Le muscle cardiaque est un organe qui s'adapte à différents stress hémodynamiques en activant la synthèse protéique et en augmentant la taille des cardiomyocytes, résultant sur le développement d'une hypertrophie cardiaque. L'objectif de cette thèse est d'étudier le rôle potentiel du canal TRPM4 dans différents types d'hypertrophie cardiaque. Une altération du Ca2+ diastolique est à l'origine du signal initial activant les voies de signalisation d'une hypertrophie cardiaque délétère de type pathologique telle que la voie de la calcineurine-NFAT et la ré-expression de gènes fœtaux. Cette hypertrophie est alors compensatrice et vise à préserver la fonction de pompe du myocarde. Cette altération peut être conduite par divers stimulis tels qu'une augmentation de l'angiotensine II ou par des pathologies cardiovasculaires telles que l'infarctus du myocarde et l'hypertension. Cependant, une hypertrophie cardiaque bénéfique est également décrite dans la littérature, notamment lors des stades de développement du myocarde lors de l'embryogénèse ou en encore en réponse à une activité physique modérée régulière. Elle se caractérise par l'activation d'une toute autre voie de signalisation qu'est la voie de l'IGF-1-PI3K-Akt engendrée par une augmentation du taux de facteur de croissance qu'est l'insulin growth factor-1. Ces voies de signalisation ont été largement décrites dans la littérature et s'entrecroisent. Le canal TRPM4 est un canal cationique non sélectif perméable de manière égale aux ions Na+ et au K+, imperméables au Ca2+, mais activé par le Ca2+ intracellulaire. Dans le système immunitaire, il régule négativement l'entrée de Ca2+ et ce canal apparaît donc impliqué dans de nombreuses fonctions cellulaires dépendantes du Ca2+ dans différents types cellulaires. Par l'utilisation de deux modèles d'hypertrophie cardiaque, un physiologique généré par quatre semaines d'entraînement en endurance et un pathologique suite à un infarctus du myocarde induit par la ligature de l'artère coronaire gauche sur des souris wild-type et knock-out (KO) pour le canal TRPM4, nous avons mis en évidence une augmentation d'expression fonctionnelle du canal TRPM4 au sein du ventricule gauche associée à une régulation négative d'entrée de Ca2+. Le canal TRPM4 étant un régulateur de l'homéostasie calcique des cardiomyocytes, son expression fonctionelle après l'infarctus du myocarde ainsi que l'entraînement favorise l'activation de la voie de l'IGF-1-PI3K-Akt et prévient partiellement l'activation de la voie de la Calcineurine-NFAT et le développement d'une hypertrophie cardiaque pathologique, notamment dans le modèle d'infarctus du myocarde. En effet, en absence d'expression du canal, l'entrée de Ca2+ n'étant plus régulée, la voie de la Calcineurin-NFAT est favorisée. Mots clés : TRPM4, hypertrophie cardiaque, entraînement, IGF-1-PI3K-Akt, Calcineurine / Abstract: Cardiac muscle is an organ that adapts to different hemodynamic stress by activating protein synthesis and increasing cardiomyocytes size, resulting in cardiac hypertrophy. The objective of this PhD is to study the potential role of TRPM4 channel in different types of cardiac hypertrophy. Impaired diastolic Ca2+ is responsible for the initial signal activating signaling pathways in a deleterious cardiac hypertrophy pathological type such as Calcineurin-NFAT pathway and the re-expression of fetal genes. This hypertrophy is first compensatory and preserves the myocardial pump function. This alteration can be carried out by various stimuli such as increased angiotensin II or by cardiovascular diseases such as myocardial infarction and hypertension.However, a beneficial cardiac hypertrophy is also described in the literature, especially during development stages during embryogenesis or even in response to regular moderate physical activity. It is characterized by the activation one different signaling pathway, the IGF-1 - PI3K –Akt, generated by an increase in growth factor levels that is the insulin growth factor -1. These signaling pathways have been widely described in the literature and cross-talking. TRPM4 channel is a nonselective cation channel permeable equally to Na+ and K+, impermeable to Ca2+ but activated by the intracellular Ca2+. In the immune system, it downregulates Ca2+ entry and therefore appears to be involve in many Ca2+-dependent cellular functions in different cell types. By the use of two models of cardiac hypertrophy, a physiological generated by four weeks of training in endurance and pathological after myocardial infarction induced by ligation of the left coronary artery on wild-type and knockout mice -out (KO) for TRPM4 channel, we have demonstrated a functional expression increased TRPM4 channel within the left ventricle associated with down-regulation of Ca2 + entry. TRPM4 the channel being a regulator of calcium homeostasis in cardiomyocytes functional expression after myocardial infarction as well as the drive promotes the activation of the pathway of IGF-1-PI3K-Akt and partially prevents the pathway activation of the NFAT-calcineurin and the development of pathological cardiac hypertrophy, in particular myocardial infarction model. Indeed, in the absence of expression of the channel, the Ca2 + is not regulated, the path of Calcineurin-NFAT is favored. Keywords: TRPM4, cardiac hypertrophy, training, IGF-1-PI3K-Akt, calcineurin

