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

Abstract

Recently, we identified a novel signaling pathway involving Epac, Rap, and phospholipase C (PLC)ε 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 PLCε phosphatidylinositol 4,5-bisphosphate hydrolytic activity and PLCε-stimulated Rap1 GEF activity are both required for PLCε-mediated enhancement of sarcoplasmic reticulum Ca2+ release and that PLCε significantly enhances Rap activation in response to βAR stimulation in the heart. Downstream ofPLCε hydrolytic activity, pharmacological inhibition of PKC significantly inhibited both βAR- and Epac-stim-ulated increases in CICR in PLCε+/+ myocytes but had no effect in PLCε-/- myocytes. βAR and Epac activation caused membrane translocation of PKCε in PLCε+/+ but not PLCε-/- myocytes and small interfering RNA-mediated PKCε knockdown significantly inhibited both βAR and Epac-mediated CICR enhancement. Further downstream, the Ca2+/calmodulin-de-pendent protein kinase II (CamKII) inhibitor, KN93, inhibited βAR- and Epac-mediated CICR in PLCε+/+ but not PLCε-/- myocytes. Epac activation increased CamKII Thr286 phospho-rylation and enhanced phosphorylation at CamKII phosphoryl-ation sites on the ryanodine receptor (RyR2) (Ser2815) and phos-pholamban (Thr17) in a PKC-dependent manner. Perforated patch clamp experiments revealed that basal and βAR-stimulated peak L-type current density are similar in PLCε+/+ and PLCε-/- myocytes suggesting that control of sarcoplasmic reticulum Ca2+ release, rather than Ca 2+ influx through L-type Ca2+ channels, is the target of regulation of a novel signal transduction pathway involving sequential activation of Epac, PLCε, PKCε, and CamKII downstream of εAR activation. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.