学専攻 / (主査)教授 山下 潤, 教授 羽賀 博典, 教授 瀬原 淳子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Glucocorticoids (GCs) are frequently prescribed pharmacological agents most notably for their immunosuppressant effects. Endogenous GCs mediate biological processes such as energy metabolism and tissue development. At the cellular level, GCs bind to the Glucocorticoid Receptor (GR), a cytosolic receptor that translocates to the nuclei upon ligand binding and alters gene transcription. Among a long list of genes activated by GCs is the Glucocorticoid Induced Leucine Zipper (GILZ). Although GC induced GILZ expression has been well established in lymphocytes, little is known whether cardiomyocytes respond to GCs by inducing GILZ. Unlike lymphocytes, in which GCs induce apoptosis and GILZ mediates GC induced apoptosis, cardiomyocytes respond to GCs by gaining resistance against apoptosis. We determined GILZ expression pattern in cardiomyocytes in vivo and in vitro. Our data demonstrate GILZ induction in cardiomyocytes both in vivo and in vitro by GCs and point to H9C2 cell line as a valid model for studying the biological function of GILZ in cardiomyocytes. I have also determined GILZ functions as GC induced cytoprotective protein against the known cardiac toxicant Doxorubicin. Finally I have determined GILZ stabilizes Bcl-xL pro-survival protein, providing a possible mechanism of cytoprotection in cardiomyocytes.

CONTEXTE: Au cours de l'infarctus du myocarde, l'homéostasie du calcium entre leréticulum sarcoplasmique (SR), les mitochondries et le cytosol est altérée chez lescardiomyocytes (CM) et conduit à la mort cellulaire. Les canaux de fuite de calcium sontconsidérés comme des régulateurs clés de l'homéostasie calcique réticulaire. Le translocon(TLC), un composant majeur de la machine de la traduction protéique, est un important canalde fuite calcique réticulaire.METHODES: Par des moyens optiques, nous avons d'abord évalué l'organisation spatiale etla fonction du TLC dans le SR de souris adultes CM. Dans un second temps, nous avonsinterrogé si et comment la modulation pharmacologique TLC pourrait réduire les lésionsd'ischémie/reperfusion (I/R) cardiaque dans un modèle d'infarctus du myocarde de souris.RÉSULTATS: Nos données montrent que le TLC est spécifiquement localisée dans le SRlongitudinale des CM chez la souris adulte. Nous démontrons que la puromycine (activateurpharmacologique du TLC) induit une réduction partielle des réserves de calcium dans le SRlongitudinale, alors que nous n'observons aucune altération des réserves de calcium dépendantdu récepteur ryanodine dans le SR jonctionnelle. Le préconditionnement de la souris par lapuromycine, soumis à un infarctus du myocarde, diminue significativement la zone d'infarctusde près de 30,9±6,3%. Ceci est corrélé à une diminution de l'activation des protéines proapoptotiquesmitochondriales et à une augmentation d'un mécanisme de pro-survie:l'autophagie. Nous avons également démontré que le préconditionnement de la puromycinediminue la vitesse d'augmentation du calcium dans le cytosol du CM adulte pendant la duréede l'ischémie en corrélation avec la diminution de l'activation des calpains calciques.CONCLUSIONS: Dans cette étude, nous avons caractérisé le TLC comme un canal de fuitespécifiquement situé dans le compartiment longitudinale du SR dans les CM de souris adultes.Nous avons constaté que l'activation pharmacologique de la TLC avant l'infarctus dumyocarde exerce un effet de préconditionnement sur le myocarde sans altérer les réserves de calcium dépendant de la ryanodine. Dans l'ensemble, ces résultats mettent l'accent sur les connaissances actuelles sur la dualité entre le SR jonctionnelle et le SR longitudinale et ouvrent de nouvelles perspectives thérapeutiques / BACKGROUND: During myocardial infarction, alteration of calcium homeostasis between sarcoplasmic reticulum (SR), mitochondria and cytosol occurs in cardiomyocytes (CM) and leads to cell death. Calcium leak channels are thought to be key regulators of the reticular calcium homeostasis. Translocon (TLC), a major component of the translation machinery, is a major reticular calcium leak channel.METHODS: By the mean of