A Novel Ryanodine Receptor Mutation Linked to Sudden Death Increases Sensitivity to Cytosolic Calcium

Abstract

Rationale Mutations in the cardiac type 2 ryanodine receptor (RyR2) have been linked to catecholaminergic polymorphic ventricular tachycardia (CPVT). CPVT-associated RyR2 mutations cause fatal ventricular arrhythmias in young individuals during beta-adrenergic stimulation. Objective This study sought to determine the effects of a novel identified RyR2-G230C mutation and whether this mutation and RyR2-P2328S alter the sensitivity of the channel to luminal calcium (Ca2+). Methods Functional characterizations of recombinant human RyR2-G230C channels were performed under conditions mimicking stress. Human RyR2 mutant channels were generated by site-directed mutagenesis and heterologously expressed in HEK293 cells together with calstabin2 (FKBP12.6). RyR2 channels were measured in order to examine the regulation of the channels by cytosolic vs. luminal SR Ca2+. Results A 50 year-old Caucasian male with repeated syncopal episodes after exercise had a cardiac arrest and harbored the mutation RyR2-G230C. PKA-phosphorylated RyR2-G230C channels exhibited a significantly higher open probability (Po) at diastolic Ca2+ concentrations, associated with a depletion of calstabin2. The luminal Ca2+ sensitivities of RyR2-G230C and RyR2-P2328S channels were WT-like. Conclusions The RyR2-G230C mutant exhibits similar biophysical defects compared to previously characterized CPVT mutations: decreased binding of the stabilizing subunit calstabin2 and a leftward shift in the Ca2+-dependence for activation under conditions that simulate exercise, consistent with a “leaky” channel. Both RyR2-G230C and RyR2-P2328S channels exhibit normal luminal Ca2+ activation. Thus, diastolic SR Ca2+ leak caused by reduced calstabin2 binding and a leftward shift in the Ca2+-dependence for activation by diastolic levels of cytosolic Ca2+ is a common mechanism underlying CPVT.

Non-ventricular, Clinical, and Functional Features of the RyR2R420Q Mutation Causing Catecholaminergic Polymorphic Ventricular Tachycardia

“…33,34 So far, most CPVT mutations in which functional studies have been performed behave as gain-of-function. 35,38 Increased Ca 2+ sensitivity (luminal or cytosolic), 15,24,38,39 an altered interdomain interaction between the N-terminal and central domains, 40,41 and diminished FKBP12.6 binding 38,39 account for the most accepted mechanisms. Very recently, a disrupting effect of the RyR2 R420Q has been observed in the crystal structure of the N-terminal region, since it ablates chloride binding, disturbing its domain folding 42,43 even though, in linear terms, the 420 residue is very far from the proposed molecular region involved in Ca 2+ -dependent activation (residues 4485-4494).…”

Utility of the Exercise Electrocardiogram Testing in Sudden Cardiac Death Risk Stratification

“…[13][14][15][16][17] Exercise ECG testing is often the most useful procedure to establish the diagnosis of CPVT. In fact, exercise ECG testing unmasks the arrhythmia susceptibility of patients with CPVT with a progressive worsening of supraventricular and VAs that parallel the increase in workload during exercise ECG testing.…”

Circulation Research Thematic Synopsis

“…A Novel Ryanodine Receptor Mutation Linked to Sudden Death Increases Sensitivity to Cytosolic Calcium; Meli et al 46 What Is Known?…”