La contraction des muscles stries est sous la dependance du Complexe de Relâchement du Calcium (CRC). Ce complexe proteique est constitue principalement de deux canaux calciques, le recepteur des dihydropyridines, un canal sensible au voltage localise dans la membrane des tubules-T et le recepteur de la ryanodine (RyR) situe dans la membrane du RS. Le CRC comprend egalement de nombreuses proteines regulatrices comme la triadine, la calsequestrine, la junctine et FKBP. Des mutations dans les genes codant les proteines du CRC conduisent a des pathologies rares et souvent severes. Cette these porte sur l'etude des mecanismes physiopathologiques induits par quelques unes de ces mutations pour decrypter les mecanismes pathologiques mis en œuvre mais egalement pour comprendre le fonctionnement global du CRC dans les muscles squelettique et cardiaque. La premiere partie de cette etude concerne RYR1, le gene codant l'isoforme squelettique du RyR qui est une cible importante de mutations chez des patients atteints de myopathies congenitales a cores. L'effet fonctionnel de ces mutations, reparties sur toute la sequence de RYR1, est peu connu. Ces mutations pourraient modifier la fonction canal de RyR1 mais egalement son adressage a la triade ou sa regulation par d'autres proteines du CRC. Parmi ces hypotheses, la modification de la localisation de RyR1 et sa regulation par une proteine regulatrice (la caveoline-3) ont ete revelees par l'etude de deux mutations de RyR1. La deuxieme partie de cette etude concerne la tachycardie ventriculaire polymorphe catecholaminergique (TVPC), une pathologie liee a des defauts du CRC cardiaque, pour laquelle des recherches de mutations sont effectuees sur l'isoforme cardiaque du RyR, RYR2, puis dans les autres proteines du complexe. Nous avons identifie au laboratoire les premieres mutations dans le gene de la triadine chez un de ces patients. L'impact d'une de ces mutations sur le fonctionnement du complexe a ete etudie et nous avons pu caracteriser le mecanisme physiopathologique mis en œuvre et conduisant a la TVPC chez ces patients.
Atrial fibrillation is the most common clinical arrhythmia and may be due in part to metabolic stress. Atrial specific deletion of the master metabolic sensor, AMP-activated protein kinase (AMPK), induces atrial remodeling culminating in atrial fibrillation in mice, implicating AMPK signaling in the maintenance of atrial electrical and structural homeostasis. However, atrial substrate preference for mitochondrial oxidation and the role of AMPK in regulating atrial metabolism are unknown. Here, using LC-MS/MS methodology combined with infusions of [
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Background SERCA 2a gene transfer ( GT ) improves mechano-electrical function in animal models of nonischemic heart failure Whether SERCA 2a GT reverses pre-established remodeling at an advanced stage of ischemic heart failure is unclear. We sought to uncover the electrophysiological effects of adeno-associated virus serotype 1. SERCA 2a GT following myocardial infarction ( MI ). Methods and Results Pigs developed mechanical dysfunction 1 month after anterior MI , at which point they received intracoronary adeno-associated virus serotype 1. SERCA 2a ( MI + SERCA 2a) or saline ( MI ) and were maintained for 2 months. Age-matched naive pigs served as controls (Control). In vivo ECG -and-hemodynamic properties were assessed before and after dobutamine stress. The electrophysiological substrate was measured using optical action potential ( AP ) mapping in controls, MI , and MI + SERCA 2a preparations. In vivo ECG measurements revealed comparable QT durations between groups. In contrast, prolonged QRS duration and increased frequency of R' waves were present in MI but not MI + SERCA 2a pigs relative to controls. SERCA 2a GT reduced in in vivo arrhythmias in response to dobutamine. Ex vivo preparations from MI but not MI + SERCA 2a or control pigs were prone to pacing-induced ventricular tachycardia and fibrillation. Underlying these arrhythmias was pronounced conduction velocity slowing in MI versus MI + SERCA 2a at elevated rates leading to ventricular tachycardia and fibrillation. Reduced susceptibility to ventricular tachycardia and fibrillation in MI + SERCA 2a pigs was not related to hemodynamic function, contractile reserve, fibrosis, or the expression of Cx43 and Nav1.5. Rather, SERCA 2a GT decreased phosphoactive CAMKII -delta levels by >50%, leading to improved excitability at fast rates. Conclusions SERCA 2a GT increases conduction velocity reserve, likely by preventing CAMKII overactivation. Our findings suggest a primary effect of SERCA 2a GT on myocardial excitability, independent of altered mechanical function.
Pharmacotherapy for atrial fibrillation (AF) is confounded by ventricular proarrhythmia. Vernakalant (VER) belongs to a class of atrial selective channel blockers developed in attempts to convert AF safely. Yet, robust analysis of the effects of VER in human based tissues has not been performed. We hypothesized that engineered heart tissues (EHT) composed of atrial (A) or ventricular (V) human iPSCs would provide a robust platform for quantitative analysis of chamber specific electrophysiological (EP) effects of VER. Methods: iPSCs were differentiated into cardiomyocytes using biphasic Wnt signaling, insulin, and lactate purification. Atrial-like myocytes were enriched by adding 0.75 μM retinoic acid on days 4-6 to promote atrial gene expression and specification. Myocytes were seeded onto cryosectioned, laser cut and decellularized myocardial tissues and cultured under isometric conditions. EHTs were stained with high dose, water soluble di-2-ANEPEQ to enhance signal-to-noise while avoiding toxicity. Results: Staining with 25 μM di-2-ANEPEQ provided high fidelity optical AP signals that allowed comprehensive characterization of the EP substrate. A-EHT exhibited significantly shorter AP durations than V-EHT reflecting greater repolarizing drive (APD80, 180 ms vs 367 ms; p < 0.0001, n = 4-5). Consistent with its role as an atrial-selective blocker, VER increased APD80 only in A-EHTs (180 ms vs 258 ms, p = 0.0027, n = 5). This beneficial property for combatting AF was countered by marked slowing of A-EHT conduction and AP upstroke velocity (20.6 cm/s vs 11.7 cm/s, p = 0.027, n = 5). Conclusions: Optical mapping of chamber specific EHTs confirms atrial specific nature of VER as an atrial repolarization prolonging agent but raises unexpected concerns regarding potential proarrhythmic toxicity related to atrial conduction slowing.
Paradigm by which CDC-derived exosomes may harness the advantages and avoid the disadvantages of standard gene and cell therapies to suppress post-MI arrhythmias.
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