photonics, we first assessed the spatial organization and the function of TLC in the SR of adult mouse CM. In a second time, we questioned if and how the pharmacological TLC modulation could reduce ischemia/reperfusion (I/R)-mediated heart injury in a model of mouse myocardial infarction.RESULTS: Our data show that TLC is specifically located in the longitudinal SR in adult mouse CM. We demonstrate that puromycin induces a partial reduction of calcium stores in the longitudinal SR, while we observe no alteration in the ryanodine receptor-dependent calcium stores in the junctional SR. Puromycin preconditioning of mouse subjected to myocardial infarction significantly decreases the infarct area by near 30.9±6.3%. This is correlated with a decrease in the activation of mitochondrial pro-apoptotic proteins and an increase of a pro-survival mechanism: autophagy. We further demonstrated that puromycin preconditioning decreases the rate of calcium increase in the cytosol of adult CM during the ischemia duration in correlation with the decreased activation of calcium-dependent calpains.CONCLUSIONS: In this study, we characterized TLC as a leak channel specifically located in the longitudinal SR compartment of adult mouse CM. We found that the pharmacological activation of TLC before myocardial infarction exerts a preconditioning effect on myocardium without altering the ryanodine-dependent calcium stores. Altogether, these findings emphasize the present knowledge on the duality between junctional and longitudinal SR in CM and open up new therapeutic perspectives

L'infarctus du myocarde, une des causes majeures de mortalité à travers le monde, engendre une mort irréversible du muscle cardiaque suite à une ischémie. Cette ischémie, c'est-à-dire une privation de dioxygène et de nutriments, déclencher un stress réticulaire qui perturbe l'équilibre calcique de la cellule cardiaque. Plusieurs pompes et canaux calciques situés à la membrane plasmique ou réticulaire sont des intervenants clés dans le maintien de l'homéostasie calcique. Parmi eux, il existe des canaux de fuites calciques passives, comme les TRPs, et peu d'informations sont actuellement connus à propos de leur rôle précis au cours de l'infarctus du myocarde.TRPV1 est un canal cationique non sélectif qui est activé par la capsaïcine, le pH et la chaleur nocive (>42°C). Dans le muscle squelettique, nous avions démontré que TRPV1 est situé dans la partie longitudinale du réticulum sarcoplasmique et qu'il répond à différentes stimulations physiologiques et pharmacologiques (Lotteau et al., 2013). Ici, nous nous interrogeons sur un éventuel rôle similaire de TRPV1 dans la physiologie cardiaque. Des analyses biochimiques et des mesures de calcium intracellulaire furent réalisées sur des cardiomyocytes issus de souris WT et KO TRPV1. Nos résultats in vitro montrent que: (a) TRPV1 est exprimé dans les cellules cardiaques; (b) une activation de TRPV1 engendre une réduction de la concentration calcique réticulaire et que (c) TRPV1 pourrait être une cible directe de l'isoflurane.Dans la mesure où TRPV1 peut être modulé par de nombreuses molécules pharmacologiques, il pourrait constituer une cible thérapeutique pour réduire la taille d'infarctus. De nombreuses études antérieures ont déjà mis en évidence un rôle cardioprotecteur de TRPV1 dans le système nerveux entourant le cœur. Le but de cette étude est de décrire le fonctionnement des canaux TRPV1 dans des cardiomyocytes adultes / Acute myocardial infarction (MI), a leading cause of morbidity and mortality worldwide, is the irreversible death of heart muscle secondary to ischemia. This ischemia, i.e. oxygen and nutrients deprivation, triggers a reticular stress disrupting the Ca2+ balance of the cardiac cell. Several Ca2+ pumps and channels located at the sarcolemma or at the reticulum membrane are key players in this maintenance of Ca2+ homeostasis. Among them, we find passive leak channels, such as TRPs and little is known about their precise role in MI.TRPV1 represents a non-selective cation channel that is activated by capsaicin, pH and noxious heat. In skeletal muscle, we previously demonstrated that TRPV1 is located in the longitudinal