Epac and phospholipase Cepsilon regulate Ca2+ release in the heart by activation of protein kinase Cepsilon and calcium-calmodulin kinase II.

Recently, we identified a novel signaling pathway involving Epac, Rap, and phospholipase C (PLC)e that plays a critical role in maximal β-adrenergic receptor (βAR) stimulation of Ca2+-induced Ca2+ release (CICR) in cardiac myocytes. Here we demonstrate that PLCe phosphatidylinositol 4,5-bisphosphate hydrolytic activity and PLCe-stimulated Rap1 GEF activity are both required for PLCe-mediated enhancement of sarcoplasmic reticulum Ca2+ release and that PLCe significantly enhances Rap activation in response to βAR stimulation in the heart. Downstream of PLCe hydrolytic activity, pharmacological inhibition of PKC significantly inhibited both βAR- and Epac-stimulated increases in CICR in PLCe+/+ myocytes but had no effect in PLCe–/– myocytes. βAR and Epac activation caused membrane translocation of PKCe in PLCe+/+ but not PLCe–/– myocytes and small interfering RNA-mediated PKCe knockdown significantly inhibited both βAR and Epac-mediated CICR enhancement. Further downstream, the Ca2+/calmodulin-dependent protein kinase II (CamKII) inhibitor, KN93, inhibited βAR- and Epac-mediated CICR in PLCe+/+ but not PLCe–/– myocytes. Epac activation increased CamKII Thr286 phosphorylation and enhanced phosphorylation at CamKII phosphorylation sites on the ryanodine receptor (RyR2) (Ser2815) and phospholamban (Thr17) in a PKC-dependent manner. Perforated patch clamp experiments revealed that basal and βAR-stimulated peak L-type current density are similar in PLCe+/+ and PLCe–/– 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, PLCe, PKCe, and CamKII downstream of βAR activation.