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

Rationale: Ca alternans plays an essential role in cardiac alternans that can lead to ventricular fibrillation, but the mechanism underlying Ca alternans remains undefined. Increasing evidence suggests that Ca alternans results from alternations in the inactivation of cardiac RyR2 (ryanodine receptor 2). However, what inactivates RyR2 and how RyR2 inactivation leads to Ca alternans are unknown.

Objective: To determine the role of CaM (calmodulin) on Ca alternans in intact working mouse hearts.

Methods And Results: 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 Ca transients in ventricular myocytes near the adenovirus-injection sites in Langendorff-perfused intact working hearts using confocal Ca imaging. We found that CaM-wild type and CaM-M37Q promoted Ca alternans and prolonged Ca 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 Ca current but had no significant impact on sarcoplasmic reticulum Ca content. Furthermore, we developed a novel numerical myocyte model of Ca alternans that incorporates Ca-CaM-dependent regulation of RyR2 and the L-type Ca channel. Remarkably, the new model recapitulates the impact on Ca alternans of altered CaM and RyR2 functions under 9 different experimental conditions. Our simulations reveal that diastolic cytosolic Ca elevation as a result of rapid pacing triggers Ca-CaM dependent inactivation of RyR2. The resultant RyR2 inactivation diminishes sarcoplasmic reticulum Ca release, which, in turn, reduces diastolic cytosolic Ca, leading to alternations in diastolic cytosolic Ca, RyR2 inactivation, and sarcoplasmic reticulum Ca release (ie, Ca alternans).

Conclusions: Our results demonstrate that inactivation of RyR2 by Ca-CaM is a major determinant of Ca alternans, making Ca-CaM dependent regulation of RyR2 an important therapeutic target for cardiac alternans.