Ablation of three major phospho-sites in RyR2 preserves the global adrenergic response but creates an arrhythmogenic substrate.

ABSTRACT

Background: Ryanodine receptor 2 (RyR2) is one of the first substrates undergoing phosphorylation upon catecholaminergic stimulation. Yet, the role of RyR2 phosphorylation in the adrenergic response remains debated. To date, three residues in RyR2 are known to undergo phosphorylation upon adrenergic stimulation. We generated a model of RyR2 phospho-ablation of all three canonical phospho-sites (RyR2-S2031A/S2808A/S2814A, triple phospho-mutant, TPM) to elucidate the role of phosphorylation at these residues in the adrenergic response. Methods: Cardiac structure and function, cellular Ca 2+ dynamics and electrophysiology, and RyR2 channel activity both under basal conditions and under isoproterenol (Iso) stimulation were systematically evaluated. We used echocardiography and electrocardiography in anesthetized mice, single-cell Ca 2+ imaging and whole-cell patch clamp in isolated adult cardiomyocytes, and biochemical assays. Results: Iso stimulation produced normal chronotropic and inotropic responses in TPM mice as well as an increase in the global Ca 2+ transients in isolated cardiomyocytes. Functional studies revealed fewer Ca 2+ sparks in permeabilized TPM myocytes, and reduced RyR2-mediated Ca 2+ leak in intact myocytes under Iso stimulation, suggesting that the canonical sites may regulate RyR2-mediated Ca 2+ leak. TPM mice also displayed increased propensity for arrhythmia. TPM myocytes were prone to develop early afterdepolarizations (EADs), which were abolished by chelating intracellular Ca 2+ with EGTA, indicating that EADs require SR Ca 2+ release. EADs were also blocked by a low concentration of tetrodotoxin, further suggesting reactivation of the sodium current ( I Na ) as the underlying cause. Conclusion: Phosphorylation of the three canonical residues on RyR2 may not be essential for the global adrenergic responses. However, these sites play a vital role in maintaining electrical stability during catecholamine stimulation by fine-tuning RyR2-mediated Ca 2+ leak. These findings underscore the importance of RyR2 phosphorylation and a finite diastolic Ca 2+ leak in maintaining electrical stability during catecholamine stimulation.