Epac-mediated Activation of Phospholipase Cɛ Plays a Critical Role in β-Adrenergic Receptor-dependent Enhancement of Ca2+ Mobilization in Cardiac Myocytes

Emily A Oestreich,Huan Wang,Sundeep Malik,Katherine A Kaproth-Joslin,Burns C Blaxall,Grant G Kelley,Robert T Dirksen,Alan V Smrcka

doi:10.1074/jbc.m608495200

Abstract

Recently we demonstrated that PLC(epsilon) plays an important role in beta-adrenergic receptor (betaAR) stimulation of Ca(2+)-induced Ca(2+) release (CICR) in cardiac myocytes. Here we have reported for the first time that a pathway downstream of betaAR involving the cAMP-dependent Rap GTP exchange factor, Epac, and PLC(epsilon) regulates CICR in cardiac myocytes. To demonstrate a role for Epac in the stimulation of CICR, cardiac myocytes were treated with an Epac-selective cAMP analog, 8-4-(chlorophenylthio)-2'-O-methyladenosine-3',5'-monophosphate (cpTOME). cpTOME treatment increased the amplitude of electrically evoked Ca(2+) transients, implicating Epac for the first time in cardiac CICR. This response is abolished in PLC(epsilon)(-/-) cardiac myocytes but rescued by transduction with PLC(epsilon), indicating that Epac is upstream of PLC(epsilon). Furthermore, transduction of PLC(epsilon)(+/+) cardiac myocytes with a Rap inhibitor, RapGAP1, significantly inhibited isoproterenol-dependent CICR. Using a combination of cpTOME and PKA-selective activators and inhibitors, we have shown that betaAR-dependent increases in CICR consist of two independent components mediated by PKA and the novel Epac/(epsilon) pathway. We also show that Epac/PLC(epsilon)-dependent effects on CICR are independent of sarcoplasmic reticulum loading and Ca(2+) clearance mechanisms. These data define a novel endogenous PKA-independent betaAR-signaling pathway through cAMP-dependent Epac activation, Rap, and PLC(epsilon) that enhances intracellular Ca(2+) release in cardiac myocytes.

Highlights

(␤AR)2 in cardiac myocytes [1, 2]
We show here that cAMP-stimulated Epac/Rap1-dependent activation of phospholipase C (PLC)⑀ acts in concert with the classical cAMP-dependent activation of PKA to facilitate ␤AR-mediated enhancement of intracellular Ca2ϩ release from the sarcoplasmic reticulum in cardiac myocytes
We recently reported a significant decrease in ␤-adrenergic receptor-dependent enhancement of electrically evoked Ca2ϩ release in ventricular myocytes isolated from PLC⑀Ϫ/Ϫ mice [3] and hypothesized that PLC⑀ plays a direct role in the ␤ARsignaling cascade

Summary

Introduction

(␤AR)2 in cardiac myocytes [1, 2]. Activation of ␤AR activates Gs and adenylyl cyclase resulting in the production of cAMP and subsequent activation of protein kinase A, which phosphorylates key components of the calcium handling and contractile machinery. Isolated myocytes from PLC⑀Ϫ/Ϫ mice exhibit decreased isoproterenol-dependent enhancement of electrically evoked Ca2ϩ release in the absence of effects on ␤AR density or cAMP generation. We show here that cAMP-stimulated Epac/Rap1-dependent activation of PLC⑀ acts in concert with the classical cAMP-dependent activation of PKA to facilitate ␤AR-mediated enhancement of intracellular Ca2ϩ release from the sarcoplasmic reticulum in cardiac myocytes.

Results

Conclusion