Le canal calcique de libération du Ca2+, appelé récepteur à la ryanodine (RyR) est localisé dans la membrane du réticulum sarcoplasmique des cardiomyocytes, en incluant ceux du pacemaker, et a un rôle important dans le couplage excitation contraction et la génération du rythme cardiaque. Des mutations dans leur gène sont responsables de la tachycardie catécholergique (CPVT), qui est une maladie létale, manifestée par des syncopes ou mort subite lors de stress émotionnel ou physique. Au repos, ces patients ont un électrocardiogramme normal, mais une tendance plus importante à la bradycardie.Nos collaborateurs ont identifié la mutation RyR2R420Q dans une famille espagnole atteinte de CPVT. Nous avons construit une souris portant cette mutation et étudié l’activité du nœud sinoatrial (NSA, pacemaker principale) afin d’élucider les mécanismes.Nous avons trouvé que les cellules du NSA présentent une activité spontanée plus lente que les souris sauvages (WT). Dans la cellule in situ, on peut étudier l’activité des RyRs par l’analyse des « sparks » Ca2+, qui sont des évenements élémentaires produits par l’activation d’un cluster des RyRs. Nos analyses en microscopie confocale sur des NSA disséquées on montré que la fréquence des sparks Ca2+ était légèrement augmentée. Par contre, la longueur de ces sparks est fortement prolongée dans les cellules KI. Ceci produit une libération plus importante de Ca2+ pendant la diastole dans les cellules KI qui réduit l’automatisme, en réduisant la charge en Ca2+ du réticulum sarcoplasmique et en inactivant le courant calcique type L. Donc les thérapies en étude qi favoriseraient la stabilisation du RyR2 en état fermé pourraient ne pas Être efficaces, et il faudra plutôt essayer des thérapies qui faciliteraient la fermeture du canal, une fois il est ouvert. / The cardiac type-2 ryanodine receptor (RyR2) encodes a Ca2+ release channel on sarcoplasmic reticulum (SR) membrane in cardiomyocytes, including sinoatrial node (SAN) myocytes, and releases Ca2+ required for contraction and SAN spontaneous rhythm. Its genetic defects are related to catecholaminergic polymorphic ventricular tachycardia (CPVT), which is a lethal heritable disease characterized by exercise/stress-induced syncope and/or sudden cardiac death. Interestingly, CPVT patients frequently present SAN dysfunction as bradycardia at rest.In a previous study, a novel CPVT-related RyR2 mutation (RyR2R420Q) in a Spanish family, associated with SAN dysfunction was reported. R420 is located at the N-terminal portion of the channel and seems to be an important site for maintaining a stable A/B/C domain of N-terminus in RyR2. As N-terminal mutation resultant RyR2 behaviour and SAN function are never analyzed before, we created the KI mice model bearing mutation R420Q to understand the underlying mechanism.In this thesis, we found increased Ca2+ release during diastole, indicating a gain-of-function effect of RyR2 N-terminal mutation R420Q. Interestingly, this defect may not be only an enhanced activity, as the Ca2+ sparks frequency was only slightly increased in KI, but also the closing mechanism, producing longer Ca2+ sparks. That is, the number of Ca2+ sparks is increased by the RyR2R420Q mutation, and meanwhile the amount of Ca2+ released in each Ca2+ spark is also dramatically enhanced. This increased Ca2+ release retards SR Ca2+ replenishment, disrupting the Ca2+ clock and the coupled clock, resulting in the slower SAN function. Thus favouring RyR stabilization in the closing state might not be an adequate therapy but accelerating its closure.

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Catecholaminergic polymorphic ventricular tachycardia (CPVT) and pre-diabetic cardiomyopathy (pre-DC) are two cardiac diseases characterized by dysfunction in sarcoplasmic reticulum (SR) Ca2+ release channel RyR2. Despite similar defects in RyR2 leading to aberrant Ca2+ release (ACR), CPVT and pre-DC display divergent pathological phenotypes. Recent findings suggest SR-mitochondria interplay could contribute to pathological development; therefore, the role of SR-mitochondria interplay in shaping intracellular Ca2+ signaling was examined in CPVT and pre-DC by pharmacologically modulating mitochondria Ca2+ handling. Mitochondria can affect cytosolic/global Ca2+ dynamics through at least two mechanisms: buffering cytosolic Ca2+, or generating ROS to modulate RyR2 functionality via posttranslational modifications. Our data from cellular experiments suggests that CPVT mitochondria buffer Ca2+ to alleviate ACR, while pre-DC mitochondria cannot handle excess Ca2+, generating excess ROS to exacerbate ACR. These results were further consolidated by genetic models specifically targeting mitochondria Ca2+ handling proteins, which provided additional evidence SR-mitochondria crosstalk shapes cardiac pathological phenotypes.

