6533b838fe1ef96bd12a3d1d

RESEARCH PRODUCT

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

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Rationale: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a rare disease, manifested by syncope or sudden death in children or young adults under stress conditions. Mutations in the Ca 2+ release channel/RyR2 (type 2 ryanodine receptor) gene account for about 60% of the identified mutations. Recently, we found and described a mutation in RyR2 N-terminal domain, RyR2 R420Q . 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 Ca 2+ waves during confocal Ca 2+ recording in both mouse and human RyR2 R420Q cardiomyocytes. In addition, Ca 2+ -induced Ca 2+ -release, as well as a rough indicator of fractional Ca 2+ release, were higher and Ca 2+ sparks longer in the RyR2 R420Q -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 electron microscopy, respectively. The increase in junctional sarcoplasmic reticulum width might be due to the impairment of RyR2 R420Q binding to JPH2 (junctophilin-2), as there were less junctophilin-2 coimmunoprecipitated with RyR2 R420Q . At the single current level, the RyR2 R420Q channel dwells longer in the open state at low intracellular Ca 2+ ([Ca 2+ ] 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 RyR2 R420Q catecholaminergic polymorphic ventricular tachycardia mutation modifies the interdomain interaction of the channel and weakens its association with JPH2. These defects may underlie both nanoscale disarrangement of the dyad and channel dysfunction.