Les ions Ca2+ responsables de la contraction musculaire sont extrudés du réticulum sarcoplasmique (RS) via le récepteur de la ryanodine de type 1 (RyR1). Des mutations du gène de RyR1 sont responsables chez l’homme de l’hyperthermie maligne (HM) et de la myopathie à cores centraux (MCC). Nous avons caractérisé les altérations de l’efflux calcique du RS dues à de telles mutations dans la fibre musculaire de souris par électrophysiologie et imagerie confocale. L’expression des formes Y523S, R615C et R2163H de RyR1, associées à l’HM, provoque une hypersensibilité de l’efflux vis-à-vis du potentiel membranaire alors que les formes I4897T et G4896V associées à la MCC provoquent une réduction chronique de l’efflux sans modification de densité des RyR1 s ainsi que des protéines Cav1.1 et SERCA1. L’expression de la forme R4892W associée à la MCC ne modifie pas l’efflux calcique suggérant une plus faible pénétrance fonctionnelle de cette forme. Dans tous les cas, aucune indication de changement du contenu en calcium RS n’a été observée. Les résultats suggèrent que les modifications pathologiques de l’efflux calcique sont la conséquence directe de l’altération de fonction des canaux. Le deuxième objectif du travail s’est intéressé au rôle de certains phosphoinositides (PtdInsPs) dans la régulation de l’efflux calcique du RS. La surexpression de la PtdInsPs-phosphatase Mtm 1 n’a aucun effet sur l’efflux calcique alors que l’application intracellulaire de ses deux principaux substrats inhibe l’efflux, suggérant que leur accumulation dans les fibres musculaires déficientes en Mtm1 pourrait contribuer aux altérations pathologiques associées du couplage excitation-contraction / Ca2+ ions that trigger muscle contraction are released from the sarcoplasmic reticulum (SR) through the type 1 ryanodine receptor (RyR1) channel. Mutations of the gene encoding RyR1 are responsible for malignant hyperthermia (MH) and central core disease (CCD) in human. We characterized the alterations of SR Ca2+ release due to such mutations in mouse fibers using electrophysiology and confocal imaging. Expression of each of the MH-associated Y523S, R615C and R2163H mutant forms of RyR1 increases the sensitivity of Ca2+ release to membrane potential whereas forms I4897T and G4896V that are associated to CCD provoke a chronic depression of Ca2+ release with no concurrent alteration of RyR1, Cav1.1 and SERCA1 density. Expression of the CDD-associated R4892W form of RyR1 has no effect on Ca2+ release suggesting a weaker functional penetrance of this mutant form. In all cases we found no indication for a change in SR calcium content. Results suggest that pathological changes in Ca2+ release are the direct consequence of the functional alteration of the channels. The second goal of this work focused on the role of certain phosphoinositides (PtdInsPs) in the control of SR Ca2+ release. Over-expression of the PtdInsPs-phosphatase Mtm 1 does not affect Ca2+ release whereas intracellular application of its two main substrates inhibits Ca2+ release, suggesting that accumulation of these molecules in Mtm 1-deficient fibers could contribute to the associated alterations of excitation-contraction coupling

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

