Functional Effects of the RyR2R420Q Catecholaminergic Ventricular Polymorphic Tachycardia in Mouse Cardiomyocytes

Abstract

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a genetic disease characterized by stress-induced syncope and/or sudden death. CPVT1 mutations are clustered in three regions: the N-terminal, the central, and the C-terminal domains of the cardiac Ca2+ release channel (type-2 ryanodine receptor, RyR2). We have created a knock-in (KI) mouse model bearing the RyR2R420Q mutation, which is located in the N-terminal region, found in three families diagnosed with CPVT. Electrocardiographic measurements of wake animals showed bidirectional ventricular tachycardia after epinephrine/caffeine injection in all the KI (heterozygous for RyR2R420Q) but in none of the WT (wild type) animals, validating the model. Consistent with the in-vivo phenotype, during action potential (AP) measurement by patch-clamp in the presence of 100 nM isoproterenol all KI myocytes displayed delayed afterdepolarizations (DADs) and triggered activity (TA), while the incidence of pro-arrhythmic events was significantly lower in WT myocytes. AP characteristics and potassium currents showed only minor differences between KI and WT. We measured ICa, [Ca2+]i transients and intra-SR [Ca2+] by patch-clamp and confocal microscopy. ICa density was similar between WT and KI cardiomyocytes, but the associated [Ca2+]i transient amplitude was unchanged or enhanced depending on the protocol, as well as its response to beta-adrenergic stimulation. Intact cardiac myocytes showed more Ca2+ sparks occurrence with a lowered SR Ca2+ threshold to initiate Ca2+ waves. Electron microscopy analyses indicate that Ca2+ release sites (or estimates indicate that couplons) in KI cardiomyocytes were fewer in number, significantly smaller and, hence, may contain a significantly lower number of RyR2. This morphological changes in couplons could participate to the differences in Ca2+ sparks characteristics recorded in KI cells compared to WT. Taken together, we have found a unique cellular alteration in this N terminal RyR2 mutation at structural and functional levels.