CaMKII Phosphorylation of RyR2 is Essential for Arrhythmia in CPVT

Abstract

BackgroundGain of function variants (GOF) in the intracellular calcium (Ca2+) release channel RYR2 predominately underlie the inherited arrhythmogenic syndrome catecholaminergic polymorphic ventricular tachycardia (CPVT). Patients with CPVT are susceptible to life-threatening ventricular arrhythmias triggered by emotional or physical stress. Adrenergic stimulation activates calcium/calmodulin-dependent protein kinase II (CaMKII), which phosphorylates RYR2 on serine 2814 to enhance Ca2+ release. Here we assessed the in vivo requirement of this phosphorylation event to unmask the latent arrhythmic potential of CPVT-causing variants. MethodsUsing multiplex murine genomic engineering, we mutated the CaMKII phosphorylation site RYR2-S2814 site to alanine (S2814A) in conjunction with a novel CPVT GOF variant (RYR2-R4650I). We systematically interrogated the consequences of CaMKII phosphorylation site ablation on the same (cis) or opposite (trans) allele with the CPVT variant at multiple phenotypic levels. ResultsThe novel GOF variant Ryr2-R4650I conferred a risk of adrenergically inducible ventricular arrhythmia, including bi-directional ventricular tachycardia (BiVT). Ablation of the CaMKII RYR2-S2814 phosphorylation site on the same allele as the GOF variant completely abrogated inducible arrhythmia. In contrast, ablation of this site on the opposite allele did not significantly alter in frequency, type, or length of inducible ventricular tachycardia compared to animals heterozygous for the Ryr2-R4650I variant alone. Furthermore, allelic concordance of Ryr2-R4650I and Ryr2-S2814A did not inhibit adrenergically-induced enhancement of RYR2-dependent functions, including heart rate response, augmentation of cellular Ca2+ flux, or intracellular Ca2+ release. ConclusionsOur data strongly support the hypothesis that CaMKII phosphorylation of RYR2 at S2814 is necessary and sufficient to unmask the arrhythmogenic phenotype in CPVT. Furthermore, pro-arrhythmia caused by CaMKII-S2814 phosphorylation in CPVT is an intra-molecular event, with implications for therapeutic interventions.