Protein Kinase A Phosphorylation of the Ryanodine Receptor Does Not Affect Calcium Sparks in Mouse Ventricular Myocytes

Ryanodine receptor (RyR) phosphorylation by protein kinase A (PKA) may be important in modulating resting sarcoplasmic reticulum (SR) Ca release, especially in heart failure. However, clear cellular data on PKA-dependent modulation of cardiac RyRs is limited because of difficulty in distinguishing between PKA effects on RyR, phospholamban (PLB), and Ca current. To clarify this, we measured resting Ca sparks in streptolysin-O permeabilized ventricular myocytes from wild-type (WT) and PLB knockout (PLB-KO) mice and transgenic mice expressing only double-mutant PLB (PLB-DM) that lacks the regulatory phosphorylation sites (S16A/T17A). In WT myocytes, cAMP dramatically increased Ca spark frequency (CaSpF) by 2- and 3-fold when [Ca] was clamped at 50 and 10 nmol/L (and the SR Ca content also rose by 40% and 50%). However, in PLB-KO and PLB-DM, neither CaSpF nor SR Ca load was changed by the addition of 10 μmol/L cAMP (even with phosphatase inhibition). PKA activation also increased Ca spark amplitude, duration, and width in WT, but not in PLB-KO or PLB-DM. RyR phosphorylation was confirmed by measurements of P incorporation on immunoprecipitated RyR. In intact resting myocytes, PKA activation increased CaSpF 2.8-fold in WT, but not in PLB-KO, confirming results in permeabilized myocytes. We conclude that the PKA-dependent increase in myocyte CaSpF and size is entirely attributable to PLB phosphorylation and consequent enhanced SR Ca load. PKA does not seem to have any appreciable effect on resting RyR function in these ventricular myocytes. Moreover, the data provide compelling evidence that elevated intra-SR [Ca] increases RyR gating independent of cytosolic [Ca] (which was clamped).