Imperacalcin‐Induced Reduction of Sarcoplasmic Reticulum Ca 2+ in Intact Mouse Heart

Abstract

Introduction Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited cardiac channelopathy, that often leads to sudden death due to arrhythmogenic episodes triggered by adrenergic stimulation. Previous studies have shown that CPVT is associated with mutations in the Ca2+ release channel of the sarcoplasmic reticulum (SR), the ryanodine receptor type-2 (RyR2). Loss of function mutations in the RyR2 result in abnormally low Ca2+ release which leads to an increase in SR Ca2+ content. It has been proposed that SR Ca2+ overload precipitates CPVT events by over activating the Na-Ca exchanger (NCX) which favors the development of arrhythmogenic delayed afterdepolarizations (DADs). Previous studies from our group and others have shown that the scorpion-derived peptide, imperacalcin (IpCa) is an effective agent to prevent/revert DADs in CPVT mouse models. The overall goal of this study was to test the hypothesis that the IpCa protects against CPVT by partially reducing SR Ca2+ content, therefore preventing the development of Ca2+ overload. To test this hypothesis, IpCa effect on SR Ca2+ content was evaluated by measuring SR Ca2+ restitution, which is the time-dependent recovery of SR Ca2+ release as a function of the stimulation frequency. If indeed, IpCa reduces SR Ca2+ content due to RyR2-mediated Ca2+ leak, SR Ca2+ restitution would be significantly shorter than in its absence. As a positive control, a known RyR2 agonist like caffeine, was used to define the effects of reducing SR Ca2+ content on SR Ca2+ restitution. Methods To test IpCa effects on SR Ca2+ restitution the pulsed local field fluorescence microscopy was used. Rhod-2AM-loaded hearts were perfused with either Tyrode, caffeine (1 mM), or IpCa (1 µM) and were stimulated at 2, 3, 4, 5, 6, 7, and 8 Hz while action potentials and intracellular Ca2+ transients were simultaneously recorded. SR Ca2+restitution was calculated from the ratio of the Ca2+ transient amplitude before and after the premature stimulation at the tested frequencies. Results At a constant stimulation frequency of 2 Hz, IpCa (1 µM; n=8) induced a 36% reduction of the Ca2+ transients amplitude, without affecting their kinetics or basal Ca2+. In contrast, caffeine (1 mM; n=10) affected basal Ca2+ (~3-fold increase), Ca2+ transients amplitude (~50% reduction), and their kinetics (~65% longer rise time & ~70% shorter decay & half-duration). As a function of the stimulation frequency, the SR Ca2+ exponential recovery (control t = 206±5 ms; n=6) was slightly accelerated by IpCa (t = 198±3 ms; n=4), & by caffeine (t = 183±4 ms; n=6). Conclusion Our results indicate that in intact mouse heart, the effects of IpCa on the intracellular Ca2+ dynamics are similar to those induced by caffeine in terms of reduction of SR Ca2+ content, without affecting the Ca2+ transient kinetics. Consistent with previous observations, our SR Ca2+ restitution results support the notion that IpCa partially depletes SR Ca2+ content, providing a plausible mechanism for its antiarrhythmic effects.