Les cellules murales du cerveau sont un groupe de cellules neurovasculaires qui présentent une hétérogénéité moléculaire, morphologique et fonctionnelle exceptionnelle. Celles en contact avec les plus petis vaisseaux du cerveau, les péricytes du lit capillaire moyen sont connues pour être essentielles à l'homéostasie cérébrale, bien que leur capacité contractile ait longtemps été débattue. Cependant, nombre de leurs propriétés physiologiques, telles que leurs mécanismes de signalisation calcique, n'ont pas encore été élucidées. Cette thèse vise donc à identifier les mécanismes cellulaires de la signalisation calcique des péricytes des capillaires cérébraux. Dans le chapitre 2, nous utilisons la pharmacologie et l'imagerie des péricytes cérébraux exprimant l'indicateur de calcium GCaMP6f (provenant de souris transgéniques PDGFRβ-Cre::GCaMP6f) pour découvrir ces mécanismes. Contrairement aux péricytes engainants dont la signalisation du calcique dépend des canaux calcique voltage-dépendants, nous constatons que les signaux calcique des péricytes capillaire moyen sont indépendants des canaux calcique voltage-dépendants. Au contraire, nous constatons que les signaux calciques transitoires des pericytes du lit capillaire moyen sont inhibés par l'élimination du Ca2+ extracellulaire, l'inhibition des canaux Orai opérés par les réserves, le blocage du remplissage des réserves du réticulum endoplasmique, ainsi que l'inhibition des récepteurs de la ryanodine (RyRs) et des récepteurs de l'inositol trisphosphate (IP3Rs). Nous constatons également que l'entrée de Ca2+ opérée par les réserves peut être induite par la déplétion des réserves du réticulum endoplasmique et inhibée par les bloqueurs d'Orai dans les pericytes du lit capillaire moyen, et que l'influx basal de Ca2+ est largement dépendant de la déplétion des réserves. Enfin, nous montrons que l'entrée de Ca2+ opérée par les réserves d'Orai amplifie les élévations de Ca2+ cytosolique en réponse au vasoconstricteur endothéline-1. Nous concluons que la signalisation calcique dans les pericytes du lit capillaire moyen, qu'elle soit spontanée ou induite de façon agoniste, est régulée par le couplage entre la libération des réserves du réticulum endoplasmique et les voies d'influx opérées par les réserves. / Brain mural cells are a grouping of neurovascular cells that display exceptional molecular, morphological, and functional heterogeneity. Mid-capillary pericytes, the mural cells which contact the smallest vessels of the brain, are known to be critical to brain homeostasis, and their contractile ability has long been debated. However, many of their physiological properties, such as their Ca2+ signaling mechanisms, have not been elucidated. This thesis aims to uncover the cellular mechanisms of brain mid-capillary pericyte Ca2+ signaling. In chapter 2, we harness pharmacology and imaging of brain pericytes expressing the calcium indicator GCaMP6f (from transgenic PDGFRβ-Cre::GCaMP6f mice) to uncover these mechanisms. In contrast to ensheathing pericytes whose Ca2+ signaling is dependent on voltage-gated Ca2+ channels (VGCCs), we find that mid-capillary pericyte Ca2+ signals are independent of VGCCs. Instead, we find that mid-capillary pericyte Ca2+ transients are inhibited by removal of extracellular Ca2+, inhibition of store-operated Orai channels, blockade of endoplasmic reticulum store filling, as well as inhibition of ryanodine receptors (RyRs) and inositol triphosphate receptors (IP3Rs). We further find that store-operated Ca2+ entry can be induced by endoplasmic reticulum store depletion and inhibited by Orai blockers in mid-capillary pericytes, and that basal Ca2+ influx is largely dependent on store depletion. Finally, we show that Orai store-operated Ca2+ entry amplifies cytosolic Ca2+ elevations in response to the vasoconstrictor endothelin-1. We conclude that both spontaneous and Gq-coupled protein receptor agonist-induced Ca2+ signaling in mid-capillary pericytes is regulated by coupling between endoplasmic reticulum store release and store-operated influx pathways.

No description available.

