Impaired calcium-calmodulin-dependent inactivation of Cav1.2 contributes to loss of sarcoplasmic reticulum calcium release refractoriness in mice lacking calsequestrin 2

Abstract

AimsIn cardiac muscle, Ca2+ release from sarcoplasmic reticulum (SR) is reduced with successively shorter coupling intervals of premature stimuli, a phenomenon known as SR Ca2+ release refractoriness. We recently reported that the SR luminal Ca2+ binding protein calsequestrin 2 (Casq2) contributes to release refractoriness in intact mouse hearts, but the underlying mechanisms remain unclear. Here, we further investigate the mechanisms responsible for physiological release refractoriness. Methods and resultsGene-targeted ablation of Casq2 (Casq2 KO) abolished SR Ca2+ release refractoriness in isolated mouse ventricular myocytes. Surprisingly, impaired Ca2+-dependent inactivation of L-type Ca2+ current (ICa), which is responsible for triggering SR Ca2+ release, significantly contributed to loss of Ca2+ release refractoriness in Casq2 KO myocytes. Recovery from Ca2+-dependent inactivation of ICa was significantly accelerated in Casq2 KO compared to wild-type (WT) myocytes. In contrast, voltage-dependent inactivation measured by using Ba2+ as charge carrier was not significantly different between WT and Casq2 KO myocytes. Ca2+-dependent inactivation of ICa was normalized by intracellular dialysis of excess apo-CaM (20μM), which also partially restored physiological Ca2+ release refractoriness in Casq2 KO myocytes. ConclusionsOur findings reveal that the intra-SR protein Casq2 is largely responsible for the phenomenon of SR Ca2+ release refractoriness in murine ventricular myocytes. We also report a novel mechanism of impaired Ca2+-CaM-dependent inactivation of Cav1.2, which contributes to the loss of SR Ca2+ release refractoriness in the Casq2 KO mouse model and, therefore, may further increase risk for ventricular arrhythmia in vivo.