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.

Background: Ventricular fibrillation (VF) is associated with dysfunctional cardiac calcium cycling and poor survival. We hypothesized dantrolene improves survival following VF by stabilizing calcium dysregulation. Methods: VF was induced in 26 healthy Yorkshire pigs and left untreated for 4 min followed by 3 min of CPR and defibrillation. Dantrolene was infused during CPR. Rabbit hearts (n=14) were studied to evaluate the effect of dantrolene on VF-induced calcium cycling dysfunction. Results: Survival was higher in the dantrolene group. (85% vs. 39%, P=0.01) Dantrolene-treated pigs required significantly lower defibrillation energy level. (150J vs. 650J, P<0.05) Systolic pressure was significantly higher during the post-defibrillation period in the dantrolene group. (P=0.001) In rabbit hearts, dantrolene significantly mitigated the amplitude of VF-induced diastolic calcium elevations and increased the calcium alternans threshold. (P<0.05) Conclusion: Our findings suggest dantrolene facilitates successful defibrillation, prevents myocardial stunning and improves survival following VF. The effects are mediated through normalizing the VF-induced dysfunctional calcium cycling.

Background: Ventricular fibrillation (VF) is associated with dysfunctional cardiac calcium cycling and poor survival. We hypothesized dantrolene improves survival following VF by stabilizing calcium dysregulation. Methods: VF was induced in 26 healthy Yorkshire pigs and left untreated for 4 min followed by 3 min of CPR and defibrillation. Dantrolene was infused during CPR. Rabbit hearts (n=14) were studied to evaluate the effect of dantrolene on VF-induced calcium cycling dysfunction. Results: Survival was higher in the dantrolene group. (85% vs. 39%, P=0.01) Dantrolene-treated pigs required significantly lower defibrillation energy level. (150J vs. 650J, P<0.05) Systolic pressure was significantly higher during the post-defibrillation period in the dantrolene group. (P=0.001) In rabbit hearts, dantrolene significantly mitigated the amplitude of VF-induced diastolic calcium elevations and increased the calcium alternans threshold. (P<0.05) Conclusion: Our findings suggest dantrolene facilitates successful defibrillation, prevents myocardial stunning and improves survival following VF. The effects are mediated through normalizing the VF-induced dysfunctional calcium cycling.

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Trichloroethylene (TCE) is among the most common water contaminant in the United States and around the world. It is estimated that between 9% and 34% of all drinking water sources contain some TCE. The EPA set a drinking water standard for TCE at 5 parts per billion (ppb) in 1989, however since this date, many studies have shown TCE is dangerous to the health of adults and unborn children, even at low-level exposures. These studies reveal exposure to TCE can cause multi-organ damage, especially for the kidney, liver, reproductive and development systems. We investigated how TCE can effect embryonic heart development by identifing possible target mechanisms changing after exposure. Acute and chronic exposure to rat cardiomyocytes produced altered calcium flow and significant changes with TCE doses as low as 10ppb. Embryonic carcinoma cells, rat cardiomyocytes and fetal heart tissue all showed global changes in gene expression after low-dose TCE exposure, including critical ion channels that drive calcium flux. High levels of folic acid supplementation in combination with 10ppb TCE exposure in maternal diets caused significant genetic modifications in mRNA expression levels of Day 10 embryonic mouse cardiac tissue. We also found both high and low folate maternal diets leads to similar phenotypic outcomes in embryo development.

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Heart failure resulting from different forms of cardiomyopathy is defined as the inability of the heart to pump sufficient blood to meet the body's metabolic demands. It is a major disease burden worldwide and the statistics show that 50% of the people who have the heart failure will eventually die from sudden cardiac death (SCD) associated with an arrhythmia. The central cause of disability and SCD is because of ventricular arrhythmias. Genetic mutations and acquired modifications to RyR2, the calcium release channel from sarcoplasmic reticulum, can increase the pathologic SR Ca2+ leak during diastole, which leads to defects in SR calcium handling and causes ventricular arrhythmias. The mechanism of RyR2 dysfunction includes abnormal phosphorylation, disrupted interaction with regulatory proteins and ions, or altered RyR2 domain interactions. Many pharmacological strategies have shown promising prospects to modulate the RyR2 as a therapy for treating cardiac arrhythmias. Here, we are trying to establish a novel approach to designing new drugs to treat heart failure and cardiac arrhythmias. Previously, we demonstrated that all pharmacological inhibitors of RyR channels are electron donors while all activators of RyR channels are electron acceptors. This was the first demonstration that an exchange of electrons was a common molecular mechanism involved in modifying the function of the RyR. Moreover, we found that there is a strong correlation between the strength of the electron donor/acceptor, and its potency as a channel inhibitor/activator, which could serve as a basis and direction for developing new drugs targeting the RyR. In this study, two new potent RyR inhibitors, 4-methoxy-3-methyl phenol (4-MmC) and the 1,3 dioxole derivative of K201, were synthesized which are derivatives of the known RyR modulators, 4-chloro-3-methyl phenol (4-CmC) and K201. The ability of K201, 1,3 dioxole derivative of K201 and 4-MmC to inhibit the cardiac calcium channel is examined and compared at the single channel level. All of these compounds inhibited the channel activity at low micromolar concentrations or sub-micromolar concentrations.

Les canalopathies cardiaques congénitales constituent la principale hypothèse diagnostique dans les cas de mort subite inexpliquée chez les sujets de moins de 35 ans. Notre travail a eu pour objectif demettre au point une stratégie de détection post-mortem des mutations sur les gènes connus pour être impliqués dans les canalopathies cardiaques, applicable en routine, à partir de la principale source d’ADN post-mortem disponible en France à savoir les prélèvements fixés au formol et inclus en paraffine (FFIP). A partir d’une cohorte de 12 cas, deux techniques de détection de variants génétiques ont été évaluées, une technique de criblage par l’analyse des courbes de fusion haute résolution et une technique de génotypage par spectrométrie de masse MALDI-TOF, respectivement sur le gène KCNQ1 et le gène RyR2. Quelle que soit la technique utilisée, il n’est pas possible de s’affranchir du séquençage de type Sanger afin d’explorer les séquences d’intérêts qui n’ont pu être optimisées avec l’une ou l’autre des méthodes à la fois sur les prélèvements congelés et FFIP. L’arrivée des séquenceurs de nouvelles générations ouvrent ainsi de nouvelles perspectives dans ce domaine. / The congenital cardiac channelopathies constitute the principal diagnostic hypothesis in autopsynegative sudden unexplained death concerning people younger than 35 years old. The present study aimed to develop a strategy of mutations detection on known genes implicated in the cardiac channelopathies. This strategy of mutations detection had to be applicable to routine and has been studied on formalin-fixed and paraffin-embedded (FFPE) tissues which are the principal DNA source available in France. On a cohort of 12 cases, two technique of sequence variants detection wereevaluated: the screening method of High Resolution Melt and the genotyping method based on a MALDI-TOF mass spectrometry, respectively on KCNQ1 and RyR2 genes. Whatever the technique, there is a necessity of resorting to the Sanger sequencing to explore the sequence of interest none optimized with one or the other technology both on FFEP and frozen tissues. That’s why the next generation sequencing method should open new perspectives in the post-mortem diagnostic of cardiac channelopathies.

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