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

Abstract

BackgroundCatecholaminergic polymorphic ventricular tachycardia (CPVT) is a familial arrhythmogenic syndrome characterized by sudden death. There are several genetic forms of CPVT associated with mutations in genes encoding the cardiac ryanodine receptor (RyR2) and its auxiliary proteins including calsequestrin (CASQ2) and calmodulin (CaM). It has been suggested that impairment of the ability of RyR2 to stay closed (ie, refractory) during diastole may be a common mechanism for these diseases. Here, we explore the possibility of engineering CaM variants that normalize abbreviated RyR2 refractoriness for subsequent viral‐mediated delivery to alleviate arrhythmias in non–CaM‐related CPVT.Methods and ResultsTo that end, we have designed a CaM protein (GSH‐M37Q; dubbed as therapeutic CaM or T‐CaM) that exhibited a slowed N‐terminal Ca dissociation rate and prolonged RyR2 refractoriness in permeabilized myocytes derived from CPVT mice carrying the CASQ2 mutation R33Q. This T‐CaM was introduced to the heart of R33Q mice through recombinant adeno‐associated viral vector serotype 9. Eight weeks postinfection, we performed confocal microscopy to assess Ca handling and recorded surface ECGs to assess susceptibility to arrhythmias in vivo. During catecholamine stimulation with isoproterenol, T‐CaM reduced isoproterenol‐promoted diastolic Ca waves in isolated CPVT cardiomyocytes. Importantly, T‐CaM exposure abolished ventricular tachycardia in CPVT mice challenged with catecholamines.ConclusionsOur results suggest that gene transfer of T‐CaM by adeno‐associated viral vector serotype 9 improves myocyte Ca handling and alleviates arrhythmias in a calsequestrin‐associated CPVT model, thus supporting the potential of a CaM‐based antiarrhythmic approach as a therapeutic avenue for genetically distinct forms of CPVT.

Ryanodine receptor–bound calmodulin is essential to protect against catecholaminergic polymorphic ventricular tachycardia

“…Liu et al recently reported that gene transfer of a mutated CaM (GSH-M37Q), which exhibited a slowed N-terminal Ca dissociation rate and prolonged RyR2 refractoriness, reduced aberrant diastolic Ca 2+ release and alleviated arrhythmias in a calsequestrin-associated CPVT model (20). These findings support the important role of CaM in regulating the RyR2 in the pathogenesis of CPVT.…”

The arrhythmogenic N53I variant subtly changes the structure and dynamics in the calmodulin N-terminal domain, altering its interaction with the cardiac ryanodine receptor

“…We show that the N53I mutation does not alter the affinity of CaM for CaMBD2, neither in the apo nor the Ca 2+ -bound state (Figure 7 and Supplementary Table 4). However, even though the N53I mutation has only a small impact on the Ca 2+ -affinity of CaM alone (30), a reduced Ca 2+ -affinity for the CaM-N53I:CaMBD2 complex was reported (29), owing to an increase in the dissociation rate of Ca 2+ from the complex (42). Single-channel ion conductivity experiments demonstrated that CaM-N53I is not as efficient in closing RyR2 as CaM-WT (28).…”

“…In addition to the missing hydrogen bonds in the Ca 2+ /CaM-N53I:RyR2-CaMBD2 crystal structure, another significant difference caused by the N53I mutation is an increased Ca 2+ dissociation rate from the N-domain in CaM-N53I, reported previously both in the absence and presence of RyR2-CaMBD2 (30,42). We therefore investigated the dynamics of both CaM-WT and CaM-N53I in absence and in presence of Ca 2+ by NMR.…”

Calmodulin Mutations Associated with Heart Arrhythmia: A Status Report

“…Interdisciplinary investigations of this type can elucidate the mechanisms CaM uses to transduces changes in [Ca 2+ ] into modified ion channel function. Such studies will aid understanding how changes in CaM protein sequence can be tolerated, cause or contribute to disease, or potentially even someday enhance physiology [61].…”