The CPVT-Associated RyR2 Mutation G230C reduces the Threshold for Store Overload-Induced Ca Release (SOICR)

Abstract

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a life-threatening arrhythmia. Many congenital mutations in both the cardiac ryanodine receptor (RyR2) and calsequestrin (CASQ2) are known to be responsible for this disorder. It is now well established that CPVT is caused by delayed afterdepolarizations (DADs)-induced triggered activities, and that DADs are caused by spontaneous sarcoplasmic reticulum (SR) Ca release during Ca overload, a process also known as store overload induced Ca release (SOICR). A large body of evidence indicates that CPVT-causing RyR2 and CASQ2 mutations enhance the propensity for SOICR and DADs by increasing the response of RyR2 to SR luminal Ca. Recently, Marks and colleagues reported that a CPVT RyR2 mutation G230C increases the cytosolic Ca sensitivity (only after PKA phosphorylation) of single RyR2 channels in lipid bilayers, but has no effect on the luminal Ca sensitivity of the channel. These observations have led to the conclusion that SOICR is not involved in the disease mechanism of the RyR2-G230C mutation. However, the cellular impact of this mutation on SOICR has yet to be determined. To this end, we generated stable, inducible HEK293 cell lines expressing RyR2-WT and the RyR2-G230C mutant. We induced SOICR in these cells by elevating extracellular Ca, and found that the RyR2-G230C mutation markedly enhances the propensity for SOICR. Further, we employed single cell luminal Ca imaging to monitor the luminal Ca dynamics in RyR2-WT- and G230C-expressing cells during store Ca overload. We found that the G230C mutation significantly reduces the luminal Ca level at which spontaneous Ca release occurs (i.e. the SOICR threshold). Therefore, these results and those of previous studies demonstrate that reduced SOICR threshold is a common defect of CPVT-associated RyR2 mutations.