<p>  </p> <p>The role of the endoplasmic reticulum (ER) Ca2+ release channels ryanodine receptor 2 (RyR2) and inositol 1,4,5-triphosphate receptor (IP3R) in pancreatic b-cell function are emerging, but are not well defined. It has been demonstrated that ER stress brought about by RyR2 dysfunction leads to impaired insulin secretion and contributes to the etiology of type 2 diabetes (T2D). Our work contributes to the understanding of the role of RyR2 in physiological pancreatic b-cell function and how loss of RyR2 contributes to the pathophysiology of T2D.</p> <p>To investigate the role of RyR2 in pancreatic b-cell function, we utilized CRISPR-Cas9 to delete RyR2 from the rat insulinoma INS-1 cell line (RyR2KO). We found that RyR2KO cells displayed an enhanced glucose-stimulated Ca2+ integral (area under the curve; AUC) and were sensitive to inhibition by the IP3R antagonist, xestospongin C. Loss of RyR2 also resulted in a reduction in IRBIT protein levels. Therefore, we deleted IRBIT from INS-1 cells (IRBITKO) and found that IRBITKO cells also displayed an increased Ca2+ AUC in response to glucose stimulation. We discovered that total cellular insulin content and secretion were reduced in RyR2KO cells, but more modestly reduced in IRBITKO cells. We found that <em>INS2</em> mRNA levels were reduced in both RyR2KO and IRBITKO cells, but <em>INS1</em> mRNA levels were specifically decreased in RyR2KO cells. Additionally, nuclear localization of S-adenosylhomocysteinase (AHCY) was increased in both RyR2KO and IRBITKO cells. DNA methylation of exon 2 of the <em>INS1</em> and <em>INS2</em> genes was more extensively methylated in RyR2KO and IRBITKO cells compared to controls. Proteomics analysis revealed that deletion of RyR2 or IRBIT resulted in differential regulation of 314 and 137 proteins, respectively, with 41 in common. Our results suggest that RyR2 regulates IRBIT levels and activity, and together maintain insulin content and secretion, and regulate the INS-1 cell proteome, perhaps via DNA methylation.</p> <p>The role of interplay between RyR2 and IP3R in Ca2+ signaling and homeostasis in pancreatic b-cell function remains understudied. Stimulation with the sulfonylurea tolbutamide resulted in markedly delayed Ca2+ transients in both RyR2KO and IRBITKO cells. Xestospongin C significantly reduced the AUC of Ca2+ in RyR2KO and IRBITKO cells. Muscarinic receptor stimulation revealed a markedly increased AUC of Ca2+ in IRBITKO cells compared to both RyR2KO and control INS-1 cells. Assessment of PLC activity revealed that basal and stimulated PLC activity were reduced in the absence of RyR2 or IRBIT. Store-operated Ca2+ entry (SOCE) following ER Ca2+ depletion revealed a decreased SOCE amplitude only in RyR2KO cells. Given evidence that phosphatidylinositol-4,5-bisphosphate (PIP2) depletion from the plasma membrane can regulate voltage-gated Ca2+ channel inhibition, we explored electrophysiological properties of all three cell lines. The frequency of glucose-stimulated action potentials was doubled in RyR2KO cells. Additionally, whole-cell voltage-gated Ca2+ current density was doubled in RyR2KO cells, and this current was more sensitive to hydrolysis of PIP2. These results evidence crosstalk between RyR2 and IP3R, and that RyR2 plays a critical role in maintaining proper Ca2+ homeostasis, PLC activity, and electrophysiological properties in pancreatic b-cells.</p>

Introdução: A miopatia centronuclear é uma doença muscular congênita com apresentação clínica heterogênea, caracterizada histologicamente pela proeminência de fibras musculares com núcleos centralizados. Três formas são reconhecidas: neonatal grave, com herança ligada ao X e envolvimento do gene MTM1; autossômica dominante, com início geralmente tardio e curso mais leve, associada a mutações no gene DNM2; e autossômica recessiva, com gravidade intermediária entre as outras formas e envolvimento dos genes BIN1, RYR1 ou TTN. Apesar da identificação dos principais genes responsáveis pela doença, os métodos usuais de diagnóstico genético não encontram mutações em cerca da metade dos casos. Objetivo: O objetivo deste estudo foi a caracterização clínica, histológica e molecular de pacientes brasileiros portadores de miopatia centronuclear. Métodos: Laudos de dois bancos de biópsia muscular foram usados para identificar pacientes com diagnóstico de miopatia centronuclear nos últimos dez anos. As lâminas das biópsias foram revisadas e analisadas, e as famílias correspondentes convocadas para aplicação de protocolo clínico e coleta de sangue periférico para extração de DNA genômico. As famílias foram estudadas para os genes conhecidos por sequenciamento Sanger, MLPA, painel de genes implicados em doenças neuromusculares