Ablation of two Major Phosphorylation Sites in RyR2 Alter Sarcoplamic Reticulum Calcium Handling and Increases the Propensity to Atrial Fibrillation

Rationale: Compelling data indicate that excessive Ca2+ leak from the sarcoplasmic reticulum (SR) contribute to atrial fibrillation (AF) pathogenesis. However, the role of RyR2 phosphorylation in Ca2+ leak in particular and cardiac diseases in general remains controversial. Aim: To investigate the effect of genetic inhibition of two major RyR2 phosphorylation sites on the channel function and atrial arrhythmogenesis. Methods and Results: We performed programmed electrical stimulation in anesthetized wild type (WT) mice and in mice harboring two RyR2 mutations that prevent PKA and CaMKII phosphorylation (S2808A and S2814A, respectively). Surprisingly, the double knock-in (DKI) mice exhibited increased incidence of AF episodes (5.2±3% WT vs 45±5% DKI, p<0.05). Confocal Ca2+ imaging showed that atrial myocytes from the DKI mouse have 30% increases in fractional Ca2+ release. Patch clamping in voltage mode coupled to confocal imaging revealed increased EC coupling gain in DKI myocytes (1.69±0.14 WT vs 2.53±0.28 DKI, at +10 mV). In I-current mode, DKI myocytes showed increased incidence of DADs after 2.0 Hz of pacing (5.94±0.10 min-1 WT vs 7.9±0.30 DKI min-1; p<0.05). Importantly, whereas the response of SR Ca2+ release to adrenergic stimulation was intact in DKI myocytes, the RyR2-mediated leak was higher (spark mass x spark frequency: 38.22±5.72 WT vs 115.1 ± 26.4 DKI; p<0.05). RyR2 restitution assays showed a faster recovery from refractoriness (τ = 170±12 WT vs 120±11 ms DKI, p<0.05). Moreover, [3H]ryanodine binding assays showed 2-fold increase of RyR2 activity in atrial homogenates from DKI at equivalent systolic and diastolic ]Ca2+]. Conclusion: Ablation of two major RyR2 phosphorylation sites increased the activity of the channel and was associated with increased susceptibility to AF.