Seminario de Título entregado a la Universidad de Chile en cumplimiento parcial de los requisitos para optar al Título de Ingeniero en Biotecnología Molecular. / Los canales de la superfamilia TRP son canales catiónicos no selectivos que permean principalmente Ca2+ y Na+, y comparten una arquitectura molecular general. Las subunidades de un canal TRP ensamblan en homo o heterotrámeros entre miembros de una misma o una diferente subfamilia compartiendo mecanismos de regulación y tráfico, lo que resulta en una gran diversidad de conductancias cationicas, en términos de sus propiedades regulatorias y biofísicas, por lo que han sido descritos como sensores polimodales que responden a una gran variedad de estímulos intracelulares y externos, cumpliendo roles fisiológicos esenciales en las funciones sensoriales, homeostáticas e incluso diversas funciones motiles como la contracción muscular y la migración celular. TRPC3 es un miembro de la subfamilia TRPC que está involucrado en un amplio espectro de mecanismos de señalización de Ca2+, y presenta propiedades de activación y regulación únicas que le permiten el reconocimiento e integración de múltiples estímulos. Este canal se asocia con varias proteínas permitiendo la formación de canales catiónicos diferentes en diversos tipos de células, afectando su actividad y función, por lo que es considerado un sensor multifuncional y versátil de gran relevancia fisiológica y fisiopatológica. Se ha visto que TRPC3 interacciona físicamente con TRPM4 en sistemas de expresión heterólogos, un miembro de la subfamilia TRPM impermeable a calcio, pero activado por este catión, que está involucrado en diferentes procesos fisiológicos, y cuya ganancia de función está relacionada con gran variedad de eventos fisiopatológicos como cáncer, enfermedades cardiovasculares y neurodegenerativas. En nuestro laboratorio, se encontró que las proteínas End Binding (EBs) interaccionan con un motivo ‘SxIP’ ubicado en la región amino terminal de TRPM4, y que xiii esta interacción TRPM4-EB gobierna el tráfico anterógrado del canal y su actividad. De acuerdo a estos antecedentes, se propone validar la interacción física entre TRPC3 y TRPM4, y evaluar si es que comparten mecanismos de tráfico y exportación a la superficie celular. Para ello, se realizaron experimentos de inmunoprecipitación y se evaluó el co-trafico de estos canales utilizando diferentes aproximaciones en modelos de expresión heteróloga que coexpresaban ambos canales. Se encontró que efectivamente TRPM4 interacciona con TRPC3, y además que la deleción del ‘motivo SWIP’ de TRPM4 afecta la localización y exportación de TRPC3 a la superficie celular. Debido a la creciente relevancia fisiológica de los eventos de heteroasociación y co-trafico relacionados a los canales TRP, estos datos sugieren que la interacción física entre estos miembros de diferentes sufamilias TRP involucra mecanismos de tráfico asociado a la interacción TRPM4-EBs y podría tener relevancia en tejidos que coexpresen ambos canales, como en tejido cardiovascular o neuronal. / TRP proteins are non-selective cationic channels that permeate mainly Ca2+ and Na+, and share a general molecular architecture. TRP channels subunits assemble as homo or heterotetramers between members of the same or different subfamily, sharing regulation and trafficking mechanisms which results in a great diversity of cationic conductances, in terms of their regulatory and biophysical properties, so they have been described as polymodal sensors that respond to a wide variety of intracellular and external stimuli, fulfilling essential physiological roles in sensory, homeostatic and even diverse motile functions such as muscle contraction and cell migration. TRPC3 is a member of the TRPC subfamily involved in a broad spectrum of Ca2+ signaling mechanisms, and has unique activation and regulation properties that allow recognition and integration of multiple stimuli. These channels are associated with several proteins that allow it to form different cation channels in different types of cells, affecting the activity and function of the channel, so it is considered a multifunctional and versatile sensor of great physiological and physiopathological relevance. TRPC3 physically interacts with TRPM4, a member of the TRPM subfamily impermeable by calcium, but activated by this cation, which is involved in different physiological processes, and whose gain of function is related to a great variety of pathophysiological