ou sequenciamento de exoma. Resultados: Foram convocados 24 pacientes provenientes de 21 famílias, em 16 das quais foi possível estabelecer o diagnóstico molecular. As 7 famílias com a forma neonatal grave constituíam um grupo homogêneo clínica e histologicamente, e mutações novas e conhecidas foram encontradas no gene MTM1 em 6 destas. Dois meninos deste grupo, com evolução estável, tiveram óbito súbito por choque hipovolêmico subsequente a rompimento de cisto hepático. O gene MTM1 também foi implicado em uma menina portadora manifestante, com quadro mais leve, na forma de uma macrodeleção em heterozigose, detectada por MPLA. Duas famílias em cuja histologia foram encontradas fibras com aspecto em \"roda de carroça\" apresentaram mutações no gene DNM2, uma das quais, p.Phe372Cys, nunca havia sido descrita. Em 7 famílias, o gene RYR1 foi o responsável, em todas sob a forma de heterozigose composta, com 14 mutações, das quais 13 novas, encontradas ao longo de todo o gene. Este grupo, apesar de heterogêneo clinicamente, apresentou em comum a presença de falhas focais na atividade oxidativa das fibras musculares na maioria dos indivíduos. O gene TTN está provavelmente implicado em uma família com um único afetado, no qual o sequenciamento de exoma mostrou mutação nula em heterozigose composta. Nesta coorte de pacientes brasileiros, não houve famílias com alterações no gene BIN1, e três famílias seguem sem diagnóstico molecular, com prováveis novos genes implicados. Conclusões: Os achados clínicos e histológicos de pacientes brasileiros com miopatia centronuclear seguem os padrões descritos na literatura, e em conjunto podem direcionar o estudo molecular adequado. Nesta coorte de pacientes, o gene RYR1, estudado por sequenciamento de alto débito de exoma, foi o mais frequentemente acometido, sugerindo que sua implicação na miopatia centronuclear vem sendo subestimada. Novas mutações encontradas nos genes MTM1, DNM2 e RYR1 contribuíram para confirmar regiões de patogenicidade e ampliar o espectro de alterações nestes genes / Introduction: Centronuclear myopathy is a heterogeneous congenital muscle disease, characterized by the prominence of centralized nuclei in muscle fibers. Three disease forms are recognized: a severe neonatal, X-linked form caused by mutations in the MTM1 gene; an autosomal dominant, late-onset milder form, associated to the DNM2 gene; and an autosomal recessive form, with intermediate severity, so far with the BIN1, RYR1 or TTN genes implicated. In spite of the identification of these genes, usual molecular diagnostic methods don\'t yield a molecular diagnosis in about half of cases. Objetives: The aim of this work was to study clinical, histological, and molecular aspects of centronuclear myopathy Brazilian patients. Methods: Reports taken from two muscle biopsy banks were used to identify centronuclear myopathy patients in the last ten years. Biopsy slides were reviewed and analyzed, and corresponding families recruited to apply a clinical protocol and to draw peripheral blood to extract genomic DNA. Families were studied for known genes via Sanger sequencing, MLPA, panel of genes implicated in neuromuscular diseases, or exome sequencing. Results: Twentyfour patients out of 21 families were recruited, and in 16 families molecular diagnosis was established. The 7 families with the severe neonatal form amounted to a clinically and histologically homogeneous group, and mutations, both known and novel, were found in the MTM1 gene in 6 of these. Two boys of this group, with a stable course, died suddenly of hypovolemic shock due to a hepatic cyst rupture. The MTM1 gene was also implicated in the case of a mild manifesting carrier girl with a heterozygous macrodeletion detected via MLPA. Two families whose histology contained fibers with a \"spoke of wheels\" aspect had mutations in the DNM2 gene, one of which, p.Phe372Cys, had never been described. In 7 families, the RYR1 gene was the culprit, in all of them in a compound heterozygous state, with 14 mutations, 13 of which novel, found throughout the length of the gene. This group, despite clinically heterogeneous, had in common the presence of focal disruptions in the oxidative activity of muscle fibers in the majority of individuals. The TTN gene is probably implicated in a family with a single affected, whose exome sequencing showed compound heterozygous null mutations. In this cohort of Brazilian patients, no family was found to have alterations in the BIN1 gene, and three families remain without molecular diagnosis, with probable new implicated genes. Conclusions: Clinical and histological findings of Brazilian patients with centronuclear myopathy follow patterns already described in the literature, and taken as a whole can direct the adequate molecular study. In this patient cohort, the RYR1 gene, sequenced though hight-throughput techniques, was the most frequently involved, suggesting that its implication in centronuclear myopathy is underestimated. Novel mutations found in the MTM1, DNM2 and RYR1 genes contributed to confirm pathogenic regions and expand the spectrum of alterations in these genes

