Regulation of cardiac ryanodine receptor function by the cyclic-GMP dependent protein kinase G

Abstract

Background: The cGMP-dependent protein kinase G (PKG) phosphorylates the cardiac ryanodine receptor (RyR2) in vitro. We aimed to determine whether modulation of endogenous PKG alters RyR2-mediated spontaneous Ca²⁺ release and whether this effect is linked to a change in RyR2 phosphorylation. Methods: & Results: Human embryonic kidney (HEK293) cells with inducible RyR2 expression were treated with the cGMP analogue 8-Br-cGMP (100 μM) to activate endogenous PKG. In cells transfected with luminal Ca²⁺ sensor, D1ER, PKG activation significantly reduced the threshold for RyR2-mediated spontaneous Ca²⁺ release (93.9 ± 0.4% of store size with vehicle vs. 91.7 ± 0.8% with 8-Br-cGMP, P = 0.04). Mutation of the proposed PKG phosphorylation sites, S2808 and S2030, either individually or as a combination, prevented the decrease in Ca²⁺ release threshold induced by endogenous PKG activation. Interestingly, despite a functional dependence on expression of RyR2 phosphorylation sites, 8-Br-cGMP activation of PKG did not promote a detectable change in S2808 phosphorylation (P = 0.9). Paradoxically, pharmacological inhibition of PKG with KT 5823 (1 μM) also reduced the threshold for spontaneous Ca²⁺ release through RyR2 without affecting S2808 phosphorylation. Silencing RNA knockdown of endogenous PKG expression also had no quantifiable effect on RyR2 S2808 phosphorylation (P = 0.9). However, unlike PKG inhibition with KT 5823, PKG knockdown did not alter spontaneous Ca²⁺ release propensity or luminal Ca²⁺ handling. Conclusion: In an intact cell model, activation of endogenous PKG reduces the threshold for RyR2-mediated spontaneous Ca²⁺ release in a manner dependent on the RyR2 phosphorylation sites S2808 and S2030. This study clarifies the regulation of RyR2 Ca²⁺ release by endogenous PKG and functionally implicates the role of RyR2 phosphorylation.