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(2+)/calmodulin kinase type-2-specific site. To examine the contribution of these phosphosites to Ca(2+) signalling, we generated double knock-in (DKI) mice in which Ser2808 and Ser2814 phosphorylation sites have both been replaced by alanine (RyR2-S2808A/S2814A).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(2+) imaging and electrophysiology experiments. Isoprenaline was used to stimulate β-ARs. Measurements of Ca(2+) waves and latencies in myocytes revealed an increased propensity for spontaneous Ca(2+) 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(2+) inside the sarcoplasmic reticulum, suggesting higher Ca(2+) sensitivity of the RyRs. The refractoriness of Ca(2+) spark triggering depends on the Ca(2+) sensitivity of the RyR2. We found that RyR2-S2808A/S2814A channels were more Ca(2+) 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(2+) 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(2+)-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(2+) releases, as previously suggested for hyperphosphorylated RyRs. The intra-sarcoplasmic reticulum Ca(2+) threshold for spontaneous Ca(2+) wave generation is lower in RyR2-double-knock-in cells. The RyR2 from double-knock-in cells exhibits increased Ca(2+) sensitivity. Phosphorylation of Ser2808 and Ser2814 might be important for basal activity of the channel. Phosphorylation of Ser2030 might be sufficient for a β-adrenergic response.