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

Abstract

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 ryanodine receptor (RyR2). However, what inactivates RyR2 and how RyR2 inactivation leads to Ca2+ alternans are unknown. Objective: To determine the role of calmodulin (CaM) 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 (CaM-WT), 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 WT 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-WT and CaM-M37Q promoted Ca 2+ alternans and prolonged Ca 2+ transient recovery in intact RyR2 WT 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. Further, 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 SR Ca 2+ release, which in turn reduces diastolic cytosolic Ca 2+ , leading to alternations in diastolic cytosolic Ca 2+ , RyR2 inactivation, and SR Ca 2+ release (i.e. 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.

L‐type Ca 2+ channel recovery from inactivation in rabbit atrial myocytes

“…LCC RFI in ventricular myocytes was demonstrated to be faster than recovery of intracellular Ca 2+ release (Sun et al, 2018; Wei et al, 2021). Here, we report a similar observation in atrial cells (Figure 6).…”

Role of Reduced Sarco-Endoplasmic Reticulum Ca2+-ATPase Function on Sarcoplasmic Reticulum Ca2+ Alternans in the Intact Rabbit Heart

“…However, as pacing frequency increases, SR Ca 2+ load also increases ( Wang et al, 2014 ), which in turn would increase the magnitude of SR Ca 2+ release and make alternans more apparent. Alternatively, rapid pacing may cause diastolic cytosolic Ca 2+ elevation, which in turn triggers Ca 2+ -calmodulin-dependent inactivation of RyR and, consequently, an imbalance of SR Ca 2+ release and reuptake ( Wei et al, 2020 ). Thus, the role of SERCA activity in either suppressing or promoting SR Ca 2+ alternans may depend on relative changes in SR Ca 2+ content at various pacing frequencies.…”

Ventricular voltage‐gated ion channels: Detection, characteristics, mechanisms, and drug safety evaluation

“…For example, CaM 91 inactivates Ryr2 during diastolic cytosolic calcium elevation, thus playing an important role in Ca 2+ alternans. 113 The CaM binding sites on cytosolic sites of Ryr2 will be shifted and dependent on Ca 2+ concentration binding to CaM. 112 Enhancement of CaM function promotes, whereas impairment of CaM function suppresses Ca 2+ alternans.…”

“… 112 Enhancement of CaM function promotes, whereas impairment of CaM function suppresses Ca 2+ alternans. 113 Several enzymes, such as PKA, CaMKII, PP1, and PP2A, interact with Ryr2 and exert phosphorylation/dephosphorylation effects on Ryr2. 114 The hyperphosphorylation of RyR2 by PKA 114 and/or by CaMKII 115 causes abnormal Ca 2+ leakage from the SR. RyR2 is also coupled to proteins at the luminal SR surface, such as type 2 calsequestrin (CASQ2), 116 , 117 which increases the open probability and facilitates high rates of Ca 2+ efflux during systole.…”