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

Abstract

Ryanodine receptor (RyR) phosphorylation by protein kinase A (PKA) may be important in modulating resting sarcoplasmic reticulum (SR) Ca 2+ 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 2+ current. To clarify this, we measured resting Ca 2+ 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 2+ spark frequency (CaSpF) by 2- and 3-fold when [Ca 2+ ] was clamped at 50 and 10 nmol/L (and the SR Ca 2+ content also rose by 40% and 50%). However, in PLB-KO and PLB-DM, neither CaSpF nor SR Ca 2+ load was changed by the addition of 10 μmol/L cAMP (even with phosphatase inhibition). PKA activation also increased Ca 2+ spark amplitude, duration, and width in WT, but not in PLB-KO or PLB-DM. RyR phosphorylation was confirmed by measurements of 32 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 2+ 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 2+ ] increases RyR gating independent of cytosolic [Ca 2+ ] (which was clamped).