La cascade d’évènements permettant la contraction de la fibre musculaire striée squelettique en réponse à l’activité électrique de sa membrane plasmique est regroupée sous le terme de couplage excitation-contraction (EC). Le couplage EC a lieu au niveau des triades, domaines nanoscopiques au niveau desquels les invaginations transversales de la membrane plasmique (tubules-T) sont en contact étroit avec deux citernes terminales adjacentes de réticulum sarcoplasmique (RS). Plus précisément, lors de l’excitation d’une fibre musculaire, un potentiel d’action se propage dans toute la surface de la membrane plasmique et en profondeur de la cellule via les tubules-T. Cette dépolarisation y est détectée par les protéines membranaires sensibles au potentiel Cav1.1 qui en retour, par couplage mécanique, déclenchent l’ouverture des canaux calciques du RS que sont les récepteurs de la ryanodine de type 1 (RYR1s). Ceci est à l’origine de l’augmentation massive de Ca2+ intracellulaire qui déclenche l’activation des myofilaments et donc la contraction. La compréhension des mécanismes de contrôle et de régulation des canaux RYR1s reste encore aujourd’hui limitée. En particulier, la mesure de l’activité physiologique de ces canaux dans la fibre musculaire intacte est toujours réalisée de manière très indirecte. Par ailleurs le rôle éventuel de variations de potentiel de la membrane du RS pendant l’activité musculaire n’a jamais été révélé. Une connaissance approfondie de ces phénomènes est pourtant essentielle à la compréhension de la fonction musculaire squelettique normale et pathologique. Dans ce contexte, l’objectif général de mon projet de thèse a été de mettre au point et utiliser des biosenseurs fluorescents localisés spécifiquement à la membrane des citernes terminales du RS de fibres musculaires différenciées – par leur fusion à une séquence d’adressage appropriée. Grâce à la combinaison des techniques d’électrophysiologie et d’imagerie de la fluorescence des biosenseurs sur fibres musculaires isolées, nous avons pu étudier l’activité du RS au cours de la fonction musculaire. Plus particulièrement, mon travail de thèse aborde deux problèmes biologiques principaux : le potentiel de membrane du RS et la signalisation calcique du RS au cours du couplage EC. Le premier objectif a visé à caractériser les changements de potentiel de la membrane du RS pendant l’activation du couplage EC. Pour cela, nous avons utilisé des biosenseurs de FRET de la famille Mermaid. Nos résultats montrent qu’il n’y a pas de changement substantiel du potentiel transmembranaire du RS pendant l’activation du couplage EC. Ces données confirment – pour la première fois en condition physiologique – que le flux de Ca2+ à travers les canaux RYR1s est équilibré par des contre-flux ioniques compensatoires qui permettent le maintien du potentiel de membrane du RS. Ceci assure la pérennité du flux de Ca2+ et contribue à l’efficacité du couplage EC. Le deuxième objectif a visé à détecter les variations de concentration en Ca2+ à proximité immédiate des canaux RYR1s. Pour cela, nous avons utilisé le biosenseur fluorescent sensible au Ca2+ GCamP6f. Le biosenseur adressé à la membrane du RS fournit un accès unique à l’activité individuelle de populations distinctes de canaux RYR1s au sein de différentes triades d’une même fibre musculaire. Au-delà de la caractérisation détaillée des propriétés des sondes GCaMP6f dans cette préparation, nos résultats montrent la stupéfiante synchronisation de l’activité de libération de Ca2+ des triades d’une même fibre musculaire au cours du couplage EC. Les résultats ouvrent des perspectives particulièrement intéressantes pour les études de situations pathologiques d’altération de l’activité des canaux RYR1s / Excitation-contraction (EC) coupling in skeletal muscle corresponds to the sequence of events through which muscle fiber contraction is triggered in response to plasma membrane electrical activity. EC coupling takes place at the triads; these are nanoscopic domains in which the transverse invaginations (t-tubules) of the surface membrane are in closed apposition with two adjacent terminal cisternae of the sarcoplasmic reticulum membrane (SR). More precisely, EC coupling starts with action potentials fired at the endplate, propagating throughout the surface membrane