In Vitro Characterization of a Novel N-Terminal CPVT RyR Mutation

Abstract

Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) is an arrhythmogenic disease characterized by stress-triggered syncope and sudden death. Most of the CPVT mutations are concentrated in RyR “hotspots”: the C terminus, the central and the N terminal domains. Although the phenotypic manifestations are common, it is likely that the intrinsic mechanism of RyR dysfunction varies with the location of the mutation within the RyR. Previously, we characterized RyR dysfunction in a CPVT mutation located at the C terminal domain, identifying an increased cytosolic Ca2+ sensitivity as the causative of a gain-of-funcion defect. Recently, we have identified a mutation in a family with CPVT syndrome, which is located in the N terminal portion: RyR2R420Q. To analyze the function of the mutated RyR channel, we have created an expression plasmids of RyR2R420Q and RyR2WT. Heterologous expression in HEK cells showed decreased caffeine sensitivity, suggesting loss-of-function defect. In order to be able to further characterize this mutation in a cardiac context, we expressed both RyR2R420Q and RyR2WT in cultured neonatal rat cardiac myocytes and analyzed the amplitude and frequency of spontaneous [Ca2+]i transients and Ca2+ sparks by confocal microscopy. We found that the spontaneous [Ca2+]i transients were more frequent but of lower amplitude in RyR2R420Q expressing cells. Further experiments are being conducted to fully understand the underlying mechanism.