PKA phosphorylation of cardiac ryanodine receptor modulates SR luminal Ca2+ sensitivity

During physical exercise and stress, the sympathetic system stimulates cardiac contractility via β-adrenergic receptor activation, resulting in protein kinase A (PKA)-mediated phosphorylation of the cardiac ryanodine receptor, RyR2, at Ser2808. Hyperphosphorylation of RyR2-S2808 has been proposed as a mechanism contributing to arrhythmogenesis and heart failure. However, the role of RyR2 phosphorylation during β-adrenergic stimulation remains controversial. We examined the contribution of RyR2-S2808 phosphorylation to altered excitation-contraction coupling and Ca(2+) signaling using an experimental approach at the interface of molecular and cellular levels and a transgenic mouse with ablation of the RyR2-S2808 phosphorylation site (RyR2-S2808A). Experimentally challenging the communication between L-type Ca(2+) channels and RyR2 led to a spatiotemporal de-synchronization of RyR2 openings, as visualized using confocal Ca(2+) imaging. β-Adrenergic stimulation re-synchronized RyR2s, but less efficiently in RyR2-S2808A than in control cardiomyocytes, as indicated by comprehensive analysis of RyR2 activation. In addition, spontaneous Ca(2+) waves in RyR2-S2808A myocytes showed significantly slowed propagation and complete absence of acceleration during β-adrenergic stress, unlike wild type cells. Single channel recordings revealed an attenuation of luminal Ca(2+) sensitivity in RyR2-S2808A channels upon addition of PKA. This suggests that phosphorylation of RyR2-S2808 may be involved in RyR2 modulation by luminal (intra-SR) Ca(2+) ([Ca(2+)](SR)). We show here by three independent experimental approaches that PKA-dependent RyR2-S2808 phosphorylation plays significant functional roles at the subcellular level, namely, Ca(2+) release synchronization, Ca(2+) wave propagation and functional adaptation of RyR2 to variable [Ca(2+)](SR). These results indicate a direct mechanistic link between RyR2 phosphorylation and SR luminal Ca(2+) sensing.