events, such as cancer, cardiovascular and neurodegenerative diseases. In our laboratory, we found that End Binding proteins (EBs) interact with a 'SxIP' motif located in the amino terminal region of TRPM4, and this TRPM4-EB interaction governs the anterograde trafficking of the channel and its activity. Accordingly, we achieved to validate the physical interaction between TRPC3 and TRPM4, and to evaluate whether these channels share trafficking and exporting mechanisms to the cell surface. To do that, we performed immunoprecipitation assays and the co-trafficking assays in heterologous systems that coexpressed both channels. We found that TRPM4 interacts with TRPC3. Moreover, we observed that the deletion of the 'SWIP motif' of TRPM4 affects the localization and exporting of TRPC3 to the cell surface. Due to the increasing physiological relevance of hetero-association and co-trafficking events related to TRP channels, these data suggest that the physical interaction between these members of different TRP subfamilies involve trafficking mechanisms associated with TRPM4-EBs interaction. These data could have relevance in tissues that coexpress both channels, as in cardiovascular or neuronal tissues. / Este Seminario de Título fue financiado por el proyecto FONDECYT 1160518 y Núcleo Milenio de Enfermedades Asociadas a Canales Iónicos (IR: Oscar Cerda A.),

It is well established that intracellular calcium ([Ca2+]i) controls the inotropic state of the myocardium, and evidence mounts that a "Ca2+ clock" controls the chronotropic state of the heart. Recent findings describe a calcium-activated nonselective cation channel (NSCCa) in various cardiac preparations sharing hallmark characteristics of the transient receptor potential melastatin 4 (TRPM4). TRPM4 is functionally expressed throughout the heart and has been implicated as a NSCCa that mediates membrane depolarization. However, the functional significance of TRPM4 in regards to Ca2+ signaling and its effects on cellular excitability and pacemaker function remains inconclusive. Here, we show by Fura2 Ca-imaging that pharmacological inhibition of TRPM4 in HL-1 mouse cardiac myocytes by 9-phenanthrol (10μM) and flufenamic acid (10 and 100μM) decreases Ca2+ oscillations followed by an overall increase in [Ca2+]i. The latter occurs also in HL-1 cells in Ca2+-free solution and after depletion of sarcoplasmic reticulum Ca2+ with thapsigargin (10μM). These pharmacologic agents also depolarize HL-1 cell mitochondrial membrane potential. Furthermore, by on-cell voltage clamp we show that 9-phenanthrol reversibly inhibits membrane current; by fluorescence immunohistochemistry we demonstrate that HL-1 cells display punctate surface labeling with TRPM4 antibody; and by immunoblotting using this antibody we show these cells express a 130-150kDa protein, as expected for TRPM4. We conclude that 9-phenanthrol inhibits TRPM4 ion channels in HL-1 cells, which in turn decreases Ca2+ oscillations followed by a compensatory increase in [Ca2+]i from an intracellular store other than the sarcoplasmic reticulum. We speculate that the most likely source is the mitochondrion.

It is well established that intracellular calcium ([Ca2+]i) controls the inotropic state of the myocardium, and evidence mounts that a "Ca2+ clock" controls the chronotropic state of the heart. Recent findings describe a calcium-activated nonselective cation channel (NSCCa) in various cardiac preparations sharing hallmark characteristics of the transient receptor potential melastatin 4 (TRPM4). TRPM4 is functionally expressed throughout the heart and has been implicated as a NSCCa that mediates membrane depolarization. However, the functional significance of TRPM4 in regards to Ca2+ signaling and its effects on cellular excitability and pacemaker function remains inconclusive. Here, we show by Fura2 Ca-imaging that pharmacological inhibition of TRPM4 in HL-1 mouse cardiac myocytes by 9-phenanthrol (10μM) and flufenamic acid (10 and 100μM) decreases Ca2+ oscillations followed by an overall increase in [Ca2+]i. The latter occurs also in HL-1 cells in Ca2+-free solution and after depletion of sarcoplasmic reticulum Ca2+ with thapsigargin (10μM). These pharmacologic agents also depolarize HL-1 cell mitochondrial membrane potential. Furthermore, by on-cell voltage clamp we show that 9-phenanthrol reversibly inhibits membrane current; by fluorescence immunohistochemistry we demonstrate that HL-1 cells display punctate surface labeling with TRPM4 antibody; and by immunoblotting using this antibody we show these cells express a 130-150kDa protein, as expected for TRPM4. We conclude that 9-phenanthrol inhibits TRPM4 ion channels in HL-1 cells, which in turn decreases Ca2+ oscillations followed by a compensatory increase in [Ca2+]i from an intracellular store other than the sarcoplasmic reticulum. We speculate that the most likely source is the mitochondrion.

