Abstract

The cardiac Ca2+ release channel (RyR2) governs the release of Ca2+ from the sarcoplasmic reticulum, essential for cardiac muscle contraction. Naturally occurring mutations in RyR2 have been linked to several forms of cardiac arrhythmias (CPVT, ARVC) that require a vulnerable substrate characterized by structural or electrical abnormalities and an acute initiating event. Recently, a RyR2V2475F mutation has been identified post-mortem on a young boy whose death has been associated with arrhythmias, related to higher Ca2+ sensitivity of the mutated RyR2. Our study aims to determine the arrhythmogenic mechanisms of the RyR2V2475F mutation by analyzing its impact on the ventricular action potential duration and Ca2+ homeostasis in a Knock-In rabbit model. Action Potentials (APs) were recorded using patch-clamp. Ca2+ sparks, SR Ca2+ load and Ca2+ transients have been analyzed using confocal microscopy. Ion channels expression was evaluated using RT-qPCR. With EGTA in the internal solution, APs of endocardial cardiomyocytes were shortened in RyR2V2475F mutants, with reduced APD20 and APD50 values compared to WT animals. This causes an inversion of the transmural repolarization gradient. The decrease of AP duration in endocardial cardiomyocytes was abolished in presence of BAPTA (a stronger Ca2+ chelator) in the internal solution. RyR2V2475F mutation did not alter K+ channel transcript levels. Ca2+ handling analysis showed a reduced diastolic Ca2+ sparks frequency, SR Ca2+ load and Ca2+ transients amplitudes. These results suggest a role of altered Ca2+ handling in AP lengthening that could represent the vulnerable substrate that favors the apparition of arrhythmias.