Rationale Calmodulin (CaM) mutations are associated with an autosomal-dominant syndrome of ventricular arrhythmia and sudden death that can present with divergent clinical features of catecholaminergic polymorphic ventricular tachycardia (CPVT)or long QT syndrome (LQTS).CaM binds to and inhibits RyR2 Ca release channels in the heart, but whether arrhythmogenic CaM mutants alter RyR2 function is not known. Objective To gain mechanistic insight into how human CaM mutations affect RyR2 Ca channels. Methods and Results We studied recombinant CaM mutants associated with CPVT (N54I, N98S) or LQTS (D96V, D130G, F142L). As a group, all LQTS-associated CaM mutants(LQTS-CaMs) exhibited reduced Ca affinity, whereas CPVT-associated CaM mutants(CPVT-CaMs) had either normal or modestly lower Ca affinity. In permeabilized ventricular myocytes, CPVT-CaMs at a physiological intracellular concentration (100nM) promoted significantly higher spontaneous Ca wave and spark activity, a typical cellular phenotype of CPVT. Compared to wild-type (WT) CaM, CPVT-CaMs caused greater RyR2 single channel open probability and showed enhanced binding affinity to RyR2. Even a 1:8 mixture of CPVT-CaM:WT-CaM activated Ca waves, demonstrating functional dominance. By contrast, LQTS-CaMs did not promote Ca waves and exhibited either normal regulation of RyR2 single channels (D96V) or lower RyR2 binding affinity (D130G, F142L). None of the CaM mutants altered Ca/CaM binding to CaM-kinase II. Conclusions A small proportion of CPVT-CaM is sufficient to evoke arrhythmogenic Ca disturbances, whereas LQTS-CaMs do not. Our findings explain the clinical presentation and autosomal dominant inheritance of CPVT-CaM mutations and suggest that RyR2-interactions are unlikely to explain arrhythmogenicity of LQTS-CaM mutations.
Background Catecholaminergic polymorphic ventricular tachycardia (CPVT) is caused by mutations in the cardiac ryanodine receptor (RyR2) or calsequestrin (Casq2) and can be difficult to treat. The class Ic antiarrhythmic drug flecainide blocks RyR2 channels and prevents CPVT in mice and humans. It is not known whether other class I antiarrhythmic drugs also block RyR2 channels and to what extent RyR2 channel inhibition contributes to antiarrhythmic efficacy in CPVT. Methods and Results We first measured the effect of all class I antiarrhythmic drugs marketed in the United States (quinidine, procainamide, disopyramide, lidocaine, mexiletine, flecainide, and propafenone) on single RyR2 channels incorporated into lipid bilayers. Only flecainide and propafenone inhibited RyR2 channels, with the S-enantiomer of propafenone having a significantly lower potency than R-propafenone or flecainide. In Casq2−/− myocytes, the propafenone enantiomers and flecainide significantly reduced arrhythmogenic Ca2+ waves at clinically relevant concentrations, whereas Na+ channel inhibitors without RyR2 blocking properties did not. In Casq2−/− mice, 5 mg/kg R-propafenone or 20 mg/kg S-propafenone prevented exercise-induced CPVT, whereas procainamide (20 mg/kg) or lidocaine (20 mg/kg) were ineffective (n=5 to 9 mice, P<0.05). QRS duration was not significantly different, indicating a similar degree of Na+ channel inhibition. Clinically, propafenone (900 mg/d) prevented ICD shocks in a 22-year-old CPVT patient who had been refractory to maximal standard drug therapy and bilateral stellate ganglionectomy. Conclusions RyR2 cardiac Ca2+ release channel inhibition appears to determine efficacy of class I drugs for the prevention of CPVT in Casq2−/− mice. Propafenone may be an alternative to flecainide for CPVT patients symptomatic on β-blockers.
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