Calcium Signaling in Mouse Ventricular Myocytes with Impaired Calmodulin Regulation of Ryr2

Abstract

The cardiac muscle ryanodine receptor ion cannel (RyR2) is a 560 kDa homotetramer that is regulated by calmodulin (CaM), which decreases the open probability of RyR2 at diastolic and systolic Ca2+ concentrations. Point mutations in the CaM binding domain of RyR2 (W3587A/L3591D/F3603A, RyR2ADA) result in severe cardiac hypertrophy, reduced RyR2 concentration, and death by postnatal day 16, which suggests that CaM inhibition of RyR2 is required for normal cardiac function. Little is known, however, about Ca2+ signaling of the mutant mouse hearts. Here, we report on Ca2+ signaling of enzymatically isolated cardiac myocytes of 11-15 day old wild type and homozygous RyR2ADA/ADA mouse hearts. Cardiomyocytes, dialyzed with the fluorescence probe Fluo-4, were voltage-clamped and subjected to 2-dimensional rapid confocal imaging. At holding potentials of −80 mV, the Ca2+ spark frequency was 7-fold lower in mutant (0.61±0.40 sparks/sec, n=7) compared to wild type (4.04±1.07/sec, n=7) myocytes. Further, preliminary data indicate that the amplitude of the Ca2+ transients, triggered by membrane depolarization, was lower in voltage-clamped mutant cells than in wild type cells, even though caffeine-triggered Ca2+ release was larger in mutant cardiomyocytes. The data suggest that RyR2ADA mutation reduces resting Ca2+ spark frequency and depolarization-induced SR Ca2+ release in presence of increased SR Ca2+ store size. The reduction of RyR2 expression in RyR2ADA/ADA hearts may, in part, contribute to this phenotype.Supported by NIH (HL16152 and HL073051), NSF (EPS-09037395) and AHA (10SDG3500001).