Ca²⁺-CaM Dependent Inactivation of RyR2 Underlies Ca²⁺ Alternans in Intact Heart.

Abstract

Ca²⁺ alternans plays an essential role in cardiac alternans that can lead to ventricular fibrillation, but the mechanism underlying Ca²⁺ alternans remains undefined. Increasing evidence suggests that Ca²⁺ alternans results from alternations in the inactivation of cardiac RyR2 (ryanodine receptor 2). However, what inactivates RyR2 and how RyR2 inactivation leads to Ca²⁺ alternans are unknown. To determine the role of CaM (calmodulin) on Ca²⁺ alternans in intact working mouse hearts. We used an in vivo local gene delivery approach to alter CaM function by directly injecting adenoviruses expressing CaM-wild type, a loss-of-function CaM mutation, CaM (1-4), and a gain-of-function mutation, CaM-M37Q, into the anterior wall of the left ventricle of RyR2 wild type or mutant mouse hearts. We monitored Ca²⁺ transients in ventricular myocytes near the adenovirus-injection sites in Langendorff-perfused intact working hearts using confocal Ca²⁺ imaging. We found that CaM-wild type and CaM-M37Q promoted Ca²⁺ alternans and prolonged Ca²⁺ transient recovery in intact RyR2 wild type and mutant hearts, whereas CaM (1-4) exerted opposite effects. Altered CaM function also affected the recovery from inactivation of the L-type Ca²⁺ current but had no significant impact on sarcoplasmic reticulum Ca²⁺ content. Furthermore, we developed a novel numerical myocyte model of Ca²⁺ alternans that incorporates Ca²⁺-CaM-dependent regulation of RyR2 and the L-type Ca²⁺ channel. Remarkably, the new model recapitulates the impact on Ca²⁺ alternans of altered CaM and RyR2 functions under 9 different experimental conditions. Our simulations reveal that diastolic cytosolic Ca²⁺ elevation as a result of rapid pacing triggers Ca²⁺-CaM dependent inactivation of RyR2. The resultant RyR2 inactivation diminishes sarcoplasmic reticulum Ca²⁺ release, which, in turn, reduces diastolic cytosolic Ca²⁺, leading to alternations in diastolic cytosolic Ca²⁺, RyR2 inactivation, and sarcoplasmic reticulum Ca²⁺ release (ie, Ca²⁺ alternans). Our results demonstrate that inactivation of RyR2 by Ca²⁺-CaM is a major determinant of Ca²⁺ alternans, making Ca²⁺-CaM dependent regulation of RyR2 an important therapeutic target for cardiac alternans.