Novel Mechanisms of CPVT1 in a RyR2-V2475F Knock-In Mouse Model

Abstract

Mutations in the cardiac ryanodine receptor (RyR2) have been implicated in Catecholaminergic Polymorphic Ventricular Tachicardia 1 (CPVT1), an arrhythmogenic syndrome triggered by emotional or physical stress in otherwise healthy individuals. At the cellular level, the adrenergic surge during stress presumably induces diastolic SR Ca2+ release, generating a favorable substrate for arrhythmias by the mechanism of delayed afterdepolarization (DAD). Increased sensitivity to luminal [Ca2+], unzipping of domain-domain interactions, and de-regulation by accessory proteins have been reported to contribute to RyR2 dysfunction in CPVT1. We generated a knock-in mouse model of CPVT1 (RyR2-V2475F) and characterized its mechanisms of arrhythmia. Expression of RyR2-V2475F in HEK293 cells induces a unique phenotype, namely, increased activity at diastolic [Ca2+] (pCa 7), which is not seen in other CPVT1 mutations. Mice homozygous for the V2475F mutation have not been detected in >500 mice, suggesting that the mutation causes catastrophic RyR2 dysfunction and embryonic lethality. Heterozygous mice, on the other hand, display increased propensity for premature ventricular complexes, bidirectional ventricular tachycardia and other forms of tachyarrythmias when injected a cocktail of norepinephrine and caffeine that is mostly innocuous in wild-type mice. Heterozygous Langendorff-perfused hearts subjected to adrenergic stimulation and tachypacing are also arrhythmogenic, indicating that the arrhythmias arise from mechanisms intrinsic to the heart and not necessarily from systemic factors. Despite this severe phenotype, ventricular cardiomyocytes isolated from RyR2-V2475F mice display only mild differences in the amplitude and kinetics of intracellular Ca2+ transients when compared to wild-type myocytes, regardless of stimulation frequency and β-adrenergic stimulation. Thus, cellular Ca2+ mishandling in ventricular myocytes of the RyR2-V2475F mice, although significant, does not appear sufficient to support the severe phenotype observed in whole hearts and intact mice. We are therefore investigating other mechanisms that may explain the arrhythmogenicity of heterozygous mice.