CPVT-related arrhythmogenic mechanisms: Ca2+-induced transmural electrophysiological gradient alteration in a RyR2-V2475F rabbit model

Abstract

Introduction

Inherited stress-induced ventricular arrhythmia, such as catecholaminergic polymorphic ventricular tachycardia (CPVT), is typically linked to excessive diastolic Ca2+ release from the cardiac sarcoplasmic reticulum (SR) via the ryanodine receptor (RyR2), triggering Na+/Ca2+ exchanger activity and delayed afterdepolarizations. However, altered Ca2+ release may also influence sarcolemmal ionic conductance, shaping cardiac electrical properties.

Objective

We investigated the impact of the RyR2-V2475F, using a rabbit knock-in model to determine the underlying arrhythmogenic mechanisms of this mutation.

Method

Action Potentials (APs) and Ca2+ currents (ICaL) were recorded using patch-clamp on left ventricle cardiomyocytes. Ca2+ sparks, SR Ca2+ load and Ca2+ transients were analyzed using confocal microscopy. Ion channels expression was studied using Western-Blotting.

Results

Analysis of intracellular Ca2+ homeostasis revealed that Ca2+ transient amplitude was reduced, correlating with smaller SR Ca2+ load in isolated ventricular RyR2-V2475F± cardiomyocytes compared to WT. This could be dependent on the activity of RyR2, as we found that the SR Ca2+ leak was increased. Nevertheless, the leak was mostly undetectable, as Ca2+ sparks frequency was depressed. These alterations were associated with a reduced window L-type Ca2+ current contributing to SR Ca2+ unloading in RyR2-V2475F± cardiomyocytes. Additionally, we noted a shortening of action potential duration of endocardial cardiomyocytes from RyR2-V2475F± rabbits. The resulting inversion of the transmural repolarization gradient was abolished in the presence of BAPTA, suggestive of a Ca2 + -dependent effect, and could represent a vulnerable substrate that favors the appearance of arrhythmias. Indeed, RyR2-V2475F± rabbits developed a CPVT-like phenotype under pharmacological stress challenge. Interestingly, this phenotype was more pronounced in males than in females, consistent with a lesser impact on APs and ICaL of V2475F± in female animals.

Conclusion

Using a rabbit RyR2-V2475F model, we uncovered a potential new mechanism for CPVT-related arrhythmias, defined by a differential impact on AP duration between endocardium and epicardium, leading to a loss of an important protection mechanism against arrhythmias.