CaMKII Phosphorylation of RyR2 is Essential for Arrhythmia in CPVT

Background: In structurally normal hearts, gain 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. At the cellular level, adrenergic stimulation activates both protein kinase A (PKA) and calcium/calmodulin-dependent protein kinase II (CaMKII) which phosphorylate selective sites on the RyR2 channel to enhance Ca2+ release. The relative contribution of PKA versus CaMKII phosphorylation of RyR2 to CPVT pathogenesis remains incompletely understood. Methods: Using multiplex genomic engineering, we ablated the CaMKII phosphorylation site on RyR2 by mutating serine 2814 to alanine (S2814A) in conjunction with a novel CPVT GOF variant (R4650I). We systematically interrogated the consequences of CaMKII phosphorylation site ablation on the same or opposite allele with the CPVT variant at multiple phenotypic levels. Results: We found that the novel GOF variant Ryr2-R4650I confers a risk of adrenergically inducible ventricular arrhythmia including bi-directional ventricular tachycardia (BiVT). Through genome-editing, inclusion of the CaMKII phosphorylation Ryr2-S2814A mutation on the same allele as the GOF variant completely abrogates inducible arrhythmia. In contrast, with the Ryr2-S2814A variant on the opposite allele, there is no difference in the frequency, type, or length of inducible ventricular tachycardia as compared to animals heterozygous for the Ryr2-R4650I variant alone. Furthermore, allelic concordance of Ryr2-R4650I and Ryr2-S2814A does not inhibit adrenergically-induced enhancement of RyR2-dependent functions including heart rate response, augmentation of cellular Ca2+ flux or intracellular Ca2+ release. Conclusions: Our data strongly supports the hypothesis that CaMKII phosphorylation of RyR2 at S2814 is necessary and sufficient to unmask the arrhythmogenic phenotype in CPVT. Furthermore, CaMKII phosphorylation is an intra-molecular event with implications for therapeutic interventions.