Impaired Binding to Junctophilin-2 and Nanostructural Alteration in CPVT Mutation

Abstract

RATIONALE: Catecholaminergic polymorphic ventricular tachycardia is a rare disease, manifested by syncope or sudden death in children or young adults under stress conditions. Mutations in the Ca2+ release channel/type 2 ryanodine receptor (RyR2) gene account for about 60% of the identified mutations. Recently, we found and described a mutation in RyR2 N-terminal domain, RyR2R420Q. OBJECTIVE: To determine the arrhythmogenic mechanisms of this mutation. METHODS AND RESULTS: Ventricular tachycardias under stress conditions were observed in both patients with catecholaminergic polymorphic ventricular tachycardia and knock-in mice. During action potential recording (by patch-clamp in knock-in mouse cardiomyocytes and by microelectrodes in mutant human induced pluripotent stem cell-derived cardiomyocytes), we observed an increased occurrence of delayed afterdepolarizations under isoproterenol stimulation, associated with increased Ca2+ waves during confocal Ca2+ recording in both mouse and human RyR2R420Q cardiomyocytes. In addition, Ca2+-induced Ca2+-release, as well as a rough indicator of fractional Ca2+ release, were higher and Ca2+ sparks longer in the RyR2R420Q-expressing cells. At the ultrastructural nanodomain level, we observed smaller RyR2 clusters and widened junctional sarcoplasmic reticulum measured by gated stimulated emission depletion super-resolution and electronic microscopy, respectively. The increase in junctional sarcoplasmic reticulum width might be due to the impairment of RyR2R420Q binding to junctophilin-2, as there were less junctophilin-2 coimmunoprecipitated with RyR2R420Q. At the single current level, the RyR2R420Q channel dwells longer in the open state at low intracellular Ca2+ ([Ca2+]i), but there is predominance of a subconductance state. The latter might be correlated with an enhanced interaction between the N terminus and the core solenoid, a RyR2 interdomain association that has not been previously implicated in the pathogenesis of arrhythmias and sudden cardiac death. CONCLUSIONS: The RyR2R420Q catecholaminergic polymorphic ventricular tachycardia mutation modifies the interdomain interaction of the channel and weakens its association with junctophillin-2. These defects may underlie both nanoscale disarrangement of the dyad and channel dysfunction. GRAPHIC ABSTRACT: An online graphic abstract is available for this article.