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BACKGROUND: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an arrhythmogenic disorder caused by mutations in the cardiac ryanodine receptor (RyR2) that increase diastolic Ca(2+) leak from the sarcoplasmic reticulum (SR). Calmodulin (CaM) dissociation from RyR2 has been associated with diastolic Ca(2+) leak in heart failure. OBJECTIVE: Determine if tetracaine-derivative, EL20, inhibits abnormal Ca(2+) release from RyR2 in a CPVT model and the underlying mechanism of inhibition. METHODS: Spontaneous Ca(2+) sparks in cardiomyocytes and inducible VT were assessed in a CPVT mouse model, RyR2-R176Q/+ (R176Q) in the presence of EL20 or vehicle. Single-channel studies using sheep cardiac SR or purified RyR2 reconstituted into proteoliposomes with and without exogenous CaM were used to assess mechanisms of inhibition. RESULTS: EL20 potently inhibits abnormal Ca(2+) release in R176Q myocytes (IC(50)=35.4nM) and diminishes arrhythmia in R176Q mice. EL20 inhibition of single-channel activity of purified RyR2 occurs in a similar range as seen in R176Q myocytes (IC(50)=8.2nM). Inhibition of single-channel activity for cardiac SR or purified RyR2 supplemented with 100nM or 1µM CaM shows a 200 to 1000-fold reduction in potency. CONCLUSION: This work provides a potential therapeutic mechanism for the development of antiarrhythmic compounds that inhibit leaky RyR2 resulting from CaM dissociation often associated with failing hearts. Our data also suggest that CaM dissociation may contribute to the pathogenesis of arrhythmias in the CPVT-linked R176Q mutation.

Background Approximately 10% of sudden infant death syndrome (SIDS) cases may stem from potentially lethal cardiac channelopathies, with approximately half of channelopathic SIDS involving the Na V 1.5 cardiac sodium channel. Recently, Na V beta subunits have been implicated in various cardiac arrhythmias. Thus, the 4 genes encoding Na V beta subunits represent plausible candidate genes for SIDS. Objective This study sought to determine the spectrum, prevalence, and functional consequences of sodium channel beta-subunit mutations in a SIDS cohort. Methods In this institutional review board–approved study, mutational analysis of the 4 beta-subunit genes, SCN1B to 4B , was performed using polymerase chain reaction, denaturing high-performance liquid chromatography, and direct DNA sequencing of DNA derived from 292 SIDS cases. Engineered mutations were coexpressed with SCN5A in HEK 293 cells and were whole-cell patch clamped. One of the putative SIDS-associated mutations was similarly studied in adenovirally transduced adult rat ventricular myocytes. Results Three rare (absent in 200 to 800 reference alleles) missense mutations (β3-V36M, β3-V54G, and β4-S206L) were identified in 3 of 292 SIDS cases. Compared with SCN5A+β3-WT, β3-V36M significantly decreased peak I Na and increased late I Na , whereas β3-V54G resulted in a marked loss of function. β4-S206L accentuated late I Na and positively shifted the midpoint of inactivation compared with SCN5A+β4-WT. In native cardiomyocytes, β4-S206L accentuated late I Na and increased the ventricular action potential duration compared with β4-WT. Conclusion This study provides the first molecular and functional evidence to implicate the Na V beta subunits in SIDS pathogenesis. Altered Na V 1.5 sodium channel function due to beta-subunit mutations may account for the molecular pathogenic mechanism underlying approximately 1% of SIDS cases.

Background Eight common (>0.5%) polymorphisms of SCN5A have been described in the US population. Every human also continuously generates two wild-type (WT) splice variants, one with a glutamine residue at position 1077 (Q1077) and one lacking this glutamine (Q1077del). One polymorphism (H558R) has been studied in both splice variants, five polymorphisms (R34C, R481W, S524Y, P1090L,V1951L) have not been previously studied, and two polymorphisms (S1103Y and R1193Q) have been studied in only one of the two splice variants. Objectives The purpose of this study was to examine the electrophysiologic molecular phenotype of the eight common polymorphisms in the two human splice variants of SCN5A. Methods Currents from 16 channels (all polymorphisms in both splice variants) were determined by voltage clamp and compared with WT after expression in HEK-293 cells. Results Six of eight polymorphisms showed a distinct phenotype that depended upon the background splice variant used for expression. Only R34C and V1951L showed no functional differences. S524Y showed a dramatic reduction in current density in the Q1077 background similar to that previously described for H558R. Four other polymorphisms (R481W, P1090L, S1103Y, R1193Q) showed shifts in activation, inactivation, or recovery that depended upon the splice variants. Shifts of a similar magnitude have been reported for arrhythmia syndrome mutations and are thought to be pathogenic. Conclusion The majority of common human SCN5A polymorphisms have a distinct molecular phenotype that depends upon the splice variant background. These findings have implications for the interpretation of previous studies of arrhythmia mutations. The significance of these findings for clinical arrhythmia remains to be elucidated.

Background SR Ca 2+ ([Ca 2+ ]SR) overload during β-adrenergic stimulation precipitates spontaneous Ca 2+ release (SCR) and tachyarrhythmias in cardiomyocytes harboring catecholaminergic polymorphic ventricular tachycardia (CPVT)-linked RyR2 mutations. We sought to determine whether imperacalcin (IpCa), a cell-permeable high-affinity agonist of RyR2 channels, prevents SCR and tachyarrhythmias in a mouse model of CPVT (RyR2-R4496C +/– ) by partially depleting [Ca 2+ ]SR during β-adrenergic stimulation. Methods Ca 2+ imaging was conducted by confocal microscopy in isolated ventricular myocytes from wild-type (WT) and RyR2-R4496C +/– mice. ECGs and left ventricular (LV) pressure were monitored in Langendorff-perfused beating WT and RyR2-R4496C +/– hearts stimulated with 300 nM isoproterenol (Iso). Cell penetration and topologic localization of IpCa was conducted in rat ventricular myocytes using Alexa546-IpCa. Results Alexa546-IpCa penetrated intact cardiomyocytes and localized in a striated pattern with ~2 μm interspacing, consistent with z-line labeling (n = 6 cells). Rapid perfusion of IpCa (1 µM) onto intact WT cardiomyocytes abruptly increased and then steadily decreased Ca 2+ transient amplitude, reflecting initial opening of RyR2 channels that gradually leads to partial depletion of [Ca 2+ ]SR (caffeine-induced Ca 2+ transient=7.6 ± 1.2 and 4.5 ± 0.82 F/Fo← before and after IpCa, respectively, n=15 cells, P = .001). The frequency of SCR waves in cardiomyocytes subjected to a "stress" protocol (5-second train of stimulations at 3 Hz under 300 nM Iso) was 3.6 ± 1.4 and 8.6 ± 5.3 waves/min in WT and RyR2-R4496C +/– cells, respectively, but decreased dramatically to 1.1 ± 0.9 and 1.6 ± 1.5 waves/min, respectively, after incubation with 1 µM IpCa (n = 10 cells each, P = 0.001). RyR2-R449C +/– hearts showed 56 ± 39 ectopic beats/min immediately ( Conclusions Partial depletion of [Ca 2+ ]SR with IpCa is a promising therapeutic alternative for Ca 2+ -triggered arrhythmias.