part of the SR and respond to pharmacological and physiological activations (Lotteau et al., 2013). We questioned here whether TRPV1 might have a similar role in heart physiology. Biochemical analysis and intracellular Ca2+ measurements were performed on cardiomyocytes from wild-type and TRPV1-KO mice. Our in vitro results show that: (i) TRPV1 is expressed in cardiac cells; (ii) an increase in intracellular calcium concentration ([Ca2+]i) is elicited under TRPV1 activation; (iii) TRPV1 could be a direct target of isoflurane. In parallel, our in vivo results indicate that a pharmacological preconditioning by isoflurane decrease the infarct size, probably though activation of TRPV1. According to the fact that TRPV1 activity can be modulated by a lot of pharmacological molecules, TRPV1 may serve as therapeutic target to reduce the infarct size. Most of published data have already evidenced this TRPV1 cardioprotective role in the peripheral heart system. The aim of the present work is to describe how TRPV1 channels behave in adult cardiomyocytes

L'objectif général de ce travail de doctorat a été centré sur l'utilisation des cellules pluripotentes induites humaines dans la modélisation et l'évaluation thérapeutique des pathologies cardiaques. Depuis leur découverte en 2006, les iPSC offrent une opportunité pour le développement de modèles cellulaires humains et spécifiques de patients pour l'étude des mécanismes physiopathologiques, l'évaluation de réponses pharmacologiques et le génération de cellules redifférenciées (ici en cardiomyocytes) pour des applications thérapeutiques cellulaires. Dans ce travail nous avons démontré que la quantité mais aussi la qualité finale des cardiomyocytes dérivés d'iPSC dépend des conditions spatiales et pharmacologiques utilisées durant les différentes étapes de différenciation. L'utilisation d'un protocole de différentiation en monocouche avec blocage simultané et transitoire de l'ensemble des voies Wnt (canoniques et non canoniques) permet d'obtenir une maturation plus importante du sarcomère, étape essentielle pour la modélisation des sarcomèropathies La différenciation des iPSC en cardiomyocytes peut aussi être obtenue par une approche moléculaire ciblée visant à activer spécifiquement un programme cardiogénique. Celle-ci est obtenue via l'utilisation d'une protéine Cas9 mutée et couplée à un système transactivateur et permettant le ciblage simultané de 3 facteurs de transcription clés de la cardiogénèse (Gata4, Mef2c et Tbx5). Cette approche moléculaire est renforcée par la combinaison avec une stimulation pharmacologique ciblant la voie Wnt. / The general objective of this work was centered on the use of human induced pluripotent cells in modeling and therapeutic evaluation of cardiac pathologies. Since their discovery in 2006, the iPSC provide an opportunity for the development of human cellular models and specific patients for the study of pathophysiological mechanisms, evaluation of pharmacological responses and the generation redifférenciées cells (cardiomyocytes here) for applications cellular therapeutic. In this work we demonstrated that the quantity but also the final quality of cardiomyocytes derived from iPSC depends on the spatial and pharmacological conditions used during the various stages of differentiation. The use of a monolayer differentiation protocol with simultaneous and transient blocking of all Wnt pathways (canonical and noncanonical) allows to obtain a higher maturation of the sarcomere, an essential step for modeling sarcomeropathies IPSC differentiation into cardiomyocytes can also be obtained by targeted molecular approach to specifically activate cardiogenic program. This is achieved through the use of a mutated Cas9 protein and coupled with transactivator system. This allows simultaneous targeting of 3 key cardiogenesis transcription factors (Gata4, MEF2C and Tbx5). This molecular approach is enhanced by the combination with a pharmacological stimulation targeting the Wnt pathway. Beyond modeling of monogenic cardiac disease, cardiomyocytes derived from iPSC can reproduce more complex and multigenic diseases

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