Dual ablation of the RyR2-Ser2808 and RyR2-Ser2814 sites increases propensity for pro-arrhythmic spontaneous Ca 2+ releases

During exercise or stress, the sympathetic system stimulates cardiac contractility via β-adrenergic receptor (β-AR) activation, resulting in phosphorylation of the cardiac ryanodine receptor (RyR2). Three RyR2 phosphorylation sites have taken prominence in excitation-contraction coupling: S2808 and S2030 are described as protein kinase A specific and S2814 as a Ca /calmodulin kinase type-2-specific site. To examine the contribution of these phosphosites to Ca signalling, we generated double knock-in (DKI) mice in which Ser2808 and Ser2814 phosphorylation sites have both been replaced by alanine (RyR2-S2808A/S2814A). These mice did not exhibit an overt phenotype. Heart morphology and haemodynamic parameters were not altered. However, they had a higher susceptibility to arrhythmias. We performed confocal Ca imaging and electrophysiology experiments. Isoprenaline was used to stimulate β-ARs. Measurements of Ca waves and latencies in myocytes revealed an increased propensity for spontaneous Ca releases in DKI myocytes, both in control conditions and during β-AR stimulation. In DKI cells, waves were initiated from a lower threshold concentration of Ca inside the sarcoplasmic reticulum, suggesting higher Ca sensitivity of the RyRs. The refractoriness of Ca spark triggering depends on the Ca sensitivity of the RyR2. We found that RyR2-S2808A/S2814A channels were more Ca sensitive in control conditions. Isoprenaline further shortened RyR refractoriness in DKI cardiomyocytes. Together, our results suggest that ablation of both the RyR2-Ser2808 and RyR2-S2814 sites increases the propensity for pro-arrhythmic spontaneous Ca releases, as previously suggested for hyperphosphorylated RyRs. Given that the DKI cells present a full response to isoprenaline, the data suggest that phosphorylation of Ser2030 might be sufficient for β-AR-mediated sensitization of RyRs. KEY POINTS: Phosphorylation of cardiac sarcoplasmic reticulum Ca -release channels (ryanodine receptors, RyRs) is involved in the regulation of cardiac function. Ablation of both the RyR2-Ser2808 and RyR2-Ser2814 sites increases the propensity for pro-arrhythmic spontaneous Ca releases, as previously suggested for hyperphosphorylated RyRs. The intra-sarcoplasmic reticulum Ca threshold for spontaneous Ca wave generation is lower in RyR2-double-knock-in cells. The RyR2 from double-knock-in cells exhibits increased Ca sensitivity. Phosphorylation of Ser2808 and Ser2814 might be important for basal activity of the