Partial and Controlled Depletion of Sarcoplasmic Reticulum (SR) Calcium Prevents Spontaneous Calcium Release and Tachyarrhythmias in a Mouse Model of CPVT

Abstract

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.