Cardiac Ryanodine Receptor Phosphorylation at Ser2808 is Involved in Intra-SR Calcium Sensing

Abstract

During stress, cardiac function is enhanced via β-adrenergic stimulation. Phosphorylation of cardiac ryanodine receptors (RyR2) has been proposed to lead to increased Ca2+ release from the sarcoplasmic reticulum (SR), but experimental findings are controversial. Using cardiomyocytes lacking the PKA phosphorylation site Ser2808 on RyR2 we previously observed that this site contributes to improved spatio-temporal synchronization of Ca2+ release during EC-coupling under β-adrenergic stimulation, presumably by a Ca2+ sensing mechanism inside the SR. Here, we further tested the hypothesis that Ser2808 phosphorylation modulates RyR2 function via its dependence on [Ca2+]SR. Cardiomyocytes from control and S2808A mice were loaded with fluo-3-AM and Ca2+ was imaged with confocal line-scans. Ca2+ waves were induced by increasing external [Ca2+] to 10mM and analyzed in control and during β-stimulation with isoproterenol (1µM). In control conditions Ca2+ transients during a wave showed a comparable amplitude and time course. However, wave speed was reduced by 25% in S2808A cells, possibly related to a lack of background phosphorylation at this site. While ISO increased wave speed by 15% in control cells (from 98 to 113µm/s), it remained unaffected in S2808A cells (from 73 to 75µm/s). This situation could be reproduced in control cells preincubated with the PKA inhibitor H89 or the CaMKII inhibitor KN93, indicating a complex regulation of CICR that is pharmacologically difficult to dissect in intact cells. Taken together our findings in S2808A myocytes reveal a lack of Ca2+ release synchronization (via an intra-SR Ca2+ sensing mechanism) and an ablation of Ca2+ wave speed modulation (presumably by the same mechanism) by β-adrenergic stimulation. Thus, Ser2808 phosphorylation may change the sensitivity of the RyR2 for intra-SR Ca2+. Hyperphosphorylation of Ser2808, as in failing hearts, may thereby render RyR2 hypersensitive and contribute to increased Ca2+ leak.