Calmodulin Potentiates RyR2 Block and Ca Wave Suppression by Flecainide

Abstract

Flecainide, a dual sodium channel and RyR2 Ca release channel blocker classified as a class Ic antiarrhythmic agent, has been used to suppress ventricular arrhythmias caused by spontaneous Ca release from the sarcoplasmic reticulum in animal models and humans. Since flecainide exhibits much lower antiarrhythmic potency in isolated cells and single RyR2 in vitro than in vivo, whether RyR2 block contributes to its antiarrhythmic mechanism of action has remained controversial. Here we test the hypothesis that loss of Calmodulin (CaM), a physiological binding partner of RyR2 Ca release channels, is responsible for the discrepancy of in vivo and in vitro results.Using single channels isolated from sheep hearts and incorporated into lipid bilayers, we constructed concentration-response curves of flecainide in the presence and absence of physiological CaM concentrations (100nM). Presence of CaM potentiated the action of flecainide on single RyR2 channels, with 3-fold increase in potency (IC50 flecainide= 8.6 µM vs. IC50 flecainide + CaM= 2.8 µM) and a modest increase in Emax. We next tested flecainide in permeabilized ventricular myocytes isolated from calsequestrin knockout mice, an established genetic model of Ca-release triggered ventricular arrhythmia, and from C57Bl6 wild-type mice. Addition of 100 nM CaM doubled both the potency (from 3 µM to 1.4 µM) and efficacy (from 40% to 80% inhibition) of Ca wave suppression by flecainide in calsequestrin KO cells. Moreover, in wild type cells, flecainide had no effect in absence of CaM, whereas in the presence of CaM, flecainide exhibited a modest effect in waves suppression with 25% inhibition of Ca waves and IC50 = 1 µM. Thus, the presence of physiological concentrations of Calmodulin is critical for flecainide's block of RyR2 channels and therefore its antiarrhythmic effect.