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

La Tachycardie Ventriculaire Polymorphe Catécholaminergique (TVPC) est une pathologie rythmique héréditaire rare et sévère, responsable de mort subite chez le sujet jeune. Les mutations des gènes RYR2 et CASQ2 sont associées respectivement à une transmission autosomique dominante et récessive de la maladie. Le canal calcique RyR2 et la protéine chélatrice du calcium Casq2 sont situés dans le réticulum sarcoplasmique (RS) où ils participent au complexe de relâchement calcique (CRC), essentiel à l'homéostasie calcique cardiaque. L'analyse de RYR2 et CASQ2 chez 214 probands ayant présenté une TVPC nous a permis d'identifier respectivement des mutations chez 75 et 11 probands. Deux cas de mosaïques germinales et somatiques ont été identifiés dans le gène RYR2. Deux mutations d'épissage du gène CASQ2 ont été validées à l'aide de minigènes. Chez 97 patients négatifs pour RYR2 et CASQ2, nous avons décidé de rechercher des mutations de trois protéines du CRC (la triadine, la junctine et FKBP12.6) en séquençant les gènes correspondants. Nous n'avons retrouvé aucune mutation de la junctine, ni de FKBP12.6. En revanche, nous avons identifié trois mutations de la triadine: une micro-délétion et une mutation non-sens entraînant un codon stop prématuré, ainsi qu'une variation faux-sens, dont la caractérisation à l'aide de modèle animal et cellulaire a montré qu'elle entraînait une dégradation massive de la protéine. Les mutations du gène TRDN seraient associées à une absence de triadine entraînant une dysfonction du CRC, à l'origine des arythmies observées. En conclusion, nos résultats confirment que RYR2 est le gène majeur impliqué dans la TVPC, CASQ2 étant rarement impliqué; et nous rapportons, pour la première fois, des mutations du gène TRDN en pathologie humaine, associée à une forme autosomique rare de TVPC. / Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a rare and severe inherited arrhythmogenic disorder, responsible for sudden death in young patients. It is a genetically heterogenous pathology with an autosomal dominant form associated with mutations of the RYR2 gene, and a recessive form associated with mutations of the CASQ2 gene. The ryanodine receptor RyR2 is a Ca2+ channel, and the calsequestrin Casq2 is the major calcium storage protein, located in the sarcoplasmic reticulum of the cardiomyocytes. They belong to the calcium release complex (CRC) that plays a central role in excitation-contraction coupling. In this work, we report the identification of RYR2 and CASQ2 mutations in 75 and 11 CPVT probands, respectively. We identified two cases of germline and somatic mosaicism in RYR2. Two splicing mutations of CASQ2 have been validated using a splicing minigene assay. We searched for mutations among 97 CPVT probands, negative for RYR2 and CASQ2, in three candidate genes: TRDN, ASPH and FKBP1B, encoding three proteins of the CRC. We did not identify any mutation of ASPH and FKBP1B genes. However, we found three mutations in the TRDN gene, encoding the cardiac triadin: a microdeletion, a nonsense mutation, both leading to a premature stop codon, and a missense mutation. We demonstrated that the missense mutation induces a drastic reduction of the protein in cellular and animal models. All the three mutations would thus be associated with the absence of triadin, leading to dysfunction of the CRC, and arythmias. In conclusion, our results confirm that RYR2 is the major gene implicated in CPVT, and CASQ2 rarely implicated. Moreover, we report mutations of the TRDN gene for the first time in pathology, as a third gene associated with a rare autosomal recessive form of CPVT.

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Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a stress-induced arrhythmia, caused by genetic defects in sarcoplasmic reticulum (SR) Ca-release channel RyR2, or its accessory proteins. Our previous studies demonstrated that CPVT mitochondria can absorb RyR2-mediated aberrant Ca release (ACR) and behave as an efficient Ca buffer which is critical in mitigating harmful consequences of ACR. In this study, we test the hypothesis that modulating mitochondrial phosphate (Pi) transport or the tethering between SR-mitochondria, known as Mitochondria-associated-membrane (MAMs), impacts arrhythmogenesis in CPVT. We found that inhibiting mitochondrial Pi carrier (PiC) exacerbated cellular arrhythmias whereas overexpressing PiC in CPVT alleviated both cellular and in vivo arrhythmias. In parallel, disrupting MAMs exacerbated arrhythmogenesis in CPVT, but promoting MAMs by overexpressing mitofusin2 tethering protein reduced cellular arrhythmias. Our study provided both pharmacological and genetic evidence that directing more Ca to mitochondria by enhancing mitochondrial Pi transport or targeting MAMs could be promising therapeutic strategies to reduce CPVT arrhythmia.

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