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Gain-of-function missense variants in the cardiac ryanodine receptor (RyR2) are linked to catecholaminergic polymorphic ventricular tachycardia (CPVT), whereas RyR2 loss-of-function missense variants cause Ca2+ release deficiency syndrome (CRDS). Recently, truncating variants in RyR2 have also been associated with ventricular arrhythmias (VAs) and sudden cardiac death. However, there are limited insights into the potential clinical relevance and in vitro functional impact of RyR2 truncating variants. We performed genetic screening of patients presenting with syncope, VAs, or unexplained sudden death and in vitro characterization of the expression and function of RyR2 truncating variants in HEK293 cells. We identified two previously unknown RyR2 truncating variants (Y4591Ter and R4663Ter) and one splice site variant predicted to result in a frameshift and premature termination (N4717 + 15Ter). These 3 new RyR2 truncating variants and a recently reported RyR2 truncating variant, R4790Ter, were generated and functionally characterized in vitro. Immunoprecipitation and immunoblotting analyses showed that all 4 RyR2 truncating variants formed heteromers with the RyR2-wildtype (WT) protein. Each of these C-terminal RyR2 truncations was non-functional and suppressed [3H]ryanodine binding to RyR2-WT and RyR2-WT mediated store overload induced spontaneous Ca2+ release activity in HEK293 cells. The expression of these RyR2 truncating variants in HEK293 cells was markedly reduced compared with that of the full-length RyR2 WT protein. Our data indicate that C-terminal RyR2 truncating variants are non-functional and can exert a dominant negative impact on the function of the RyR2 WT protein through formation of heteromeric WT/truncation complex., (© 2023 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Background: PKA (protein kinase A)-mediated phosphorylation of cardiac RyR2 (ryanodine receptor 2) has been extensively studied for decades, but the physiological significance of PKA phosphorylation of RyR2 remains poorly understood. Recent determination of high-resolution 3-dimensional structure of RyR2 in complex with CaM (calmodulin) reveals that the major PKA phosphorylation site in RyR2, serine-2030 (S2030), is located within a structural pathway of CaM-dependent inactivation of RyR2. This novel structural insight points to a possible role of PKA phosphorylation of RyR2 in CaM-dependent inactivation of RyR2, which underlies the termination of Ca 2+ release and induction of cardiac Ca 2+ alternans., Methods: We performed single-cell endoplasmic reticulum Ca 2+ imaging to assess the impact of S2030 mutations on Ca 2+ release termination in human embryonic kidney 293 cells. Here we determined the role of the PKA site RyR2-S2030 in a physiological setting, we generated a novel mouse model harboring the S2030L mutation and carried out confocal Ca 2+ imaging., Results: We found that mutations, S2030D, S2030G, S2030L, S2030V, and S2030W reduced the endoplasmic reticulum luminal Ca 2+ level at which Ca 2+ release terminates (the termination threshold), whereas S2030P and S2030R increased the termination threshold. S2030A and S2030T had no significant impact on release termination. Furthermore, CaM-wild-type increased, whereas Ca 2+ binding deficient CaM mutant (CaM-M [a loss-of-function CaM mutation with all 4 EF-hand motifs mutated]), PKA, and Ca 2+ /CaMKII (CaM-dependent protein kinase II) reduced the termination threshold. The S2030L mutation abolished the actions of CaM-wild-type, CaM-M, and PKA, but not CaMKII, in Ca 2+ release termination. Moreover, we showed that isoproterenol and CaM-M suppressed pacing-induced Ca 2+ alternans and accelerated Ca 2+ transient recovery in intact working hearts, whereas CaM-wild-type exerted an opposite effect. The impact of isoproterenol was partially and fully reversed by the PKA inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide and the CaMKII inhibitor N-[2-[N-(4-chlorocinnamyl)-N-methylaminomethyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulfonamide individually and together, respectively. S2030L abolished the impact of CaM-wild-type, CaM-M, and N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide-sensitive component, but not the N-[2-[N-(4-chlorocinnamyl)-N-methylaminomethyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulfonamide-sensitive component, of isoproterenol.

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.