Epac and Phospholipase Cε Regulate Ca2+ Release in the Heart by Activation of Protein Kinase Cε and Calcium-Calmodulin Kinase II

Recently, we identified a novel signaling pathway involving Epac, Rap, and phospholipase C (PLC)epsilon that plays a critical role in maximal beta-adrenergic receptor (beta AR) stimulation of Ca2+-induced Ca2+ release (CICR) in cardiac myocytes. Here we demonstrate that PLC epsilon phosphatidylinositol 4,5-bisphosphate hydrolytic activity and PLC epsilon-stimulated Rap1 GEF activity are both required for PLC epsilon-mediated enhancement of sarcoplasmic reticulum Ca2+ release and that PLC epsilon significantly enhances Rap activation in response to beta AR stimulation in the heart. Downstream of PLC epsilon hydrolytic activity, pharmacological inhibition of PKC significantly inhibited both beta AR- and Epac-stimulated increases in CICR in PLC epsilon(+/+) myocytes but had no effect in PLC epsilon(-/-) myocytes. beta AR and Epac activation caused membrane translocation of PKC epsilon in PLC epsilon(+/+) but not PLC epsilon(-/-) myocytes and small interfering RNA- mediated PKC epsilon knockdown significantly inhibited both beta AR and Epac-mediated CICR enhancement. Further downstream, the Ca2+/calmodulin-dependent protein kinase II (CamKII) inhibitor, KN93, inhibited beta AR- and Epac-mediated CICR in PLC epsilon(+/+) but not PLC epsilon(-/-) myocytes. Epac activation increased CamKII Thr(286) phosphorylation and enhanced phosphorylation at CamKII phosphorylation sites on the ryanodine receptor (RyR2) (Ser(2815)) and phospholamban (Thr(17)) in a PKC-dependent manner. Perforated patch clamp experiments revealed that basal and beta AR-stimulated peak L-type current density are similar in PLC epsilon(+/+) and PLC epsilon(-/-) myocytes suggesting that control of sarcoplasmic reticulum Ca2+ release, rather than Ca2+ influx through L-type Ca2+ channels, is the target of regulation of a novel signal transduction pathway involving sequential activation of Epac, PLC epsilon, PKC epsilon, and CamKII downstream of beta AR activation.