Inhibition of RYR2 Activity by Intracellular Flecainide Effectively Suppresses Arrhythmogenic Ca Waves in Intact Ventricular Myocytes from Casq2 -/- Mice

Abstract

We reported previously that flecainide suppresses Ca waves in MEMBRANE-PERMEABILIZED ventricular myocytes from Casq2-/- mice and that flecainide is more potent (i.e., lower IC50) in Casq2-/-compared to WT myocytes. Other groups reported that in INTACT myocytes flecainide acts only by blocking Na+ channels and has no effect on RyR2. Here we test the effect of flecainide on INTACT ventricular myocytes from Casq2-/- and WT littermates using voltage-clamp. To eliminate any contribution of Na channels, all solutions contained 30 µM TTX. Complete block INa was confirmed for each cell tested. Spontaneous Ca waves were elicited by a pacing train followed by a holding potential of −70mV for 45 s. 6 µM flecainide or vehicle (H2O) was added to both external and pipette solutions. Under such conditions, flecainide had no significant effect on the average number of spontaneous Ca waves in WT myocytes (Vehicle: 3.1±0.6, n=24, Flecainide: 3.0±0.8, n=20, p=NS). In Casq2-/- myocytes, however, flecainide significantly reduced Ca waves (Vehicle: 6.6±1.2, n=23; Flecainide: 2.1±0.4, n=19, p 0.5 vs. Casq2-/- vehicle), suggesting that intracellular dialysis by the patch pipette removed flecainide from the cytosol. We conclude that intracellular concentrations of 6 µM flecainide effectively inhibit arrhythmogenic Ca waves in Casq2-/- but not in WT myocytes, indicating that flecainide action is dependent on the state of RyR2. Our results further demonstrate that flecainide suppression of Ca waves is independent from its Na channel block.