Abstract Fri101: Mice with phospho-substitutions at a novel ARVC-associated residue in ryanodine receptor 2 show normal calcium handling.

Background: Ryanodine receptor 2 (RyR2) is a critical ion channel for heart function, regulated by multiple phosphorylation sites in both healthy and diseased hearts. A recent study identified increased phosphorylation of RyR2 at T2810 , a novel site, in a mouse model of arrhythmogenic right ventricular cardiomyopathy (ARVC). Our aim was to determine the impact T2810 phosphorylation on cardiac function and RyR2 regulation. Methods: We generated two CRISPR/Cas9-engineered mouse models with phospho-ablation (T2810A) and phospho-mimetic (T2810D) substitutions in RyR2 channels. Additionally, recombinant channels with the same substitutions were expressed in HEK293 cells. We assessed cardiac function, Ca 2+ handling and RyR2 activity using electrocardiography, confocal Ca 2+ imaging and [ 3 H]ryanodine binding assays. Results: [ 3 H]ryanodine binding assays in recombinant RyR2 channels revealed no differences in the Ca 2+ sensitivity or maximum activity in T2810A or T2810D channels. In vivo , T2810A mice showed no significant differences in electrocardiographic parameters under basal conditions or following a pharmacological arrhythmia challenge (2mg/kg epinephrine and 120mg/kg caffeine). Studies on T2810D are ongoing. Confocal imaging in isolated ventricular myocytes revealed no significant differences in Ca 2+ transients (1-3 Hz stimulation) or in the sarcoplasmic reticulum Ca 2+ load following a pulse of 10 mM caffeine. Conclusion: T2810 is located within RyR2 “phosphorylation hotspot,” alongside well-studied phosphorylation sites S2808 and S2814. Our data suggests that T2810 may not be critical to regulate cardiac function or RyR2 activity in basal conditions or during adrenergic stimulation. However, further investigation is necessary to elucidate the potential role of T2810 in disease, particularly ARVC.