Uptake-leak balance of SR Ca2+ determines arrhythmogenic potential of RyR2R420Q+/- cardiomyocytes

Abstract

Mutations of the RyR2 are channelopathies that can predispose to life threatening catecholaminergic polymorphic ventricular tachycardias (CPVTs) during exercise or stress. However, the cellular and molecular mechanisms that are causal for the arrhythmias downstream of the β-adrenergic receptor (β-AR) activation are not defined. They may be specific and different for each particular RyR2 mutation. Obvious possibilities are the phosphorylation of the mutated RyR2s or the stimulation of the SR Ca²⁺ pump (SERCA), which could increase SR Ca²⁺ loading. Potentially arrhythmogenic Ca²⁺ signals, such as Ca²⁺ waves, were recorded and analyzed from WT and RyR2^R420Q+/- mouse cardiomyocytes with confocal microscopy after field stimulation at 1 Hz. In RyR2^R420Q+/- cardiomyocytes we found a higher occurrence and frequency of Ca²⁺ waves, particularly upon β-AR stimulation with isoproterenol. This was accompanied by a shorter latency to the first spontaneous wave. Wave velocity from raw traces, as well as amplitude and decay time constant (τ) analyzed in de-skewed traces were comparable in both cell types. To obtain further insight into the role of the SERCA we selectively stimulated SERCA in permeabilized myocytes using Fab fragments of a PLB antibody (2D12). Surprisingly, SERCA stimulation alone resulted in considerably higher wave frequencies than when mimicking β-AR stimulation with cAMP, particularly in RyR2^R420Q+/- cardiomyocytes. This may be a consequence of some protective SR Ca²⁺ unloading resulting from the SR Ca²⁺ leak via phosphorylated RyR2s in cAMP. Spark-to-spark recovery analysis suggested a remarkably higher Ca²⁺ release sensitivity in RyR2^R420Q+/- cells, both in control and upon β-AR stimulation. Together these findings suggest that the fine balance between SR Ca²⁺ loading via SERCA and the Ca²⁺ leak via mutated and phosphorylated RyR2s is an important determinant for the overall cellular arrhythmogenicity prevailing in the RyR2^R420Q+/- myocytes.

Keywords: Arrhythmia; Calcium signaling; Catecholaminergic polymorphic ventricular tachycardia; Channelopathy; Ryanodine receptor; Sarcoplasmic reticulum calcium pump.