Novel CPVT-Associated Calmodulin Mutation in CALM3 (CALM3-A103V) Activates Arrhythmogenic Ca Waves and Sparks

Abstract

Background: Calmodulin (CaM) mutations are associated with severe forms of long QT syndrome and catecholaminergic polymorphic ventricular tachycardia (CPVT). CaM mutations are found in 13% of genotype-negative long QT syndrome patients, but the prevalence of CaM mutations in genotype-negative CPVT patients is unknown. Here, we identify and characterize CaM mutations in 12 patients with genotype-negative but clinically diagnosed CPVT.

Methods and results: We performed mutational analysis of CALM1, CALM2, and CALM3 gene-coding regions, in vitro measurement of CaM-Ca(2+) (Ca)-binding affinity, ryanodine receptor 2-CaM binding, Ca handling, L-type Ca current, and action potential duration. We identified a novel CaM mutation-A103V-in CALM3 in 1 of 12 patients (8%), a female who experienced episodes of exertion-induced syncope since age 10, had normal QT interval, and displayed ventricular ectopy during stress testing consistent with CPVT. A103V modestly lowered CaM Ca-binding affinity (3-fold reduction versus WT-CaM), but did not alter CaM binding to ryanodine receptor 2. In permeabilized cardiomyocytes, A103V-CaM (100 nmol/L) promoted spontaneous Ca wave and spark activity, a cellular phenotype of ryanodine receptor 2 activation. Even a 1:3 mixture of A103V-CaM:WT-CaM activated Ca waves, demonstrating functional dominance. Compared with long QT syndrome D96V-CaM, A103V-CaM had significantly less effects on L-type Ca current inactivation, did not alter action potential duration, and caused delayed afterdepolarizations and triggered beats in intact cardiomyocytes.

Conclusions: We discovered a novel CPVT mutation in the CALM3 gene that shares functional characteristics with established CPVT-associated mutations in CALM1. A small proportion of A103V-CaM is sufficient to evoke arrhythmogenic Ca disturbances via ryanodine receptor 2 dysregulation, which explains the autosomal dominant inheritance.

Keywords: calcium; calcium channel; calmodulin; catecholaminergic polymorphic ventricular tachycardia; ryanodine receptor.

Figure 1

Stress test for patient with A103V-CaM. A representative stress test for the patient harboring the A103V-CaM variant while on beta blocker therapy. Shown are the rhythm strips for the baseline, stage 2 with the onset of PVCs, a couplet, the peak exercise heart rate, and three minutes into recovery.

Figure 2

Ca titration curves for wild-type (WT) CPVT CaM mutant (A103V) for the CaM-C and CaM-N domain. Dissociation constants (Kd, in μM) were derived for each domain by following intrinsic Tyr and Phe fluorescence for the N-domain and C-domain, respectively, and fitting to a standard binding equation. The A103V-CaM mutant reduced Ca binding affinity of CaM C-domain (3-fold reduction compared to WT-CaM), whereas N-domain Ca binding was unchanged. Values are averages of 3 experiments, and error was determined by analysis of the curve fits.

Figure 3

A103V-CaM promotes Ca sparks activity and reduces sarcoplasmic reticulum (SR) Ca content. A. Representative line-scan images of Ca sparks in permeabilized mouse ventricular myocytes incubated for 30 minutes with WT-CaM or CPVT-CaMs (A103V, N54I and N98S) 100 nM B. Average Ca spark frequency/100μm/sec C. Ca spark amplitude D. Ca sparks full duration half max and E. Ca sparks full width half max. Data are presented as mean±SD. (n=30 *p<0.05; **p<0.01 vs. WT-CaM) F. Line scan (top) and (bottom) line plot (red arrow) examples of SR Ca content evaluated by 10 mM caffeine-evoked Ca transient in cardiomyocytes incubated with WT-CaM or CPVT-CaMs (A103V, N54I and N98S) 100 nM G. Average SR Ca content. Data are mean±SD. (n=6 *p<0.05; **p<0.01; ***p<0.001 vs. WT-CaM) free [Ca]_i = 50 nM, EGTA = 0.5 mM.

Figure 4

A103V-CaM promotes Ca waves with dominant effect. A. Representative line scans (top) from permeabilized mouse ventricular myocytes after 30 minute incubation with wild-type (WT) or mutant CaMs (100 nM) and (bottom) line plot examples (red arrow) B. Averaged Ca waves amplitude and C. frequency. A103V-CaM promoted higher Ca wave frequency and amplitude compared to WT. Data are presented as mean±SD of values normalized by WT values on each experimental day (n = 30. N=3 ***p<0.001 vs. WT-CaM). D. Representative line scans (top) from permeabilized mouse ventricular myocytes after 30 minute incubation with either wild-type (WT) or mutant CaMs 25% mixed with 75% WT-CaM (final concentration 100 nM) and (bottom) line plot examples (red arrow) E. Averaged Ca waves amplitude and F. frequency. Even 25% A103V-CaM promoted Ca waves. Data are mean±SD of values normalized by WT values on each experimental day (n = 30. N = 3 ***p<0.001 vs. WT-CaM) free [Ca]_i = 120 nM, EGTA = 100 μM.

Figure 5

The A103V mutation does not significantly change the affinity of CaM for RyR2. Both CaM species were equally effective in displacing A-CaM at nM and μM [Ca] relevant to diastolic and systolic conditions in the heart.

Figure 6

A103V-CaM showed a trend to disrupt L-type Ca channels (LTCC) inactivation. A. Representative examples of traces for each experimental group. B. Average current densities (pA/pF) obtained in cells dialyzed with either WT-CaM, A103V-CaM, N54I-CaM, N98S-CaM (CPVT mutants) or D96V-CaM (LQTS mutant). CaM mutants had no effect on the peak current density. C. Effect of A103V-CaM, N54I-CaM, N98S and D96V-CaM on inactivation time constant of the LTCC compared to WT-CaM. A103V-CaM an N98S-CaM showed a trend to impair LTCC inactivation whereas N54I-CaM did not affect it at all. However, compared to a LQTS-associated CaM mutation (D96V-CaM), A103V-CaM had a smaller effect on LTCC inactivation. Data are mean ± SD. (WT-CaM, A103V-CaM and N54I-CaM n=6; N98S-CaM and D96V-CaM n=4. *p<0.05, vs. WT-CaM; ^††p<0.01, vs. N54I-CaM)

Figure 7

A103V-CaM does not modify APD but induces spontaneous beats triggered by DADs. A. Top panel: Representative examples of AP records for each experimental group. Bottom panel: Average APD measured at 50% (APD50, left) and 90% (APD90, right) of repolarization. B. Top panel: Representative example of prolonged APs with EADs recorded from a cardiomyocyte dialyzed with D96V-CaM. Bottom panel: Percentage of cardiomyocytes exhibiting EADs during the pacing train. C. Top panel: Representative examples of DADs and triggered beats recorded from a cardiomyocyte dialyzed with A103V-CaM. Bottom panel: Averaged number of DADs (left) and triggered beats (right) during the first 45s after the pacing train in cardiomyocytes dialyzed with either WT or mutant CaMs. Data are mean ± SD. (WT-CaM n=7, A103V-CaM n=14 and D96V-CaM n=12 *p<0.05, **p<0.01 vs. WT-CaM; ^†p<0.05 vs. A103V-CaM)