En utilisant la méthode de génétique inverse, la mutation causale d'un bloc de conduction cardiaque familial a été localisée sur le bras long du chromosome 19 en 13.3 dans une grande famille libanaise. Après avoir testé 12 gènes candidats, nous avons trouvé 3 mutations différentes dans trois familles indépendantes de bloc de conduction cardiaque isolé. Les conséquences des 3 mutations ont été explorées par des études électrophysiologiques. Il s'agit dans les 3 cas d'un gain de fonction. Puis, une cohorte de 248 patients atteints de syndrome de Brugada a été étudiée. Onze mutations du gène TRPM4 ont été trouvées chez 20 patients. Les conséquences électrophysiologiques des mutations étaient diverses. Ensuite, des cohortes de patients atteints de syndrome du QT long, de fibrillation auriculaire ou de cardiomyopathie dilaté ont été étudiées. Nous avons également trouvé des mutations ou des variants prédisposants du gène TRPM4 chez ces patients. Une étude électrophysiologique doit être réalisée pour comprendre le rôle de TRPM4 dans ces autres pathologies. Enfin, notre étude immunohistochimique a démontré que TRPM4 est fortement exprimé dans le système de conduction cardiaque mais aussi plus faiblement dans les cardiomyocytes auriculaires et ventriculaires communs. Ce travail a permis d'impliquer pour la première fois le gène TRPM4 dans des maladies humaines. Ce travail donne les bases pour comprendre le rôle du canal TRPM4 dans le fonctionnement cardiaque. C'est le préalable nécessaire avant de pouvoir développer de nouvelles thérapeutiques dans le futur / By using reverse genetics, the causal mutation of a familial cardiac conduction block was localized to the long arm of chromosome 19 in 13.3. After screening 12 candidate genes, we found 3 different mutations in three independent families with isolated cardiac conduction block. The consequences of these 3 mutations were explored by electrophysiological studies. In all 3 mutations it was a gain of function. Then, a cohort of 248 patients with a Brugada syndrome was studied. Eleven mutations were found in the TRPM4 gene in 20 patients. The electrophysiological consequences of these mutations were diverse. Then, cohorts of patients with long QT syndrome, atrial fibrillation, and dilated cardiomyopathy were studied. We found also mutations or predisposing variants in these patients. An electrophysiology study should be conducted to understand the role of TRPM4 in these other pathologies. Finally, our immunohistochemical study showed that TRPM4 is highly expressed in the cardiac conduction system but also although with less intensity in common auricular and ventricular cardiomyocytes. This work implied for the first time the TRPM4 gene in human diseases. This work provides the basis to understand the role of the TRPM4 channel in cardiac function. This is a prerequisite to be able to develop novel therapies in the future

Cerebral parenchymal arterioles (PAs) play a critical role in assuring appropriate blood flow and perfusion pressure within the brain. PAs are unique in contrast to upstream pial arteries, as defined by their critical roles in neurovascular coupling, distinct sensitivities to vasoconstrictors, and enhanced myogenic responsiveness. Dysfunction of these blood vessels is implicated in numerous cardiovascular diseases. However, treatments are limited due to incomplete understanding of the fundamental control mechanisms at this level of the circulation. One of the key elements within most vascular networks, including the cerebral circulation, is the presence of myogenic tone, an intrinsic process whereby resistance arteries constrict and reduce their diameter in response to elevated arterial pressure. This process is centrally involved in the ability of the brain to maintain nearly constant blood flow over a broad range of systemic blood pressures. The overall goal of this dissertation was to investigate the unique mechanisms of myogenic tone regulation in the cerebral microcirculation. To reveal the contributions of various signaling factors in this process, measurements of diameter, intracellular Ca2+ concentration ([Ca2+]i), membrane potential and ion channel activity were performed. Initial work determined that two purinergic G protein-coupled receptors, P2Y4 and P2Y6 receptors, play a unique role in mediating pressure-induced vasoconstriction of PAs in a ligand-independent manner. Moreover, a