Impaired Ca-Calmodulin-Dependent Inactivation of CaV 1.2 Contributes to Loss of SR Ca Release Refractoriness in Mice Lacking Casq2

Abstract

In cardiac muscle, L-type Ca current (CaV 1.2, ICa) induced Ca release from sarcoplasmic reticulum (SR) is reduced with successively shorter coupling intervals of premature stimuli, a phenomenon known as SR Ca release restitution. We previously reported that myocytes lacking the SR luminal Ca binding protein calsequestrin (Casq2 KO) exhibited less SR Ca release restitution and hence largely lack Ca release refractoriness. Here, we test the hypothesis that altered CaV 1.2 channel gating contributes to altered SR Ca release restitution in Casq2 KO myocytes. Methods and Results: ICa was recorded in voltage-clamped ventricular myocytes isolated from Casq2 KO mice and wild-type (WT) littermates. Cells were pre-treated with ryanodine (50 μM) and thapsigargin (10 μM) to eliminate SR Ca release. Compared to WT, ICa peak currents were unchanged, but the inactivation time course was significantly slower in KO myocytes (tau=157 ms vs. 46 ms in WT, p<0.01). using Ba as a charge carrier abolished the differences in inactivation, suggesting that the underlying defect lies not in the voltage-dependent but rather in the Ca-dependent ICa inactivation. Addition of apo-calmodulin (CaM, 0.35 mg/ml) to highly-buffered (14 mM EGTA) pipette solution accelerated ICa inactivation in KO myocytes (tau=52 ms). To test the effect of CaM on ICa restitution, ICa was measured by applying premature stimuli (S2) at successively shorter S1-S2 coupling intervals after a 1 Hz pacing train (S1). Intracellular dialysis with apo-CaM rendered ICa restitution curves of KO myocytes similar to those obtained in WT myocytes. Conclusion: Impaired Ca-dependent inactivation of ICa in Casq2 KO mice can be reversed by excess apo-CaM, suggesting that impaired Ca-CaM-dependent inactivation of CaV 1.2 contributes to loss of SR Ca release refractoriness in Casq2 KO mice.