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

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 Ca 2+ release channel of the sarcoplasmic reticulum (SR), the ryanodine receptor type‐2 (RyR2). Loss of function mutations in the RyR2 result in abnormally low Ca 2+ release which leads to an increase in SR Ca 2+ content. It has been proposed that SR Ca 2+ 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 Ca 2+ content, therefore preventing the development of Ca 2+ overload. To test this hypothesis, IpCa effect on SR Ca 2+ content was evaluated by measuring SR Ca 2+ restitution, which is the time‐dependent recovery of SR Ca 2+ release as a function of the stimulation frequency. If indeed, IpCa reduces SR Ca 2+ content due to RyR2‐mediated Ca 2+ leak, SR Ca 2+ 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 Ca 2+ content on SR Ca 2+ restitution. Methods To test IpCa effects on SR Ca 2+ 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 Ca 2+ transients were simultaneously recorded. SR Ca 2+ restitution was calculated from the ratio of the Ca 2+ 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 Ca 2+ transients amplitude, without affecting their kinetics or basal Ca 2+ . In contrast, caffeine (1 mM; n=10) affected basal Ca 2+ (~3‐fold increase), Ca 2+ 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 Ca 2+ 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 Ca 2+ dynamics are similar to those induced by caffeine in terms of reduction of SR Ca 2+ content, without affecting the Ca 2+ transient kinetics. Consistent with previous observations, our SR Ca 2+ restitution results support the notion that IpCa partially depletes SR Ca 2+ content, providing a plausible mechanism for its antiarrhythmic effects.