The effect of PKA-mediated phosphorylation of ryanodine receptor on SR Ca2+ leak in ventricular myocytes

Abstract

Functional impact of cardiac ryanodine receptor (type 2 RyR or RyR2) phosphorylation by protein kinase A (PKA) remains highly controversial. In this study, we characterized a functional link between PKA-mediated RyR2 phosphorylation level and sarcoplasmic reticulum (SR) Ca²⁺ release and leak in permeabilized rabbit ventricular myocytes. Changes in cytosolic [Ca²⁺] and intra-SR [Ca²⁺]_SR were measured with Fluo-4 and Fluo-5N, respectively. Changes in RyR2 phosphorylation at two PKA sites, serine-2031 and -2809, were measured with phospho-specific antibodies. cAMP (10μM) increased Ca²⁺ spark frequency approximately two-fold. This effect was associated with an increase in SR Ca²⁺ load from 0.84 to 1.24mM. PKA inhibitory peptide (PKI; 10μM) abolished the cAMP-dependent increase of SR Ca²⁺ load and spark frequency. When SERCA was completely blocked by thapsigargin, cAMP did not affect RyR2-mediated Ca²⁺ leak. The lack of a cAMP effect on RyR2 function can be explained by almost maximal phosphorylation of RyR2 at serine-2809 after sarcolemma permeabilization. This high RyR2 phosphorylation level is likely the consequence of a balance shift between protein kinase and phosphatase activity after permeabilization. When RyR2 phosphorylation at serine-2809 was reduced to its "basal" level (i.e. RyR2 phosphorylation level in intact myocytes) using kinase inhibitor staurosporine, SR Ca²⁺ leak was significantly reduced. Surprisingly, further dephosphorylation of RyR2 with protein phosphatase 1 (PP1) markedly increased SR Ca²⁺ leak. At the same time, phosphorylation of RyR2 at serine 2031 did not significantly change under identical experimental conditions. These results suggest that RyR2 phosphorylation by PKA has a complex effect on SR Ca²⁺ leak in ventricular myocytes. At an intermediate level of RyR2 phosphorylation SR Ca²⁺ leak is minimal. However, complete dephosphorylation and maximal phosphorylation of RyR2 increases SR Ca²⁺ leak.

Keywords: Ca(2+) spark; Cardiomyocyte; Protein kinase A; Protein phosphatases; Ryanodine receptor; Sarcoplasmic reticulum Ca(2+) leak.