Ca 2+ -CaM Dependent Inactivation of RyR2 Underlies Ca 2+ Alternans in Intact Heart.

Rationale: Ca ²⁺ alternans plays an essential role in cardiac alternans that can lead to ventricular fibrillation, but the mechanism underlying Ca ²⁺ alternans remains undefined. Increasing evidence suggests that Ca ²⁺ alternans results from alternations in the inactivation of cardiac RyR2 (ryanodine receptor 2). However, what inactivates RyR2 and how RyR2 inactivation leads to Ca ²⁺ alternans are unknown.
Objective: To determine the role of CaM (calmodulin) on Ca ²⁺ 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 ²⁺ transients in ventricular myocytes near the adenovirus-injection sites in Langendorff-perfused intact working hearts using confocal Ca ²⁺ imaging. We found that CaM-wild type and CaM-M37Q promoted Ca ²⁺ alternans and prolonged Ca ²⁺ 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 ²⁺ current but had no significant impact on sarcoplasmic reticulum Ca ²⁺ content. Furthermore, we developed a novel numerical myocyte model of Ca ²⁺ alternans that incorporates Ca ²⁺ -CaM-dependent regulation of RyR2 and the L-type Ca ²⁺ channel. Remarkably, the new model recapitulates the impact on Ca ²⁺ alternans of altered CaM and RyR2 functions under 9 different experimental conditions. Our simulations reveal that diastolic cytosolic Ca ²⁺ elevation as a result of rapid pacing triggers Ca ²⁺ -CaM dependent inactivation of RyR2. The resultant RyR2 inactivation diminishes sarcoplasmic reticulum Ca ²⁺ release, which, in turn, reduces diastolic cytosolic Ca ²⁺ , leading to alternations in diastolic cytosolic Ca ²⁺ , RyR2 inactivation, and sarcoplasmic reticulum Ca ²⁺ release (ie, Ca ²⁺ alternans).
Conclusions: Our results demonstrate that inactivation of RyR2 by Ca ²⁺ -CaM is a major determinant of Ca ²⁺ alternans, making Ca ²⁺ -CaM dependent regulation of RyR2 an important therapeutic target for cardiac alternans.