RESUMO
BACKGROUND: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an arrhythmogenic disorder caused by mutations in the cardiac ryanodine receptor RyR2 that increase diastolic calcium cation (Ca2+) leak from the sarcoplasmic reticulum (SR). Calmodulin (CaM) dissociation from RyR2 has been associated with diastolic Ca2+ leak in heart failure. OBJECTIVE: Determine whether the tetracaine-derivative compound EL20 inhibits abnormal Ca2+ release from RyR2 in a CPVT model and investigate the underlying mechanism of inhibition. METHODS: Spontaneous Ca2+ sparks in cardiomyocytes and inducible ventricular tachycardia were assessed in a CPVT mouse model, which is heterozygous for the R176Q mutation in RyR2 (R176Q/+ mice) 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 Ca2+ release in R176Q/+ myocytes (half-maximal inhibitory concentration = 35.4 nM) 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 (half-maximal inhibitory concentration = 8.2 nM). Inhibition of single-channel activity for cardiac SR or purified RyR2 supplemented with 100-nM or 1-µM 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, which is often associated with failing hearts. Our data also suggest that CaM dissociation may contribute to the pathogenesis of arrhythmias with the CPVT-linked R176Q mutation.
Assuntos
Antiarrítmicos/farmacologia , Calmodulina/deficiência , DNA/genética , Mutação , Miócitos Cardíacos/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Taquicardia Ventricular/genética , Animais , Cálcio/metabolismo , Análise Mutacional de DNA , Modelos Animais de Doenças , Camundongos , Camundongos Mutantes , Miócitos Cardíacos/patologia , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático , Ovinos , Taquicardia Ventricular/metabolismo , Taquicardia Ventricular/patologiaRESUMO
RATIONALE: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a potentially lethal arrhythmic disorder caused by mutations in the type-2 ryanodine receptor (RyR2). Mutant RyR2 cause abnormal Ca2+ leak from the sarcoplasmic reticulum (SR), which is associated with the development of arrhythmias. OBJECTIVE: To determine whether derivatives of tetracaine, a local anesthetic drug with known RyR2 inhibiting action, could prevent CPVT induction by suppression of RyR2-mediated SR Ca2+ leak. METHODS AND RESULTS: Confocal microscopy was used to assess the effects of tetracaine and 9 derivatives (EL1-EL9) on spontaneous Ca2+ sparks in ventricular myocytes isolated from RyR2-R176Q/+ mice with CPVT. Whereas each derivative suppressed the Ca2+ spark frequency, derivative EL9 was most effective at the screening dose of 500nmol/L. At this high dose, the Ca2+ transient amplitude was not affected in myocytes from WT or R176Q/+ mice. The IC50 of EL9 was determined to be 13nmol/L, which is about 400× time lower than known RyR2 stabilizer K201. EL9 prevented the induction of ventricular tachycardia observed in placebo-treated R176Q/+ mice, without affecting heart rate or cardiac contractility. CONCLUSIONS: Tetracaine derivatives represent a novel class of RyR2 stabilizing drugs that could be used for the treatment of the potentially fatal disorder catecholaminergic polymorphic ventricular tachycardia.