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

Abstract

Background: Catecholaminergic 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 results: To 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.

Conclusions: Our 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.

Keywords: arrhythmia (mechanisms); calcium channel; calcium signaling; calmodulin; gene therapy.

Figure 1

CPVT CaM N98S increased Ca wave frequency and shortened RyR2 refractoriness. A, representative line‐scan images of spontaneous Ca waves (SCWs) in permeabilized WT myocytes exposed to 100 nmol/L wtCaM or 100 nmol/L CPVT CaM N98S. Cytosolic Ca was clamped at ≈120 nmol/L with the slow Ca buffer EGTA. B, time‐dependent recovery of Ca sparks after occurrence of SCWs. Frequency of Ca sparks (f sparks) was calculated at 200 ms intervals. Recovery of Ca sparks was fitted with mono‐exponential curve with tau of 0.25 seconds for wtCaM and 0.07 seconds for N98S CaM, respectively. Ca sparks steady state levels (f₀) were 0.7 (per 100 μm per second) for wtCaM and 1.6 (per 100 μm per second) for N98S CaM, respectively. C, Average data for SCW frequency, Ca spark frequency and refractoriness factor (RF) obtained from WT mouse ventricular myocytes permeabilized with wild‐type (wt) and CPVT (N98S) CaMs, respectively, n=38 to 58 cells, *P<0.05 vs wtCaM. Ca indicates calcium; CaM, calmodulin; CPVT, catecholaminergic polymorphic ventricular tachycardia; EGTA, ethylene glycol‐bis(β‐aminoethyl ether)‐N,N,N′,N′‐tetraacetic acid; RF, refractoriness factor; RyR2, ryanodine receptor 2; SCW, spontaneous Ca waves; WT, wild type.

Figure 2

CPVT CaM N98S increased Ca waves frequency and shortened RyR2 refractoriness in CASQ2 KO myocytes. A, representative line‐scan images of SCWs in permeabilized CASQ2 KO myocytes exposed to either 100 nmol/L of wtCaM or 100 nmol/L of N98S CaM. Cytosolic Ca was clamped at ≈120 nmol/L with the slow Ca buffer EGTA. B, Average data for SCW frequency and refractoriness factor obtained from CASQ2 KO ventricular myocytes permeabilized with 100 nmol/L wtCaM and 100 nmol/L of N98S CaM, respectively, n=31 to 38 cells, *P<0.05 vs wtCaM. Ca indicates calcium; CaM, calmodulin; CASQ2, calsequestrin; CPVT, catecholaminergic polymorphic ventricular tachycardia; EGTA, ethylene glycol‐bis(β‐aminoethyl ether)‐N,N,N′,N′‐tetraacetic acid; KO, knock out; RyR2, ryanodine receptor 2; SCW, spontaneous Ca waves.

Figure 3

The effect of CPVT CaM N54I on the rate of Ca dissociation from N‐domain of CaM. A, Ca bound N‐domain of CaM binds RyR2 and keeps it refractory following CICR. Meanwhile, CASQ2 senses luminal (Ca) decrease and contributes to RyR2 refractoriness. B, the time course of the decrease in Trp fluorescence as Ca dissociated from CaM^F20W constructs. The data traces have been staggered and normalized for clarity. C, the time course of the increase in Quin‐2 fluorescence as Ca dissociated from CaM‐pRyR2 complex. The data traces have been staggered and normalized for clarity. Ca indicates, calcium; CaM, calmodulin; CASQ2, calsequestrin; CICR, calcium induced calcium release; CPVT, catecholaminergic polymorphic ventricular tachycardia; RyR2, ryanodine receptor 2; Trp, tryptophan.

Figure 4

Engineered CaM variant D57A exhibited a faster rate of Ca dissociation and mimicked the effects of CPVT CaMs. A, the time course of the decrease in Trp fluorescence as Ca dissociated from CaM^F20W constructs. The data traces have been staggered and normalized for clarity. B, representative line‐scan images of SCWs in permeabilized WT myocytes exposed to 100 nmol/L wtCaM or 100 nmol/L D57A CaM. Cytosolic Ca was clamped at ≈120 nmol/L with the slow Ca buffer EGTA. C, Average data for SCW frequency and refractoriness factor obtained from WT mouse ventricular myocytes permeabilized with wtCaM and D57A CaM, respectively, n=40 cells, *P<0.05 vs wtCaM. Ca indicates calcium; CaM, calmodulin; cAMP, cyclic adenosine monophosphate; EGTA, ethylene glycol‐bis(β‐aminoethyl ether)‐N,N,N′,N′‐tetraacetic acid; SCW, spontaneous Ca waves; TCaM, therapeutic calmodulin; Trp, tryptophan; WT, wild type.

Figure 5

TCaM significantly reduced spontaneous Ca waves in permeabilized R33Q myocytes and prolonged refractoriness. A, the time course of the decrease in Trp fluorescence as Ca dissociated from CaM^F20W constructs. The data traces have been staggered and normalized for clarity. B, representative line‐scan images of SCWs in permeabilized R33Q myocytes exposed to cAMP. Cytosolic Ca was clamped at ≈120 nmol/L with the slow Ca buffer EGTA. C, Average data for SCW frequency (n=18–39 cells) and refractoriness factor (n=11–15 cells) obtained from R33Q ventricular myocytes permeabilized with wtCaM, GSH‐CaM, M37Q CaM, and GSH‐M37Q CaM (TCaM), *P<0.05 vs wtCaM. Ca indicates calcium; CaM, calmodulin; cAMP, cyclic adenosine monophosphate; EGTA, ethylene glycol‐bis(β‐aminoethyl ether)‐N,N,N′,N′‐tetraacetic acid; SCW, spontaneous Ca waves; TCaM, therapeutic calmodulin; Trp, tryptophan; WT, wild type.

Figure 6

TCaM infected R33Q myocytes had improved Ca handling. A, evaluation of the infection efficiency of TCaM: phase contrast and mCherry fluorescence of TCaM injected R33Q myocytes. B, representative line‐scan images of SCWs in intact myocytes isolated from mCherry or TCaM virus infected R33Q mouse. Myocytes were field‐stimulated at 0.3 Hz in the presence of 1 μmol/L isoproterenol. C, Average data for SCW frequency (n=45–50 cells) and refractoriness factor (n=15–17 cells) obtained from R33Q myocytes infected with control (mCherry) and TCaM virus, respectively, *P<0.05 vs mCherry. D, Average of Ca transient amplitude and baseline fluorescence obtained from R33Q myocytes infected with control (mCherry) and TCaM virus, respectively, *P<0.05 vs mCherry. Ca indicates calcium; TCaM, therapeutic calmodulin; SCW, spontaneous Ca waves.

Figure 7

TCaM alleviated ventricular tachycardia in vivo in CPVT mice R33Q. A, Representative surface ECG traces from WT mice, R33Q mice or R33Q mice treated with TCaM. B, Average of VT incidence, n=8 to 19, *P<0.05 vs R33Q, #P<0.05 vs WT. C, Percentage of survival after ECG n=8 to 19, *P<0.05 vs R33Q, #P<0.05 vs WT. CPVT indicates catecholaminergic polymorphic ventricular tachycardia; ECG, electrocardiogram; TCaM, therapeutic calmodulin; VT, ventricular tachycardia.

Figure 8

Multiple influences converge to impair RyR2 refractoriness and contribute to ryanopathies.57 CAM indicates calmodulin; CASQ2, calsequestrin; RyR2, ryanodine receptor 2.