Overexpression of cardiac calsequestrin as a novel gene-therapy approach to treat CPVT1: in silico and in vivo proves of principle

Abstract

Abstract Background More than 200 gain-of-function mutations in the cardiac ryanodine receptor (RyR2) cause catecholaminergic polymorphic ventricular tachycardia type 1 (CPVT1). From a molecular point of view, 3 types of mutations exist [1]: Class 1 mutants enhance RyR2 opening in response to sarcoplasmic reticulum (SR) free Ca2+; Class 2 and Class 3 mutants enhance RyR2 opening in response to both cytosolic and SR free Ca2+. Purpose The study's objective was to evaluate in silico and in vivo, in CPVT1 knock-in mice carriers of the Class 1 mutation RyR2(R4496C/+), whether overexpression of cardiac calsequestrin (CASQ2) rescues the arrhythmic phenotype in CPVT1 mice. Rationale: Several studies performed in the 2000’s have collectively demonstrated that an increase in CASQ2 promotes the closure of RyR2 channels [2]: inspired by this evidence, here we test the hypothesis that CASQ2 gene therapy might be a novel treatment for CPVT1. Methods (1) In silico study: Properties of Class 1 and Class 2 RyR2s mutants (Figure, panels A-C) were introduced into an in silico model of the murine ventricular myocyte [3]. Virtual myocytes were paced either at baseline or during simulated delivery of 100 nM Isoproterenol at 1 Hz for 180 seconds. To simulate CASQ2 overexpression, protein levels were progressively increased, and the changes in the arrhythmic burden were documented. (2) In vivo study (performed in compliance with ethical regulations): an adeno-associated viral vector containing the cDNA sequence of CASQ2 was injected into RyR2(R4496C/+) mice (tail iv, 3×10^13 vg/kg) at 4 weeks of age. Eight weeks post-infection, animals were treated with an ip bolus of caffeine (120mg/kg) and epinephrine (2 mg/kg) to elicit arrhythmias (Epi/Caff Test). Results (1) In silico: dose-response curves were simulated to correlate CASQ2 levels with arrhythmia inducibility during virtual Isoproterenol delivery. For the simulated prototypical Class 1 mutation, 1.4-fold CASQ2 expression suppressed arrhythmias. For the prototypical Class 2 mutation, 1.8-fold CASQ2 expression was needed (Figure, panel D). (2) In vivo: At 12 weeks of age, both RyR2(R4496C/+) treated and non-treated littermates received the ip Epi / Caff bolus. Arrhythmias were observed in 17/32 (53%) untreated mice and in 0/12 treated animals (p=0.0012). Conclusion(s) We provide proof-of-principle demonstrating that in mice, the overexpression of CASQ2 may be a powerful and safe therapeutic strategy for CPVT1.