Preventing the phosphorylation of RyR2 at canonical sites reduces Ca2+ leak and promotes arrhythmia by reactivating the INa current

Phosphorylation of specific sites in ryanodine receptor 2 (RyR2), a major cardiac Ca²⁺ channel, increases channel activity and promotes pathological sarcoplasmic reticulum Ca²⁺ leak and arrhythmia. RyR2 is phosphorylated during adrenergic stimulation, but the role of this phosphorylation remains debated. In this study, we generated a mouse model with phospho-ablation of the three canonical phosphorylation sites in RyR2 (S2031A/S2808A/S2814A, triple phospho-mutant (TPM)) to determine their role in the adrenergic response. TPM mice have normal basal cardiac structure and function. Isoproterenol stimulation produced normal chronotropic and inotropic responses in TPM mice and cardiomyocytes, which also showed reduced RyR2-mediated Ca²⁺ leak. However, TPM mice were susceptible to cardiac arrhythmias. These arrhythmias required systolic Ca²⁺ release and were induced by the reactivation of I_Na and early afterdepolarizations. We propose that phosphorylation of these residues in RyR2 is dispensable for chronotropy and inotropy; however, they maintain electrical stability during adrenergic stimulation by modulating a physiological RyR2-mediated Ca²⁺ leak.