Heterogeneity of Ryanodine Receptor Dysfunction in a Mouse Model of Catecholaminergic Polymorphic Ventricular Tachycardia

Abstract

Rationale Most cardiac ryanodine receptor (RyR2) mutations associated with Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) are postulated to cause one distinctive form of Ca2+ release dysfunction. Considering the spread distribution of CPVT mutations, we hypothesized that dysfunctional heterogeneity was also feasible. Objective To determine the molecular and cellular mechanism(s) by which a novel RyR2-V2475F mutation associated with CPVT in humans triggers Ca2+-dependent arrhythmias in whole hearts and intact mice. Methods and Results Recombinant channels harboring CPVT-linked RyR2 mutations were functionally characterized using [3H]ryanodine binding and single channel recordings. Homologous recombination was used to generate a knock-in mouse bearing the RyR2-V2475F mutation. Ventricular myocytes from mice heterozygous for the mutation (RyR2-V2475F+/−) and their wild-type (WT) littermates were Ca2+-imaged by confocal microscopy under conditions that mimic stress. The propensity of WT and RyR2-V2475F+/− mice to develop arrhythmias was tested at the whole heart level and in intact animals. Recombinant RyR2-V2475F channels displayed a) increased cytosolic Ca2+ activation, b) abnormal PKA phosphorylation, and c) increased activation by luminal Ca2+. The RyR2-V2475F mutation appears embryonic lethal in homozygous mice, but heterozygous mice have no alterations at baseline. Spontaneous Ca2+ release (SCR) events were more frequent and had shorter latency in isoproterenol-stimulated cardiomyocytes from RyR2-V2475F+/− hearts, but their threshold was unchanged with respect to WT. Adrenergically-triggered tachyarrhythmias were more frequent in RyR2-V2475F+/− mice. Conclusions The mutation RyR2-V2475F is phenotypically strong among other CPVT mutations and produces heterogeneous mechanisms of RyR2 dysfunction. In living mice, this mutation appears too severe to be harbored in all RyR2 channels, but remains undetected under basal conditions if expressed at relatively low levels. β-adrenergic stimulation breaks the delicate Ca2+ equilibrium of RyR2-V2475F+/− hearts and triggers life-threatening arrhythmias.

Clinical and molecular characterization of a cardiac ryanodine receptor founder mutation causing catecholaminergic polymorphic ventricular tachycardia

“…Loaiza et al 26 showed that the SOICR activity of RyR2 was not modified by the V2475F RyR2 mutation; however, beta-adrenergic stimulated ventricular myocytes from V2475F +/-mice showed an increased propensity for spontaneous Ca 2+ release events, which is very reminiscent of our observations. In the present study, in vitro experiments indicate that the mutated site, S357, itself is unlikely to be a target for PKA phosphorylation.…”

Gene Transfer of Engineered Calmodulin Alleviates Ventricular Arrhythmias in a Calsequestrin‐Associated Mouse Model of Catecholaminergic Polymorphic Ventricular Tachycardia

“…Similar results were obtained with the CPVT CaM N54I (Figure S1). Together with previous results on CASQ2 and RyR2‐mediated CPVT, these data suggests that shortened RyR2 refractoriness may be a common mechanism that promotes arrhythmias caused by both CASQ2 and CaM mutants 7, 8…”

“…Such Ca dependent binding would be expected to inhibit RyR2 specifically after Ca release lasting late into the diastolic period. Consistent with this notion, mutations in CaM have recently been linked to CPVT, a syndrome associated with a shortened RyR2 refractory period 7, 8, 21. We reasoned that if indeed certain CPVT‐causing CaM mutations act by shortening RyR2 refractoriness, then CaM variants with slowed Ca dissociation could be engineered to prolong RyR2 refractoriness in cardiac disease settings when refractoriness is pathologically shortened.…”

“…Interestingly, previous work from our and other laboratories have demonstrated that impairment of the ability of RyR2 to become refractory, ie, appropriately close after each systolic Ca release event, appears to be a common mechanism for various forms of genetic and acquired arrhythmias, including CPVT and post‐infarction ventricular fibrillation (VF) 7, 8, 9. Specifically, shortened RyR2 refractoriness results in aberrant diastolic SR Ca release that synchronizes across the myocardium to induce triggered activity, thus, precipitating malignant arrhythmias.…”

Arrhythmogenesis in a catecholaminergic polymorphic ventricular tachycardia mutation that depresses ryanodine receptor function

“…Hence, hyperactive RyR2 channels eager to release Ca 2+ on their own appear as essential components of this arrhythmogenic scheme. In fact, most RyR2-linked CPVT mutations characterized to date produce hyperactive RyR2 channels (9)(10)(11)(12). This scheme therefore appears inadequate to explain lethal arrhythmias in patients harboring RyR2 channels destabilized by loss-of-function mutations (13).…”