particular transient receptor potential (TRP) channel in the melastatin subfamily, i.e. TRPM4, was also identified as a mediator of PA myogenic responses. Notably, the observations that inhibiting TRPM4 channels substantially reduces P2Y receptor-mediated depolarization and vasoconstriction, and that P2Y receptor ligands markedly activate TRPM4 currents provide definitive evidence that this ion channel functions as an important link between mechano-sensitive P2Y receptor activation and the myogenic response in PAs. Next, the signaling cascades that mediate stretch-induced TRPM4 activation in PA myocytes were explored. Interestingly, these experiments determined that the RhoA/Rho kinase signaling pathway is involved in this mechanism by facilitating pressure-induced, P2Y receptor-mediated stimulation of TRPM4 channels, leading to subsequent smooth muscle depolarization, [Ca2+]i increase and contraction. Since Rho kinase is generally accepted as a 'Ca2+-sensitization' mediator, the present, contrasting observations point to an underappreciated role of RhoA/Rho kinase signaling in the excitation-contraction mechanisms within the cerebral microcirculation. Overall, this dissertation provides evidence that myogenic regulation of cerebral PAs is mediated by mechano-sensitive P2Y receptors, which initiate the RhoA/Rho kinase signaling pathway, subsequent TRPM4 channel opening, and concomitant depolarization and contraction of arteriolar smooth muscle cells. Revealing the unique mechanochemical coupling mechanisms in the cerebral microcirculation may lead to development of innovative therapeutic strategies for prevention and treatment of microvascular pathologies in the brain.

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.

TRPM4, un canal cationique non-sélective activé par le Ca2+ intracellulaire, est un acteur moléculaire important impliqué de la régulation du signal calcique et l’activation des lymphocytes T conventionnels mais son rôle dans la fonction des lymphocytes T régulateurs (Tregs Foxp3+) reste inconnu. Dans un modèle de souris transgéniques dans lequel le gène Trpm4 a été sélectivement invalidé dans la population des Tregs Foxp3+ (souris Foxp3(YFP)Cre+Trpm4flox/flox), nous avons démontré dans différents modèles in vivo d’inflammation aiguë et chronique que TRPM4 contrôle la fonction suppressive et la mort de ces cellules. Dans le modèle de fibrosarcome induit par le méthylcholanthrène (3-MCA) ou implanté (modèle MCA205), dans lequel le rôle des Tregs est documenté, l’absence de fonction de TRPM4 induit une diminution significative de l’incidence et de la croissance tumorale. Dans l’environnent inflammatoire chronique et hypoxique de ces tumeurs, l’expression de TRPM4 protège les Tregs infiltrant la tumeur de la mort cellulaire induit par l’ATP extracellulaire et stimule ainsi le développent et la progression tumorale. L’absence d’expression de TRPM4 dans les Tregs stimule la réponse anti-tumorale médiée par l’IFNg et induit la régression des tumeurs. En conclusion, en inhibant l’entrée de Ca2+ extracellulaire, TRPM4 régule négativement les fonctions suppressives des Tregs et protège ces cellules de la mort cellulaire induite par l’activation. / TRPM4, a Ca2+-activated non-selective cation ion channel is an important regulator of Ca2+ signaling and cell activation in conventional T cells, but its role in Foxp3+ Tregs function remains unknown. Using a model in which Trpm4 gene was selectively invalidated in Foxp3+ Tregs population (Foxp3(YFP)Cre+Trpm4flox/flox mice) we have shown in different in vivo models of acute and chronic inflammation that TRPM4 is an important regulator of Tregs functions and survival. In a model of primary carcinogenesis induced by methylcholantrene (3-MCA) or implanted fibrosarcoma (MCA205 model), in which Tregs role has been documented, lack of TRPM4 expression and function induced significantly decreased incidence and tumor growth. We found that within chronic inflammatory and hypoxic tumor microenvironment, TRPM4 protected Tregs from ATP-induced cell death and therefore promoted tumor initiation and progression. In contrast, TRPM4 deficiency in Tregs favored IFN-g-mediated spontaneous anti-tumor immune response. Thus, through inhibiting Ca2+ influx, TRPM4 acts as a negative modulator of Tregs suppressive functions and protects Tregs from activation-induced cell death.

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