and in depth into the muscle fiber through the t-tubules network. When reaching the triadic region, action potentials activate the voltage-sensing protein Cav1.1. In turns, Cav1.1 directly open up the type 1 ryanodine receptor (RYR1) in the immediately adjacent SR membrane, through intermolecular conformational coupling. This triggers RYR1-mediated SR Ca2+ release which produces an increase in cytosolic Ca2+ triggering contraction. Current understanding of the mechanisms involved in the control and regulation of RYR1 channels function is still limited. One reason is related to the fact that detection of RYR1 channel activity in intact muscle fibers is only achieved with indirect methods. Also, whether SR the membrane voltage experiences changes during muscle activity has so far never been experimentally assessed. Yet, deeper knowledge of these processes is essential for our understanding of muscle function in normal and disease conditions. In this context, the general aim of my PhD project was to design and use fluorescent protein biosensors specifically localized at the SR membrane of differentiated muscle fibers, by fusing them to an appropriate targeting sequence. Thanks to a combination of single cell physiology and biophysics techniques based on electrophysiology and biosensor fluorescence detection, we were able to study the SR activity during muscle fiber function. Specifically, my PhD work focused on two major issues: SR membrane voltage and SR calcium signaling during EC coupling. The first aim of my work was to characterize SR membrane voltage changes during muscle fiber activity. For this, we used voltage sensitive FRET-biosensors of the Mermaid family. Results show that the SR trans-membrane voltage experiences no substantial change during EC coupling. This provides the first experimental evidence, in physiological conditions, for the existence of ion counter-fluxes that balance the charge deficit associated with RYR1-mediated SR Ca2+ release. Indeed, this process is essential for maintaining the SR Ca2+ flux upon RYR1 channels opening and thus critically important for EC coupling efficiency. The second objective of my work aimed at detecting the changes in Ca2+ concentration occurring in the immediate vicinity of the RYR1 Ca2+ release channels during muscle fiber activation. For this, we took advantage of one member of the recent generation of genetically encoded Ca2+ biosensor: GCaMP6f. The SR-targeted biosensor provides a unique access to the individual activity of RYR1 channels populations within distinct triads of a same muscle fiber. Beyond allowing a detailed characterization of the biosensor properties in this preparation, results highlight the remarkable uniformity of SR Ca2+ release activation from one triad to another, during EC coupling. These results open up stimulating perspectives for the investigation of disease conditions associated with defective behavior of RYR1 channels.

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Introdução: A miopatia centronuclear é uma doença muscular congênita com apresentação clínica heterogênea, caracterizada histologicamente pela proeminência de fibras musculares com núcleos centralizados. Três formas são reconhecidas: neonatal grave, com herança ligada ao X e envolvimento do gene MTM1; autossômica dominante, com início geralmente tardio e curso mais leve, associada a mutações no gene DNM2; e autossômica recessiva, com gravidade intermediária entre as outras formas e envolvimento dos genes BIN1, RYR1 ou TTN. Apesar da identificação dos principais genes responsáveis pela doença, os métodos usuais de diagnóstico genético não encontram mutações em cerca da metade dos casos. Objetivo: O objetivo deste estudo foi a caracterização clínica, histológica e molecular de pacientes brasileiros portadores de miopatia centronuclear. Métodos: Laudos de dois bancos de biópsia muscular foram usados para identificar pacientes com diagnóstico de miopatia centronuclear nos últimos dez anos. As lâminas das biópsias foram revisadas e analisadas, e as famílias correspondentes convocadas para aplicação de protocolo clínico e coleta de sangue periférico para extração de DNA genômico. As famílias foram estudadas para os genes conhecidos por sequenciamento Sanger, MLPA, painel de genes implicados em doenças neuromusculares ou sequenciamento de exoma. Resultados: Foram convocados 24 pacientes provenientes de 21 famílias, em 16 das quais foi possível estabelecer o diagnóstico molecular. As 7 famílias com a forma neonatal grave constituíam um grupo homogêneo clínica e histologicamente, e mutações novas e conhecidas foram encontradas no gene MTM1 em 6 destas. Dois meninos deste grupo, com evolução estável, tiveram óbito súbito por choque hipovolêmico subsequente a rompimento de cisto hepático. O gene MTM1 também foi implicado em uma menina portadora manifestante, com quadro mais leve, na forma de uma macrodeleção em heterozigose, detectada por MPLA. Duas famílias em cuja histologia foram encontradas fibras com aspecto em \"roda de carroça\" apresentaram mutações no gene DNM2, uma das quais, p.Phe372Cys, nunca havia sido descrita. Em 7 famílias, o gene RYR1 foi o responsável, em todas sob a forma de heterozigose composta, com 14 mutações, das quais 13 novas, encontradas ao longo de todo o gene. Este grupo, apesar de heterogêneo clinicamente, apresentou em comum a presença de falhas focais na atividade oxidativa das fibras musculares na maioria dos indivíduos. O gene TTN está provavelmente implicado em uma família com um único afetado, no qual o sequenciamento de exoma mostrou mutação nula em heterozigose composta. Nesta coorte de pacientes brasileiros, não houve famílias com alterações no gene BIN1, e três famílias seguem sem diagnóstico molecular, com prováveis novos genes implicados. Conclusões: Os achados clínicos e histológicos de pacientes brasileiros com miopatia centronuclear seguem os padrões descritos na literatura, e em conjunto podem direcionar o estudo molecular adequado. Nesta coorte de pacientes, o gene RYR1, estudado por sequenciamento de alto débito de exoma, foi o mais frequentemente acometido, sugerindo que sua implicação na miopatia centronuclear vem sendo subestimada. Novas mutações encontradas nos genes MTM1, DNM2 e RYR1 contribuíram para confirmar regiões de patogenicidade e ampliar o espectro de alterações nestes genes / Introduction: Centronuclear myopathy is a heterogeneous congenital muscle disease, characterized by the prominence of centralized nuclei in muscle fibers. Three disease forms are recognized: a severe neonatal, X-linked form caused by mutations in the MTM1 gene; an autosomal dominant, late-onset milder form, associated to the DNM2 gene; and an autosomal recessive form, with intermediate severity, so far with the BIN1, RYR1 or TTN genes implicated. In spite of the identification of these genes, usual molecular diagnostic methods don\'t yield a molecular diagnosis in about half of cases. Objetives: The aim of this work was to study clinical, histological, and molecular aspects of centronuclear myopathy Brazilian patients. Methods: Reports taken from two muscle biopsy banks were used to identify centronuclear myopathy patients in the last ten years. Biopsy slides were reviewed and analyzed, and corresponding families recruited to apply a clinical protocol and to draw peripheral blood to extract genomic DNA. Families were studied for known genes via Sanger sequencing, MLPA, panel of genes implicated in neuromuscular diseases, or exome sequencing. Results: Twentyfour patients out of 21 families were recruited, and in 16 families molecular diagnosis was established. The 7 families with the severe neonatal form amounted to a clinically and histologically homogeneous group, and mutations, both known and novel, were found in the MTM1 gene in 6 of these. Two boys of this group, with a stable course, died suddenly of hypovolemic shock due to a hepatic cyst rupture. The MTM1 gene was also implicated in the case of a mild manifesting carrier girl with a heterozygous macrodeletion detected via MLPA. Two families whose histology contained fibers with a \"spoke of wheels\" aspect had mutations in the DNM2 gene, one of which, p.Phe372Cys, had never been described. In 7 families, the RYR1 gene was the culprit, in all of them in a compound heterozygous state, with 14 mutations, 13 of which novel, found throughout the length of the gene. This group, despite clinically heterogeneous, had in common the presence of focal disruptions in the oxidative activity of muscle fibers in the majority of individuals. The TTN gene is probably implicated in a family with a single affected, whose exome sequencing showed compound heterozygous null mutations. In this cohort of Brazilian patients, no family was found to have alterations in the BIN1 gene, and three families remain without molecular diagnosis, with probable new implicated genes. Conclusions: Clinical and histological findings of Brazilian patients with centronuclear myopathy follow patterns already described in the literature, and taken as a whole can direct the adequate molecular study. In this patient cohort, the RYR1 gene, sequenced though hight-throughput techniques, was the most frequently involved, suggesting that its implication in centronuclear myopathy is underestimated. Novel mutations found in the MTM1, DNM2 and RYR1 genes contributed to confirm pathogenic regions and expand the spectrum of alterations in these genes