The cAMP binding protein Epac modulates Ca2+ sparks by a Ca2+/calmodulin kinase signalling pathway in rat cardiac myocytes

Abstract

cAMP is a powerful second messenger whose known general effector is protein kinase A (PKA). The identification of a cAMP binding protein, Epac, raises the question of its role in Ca2+ signalling in cardiac myocytes. In this study, we analysed the effects of Epac activation on Ca2+ handling by using confocal microscopy in isolated adult rat cardiomyocytes. [Ca2+]i transients were evoked by electrical stimulation and Ca2+ sparks were measured in quiescent myocytes. Epac was selectively activated by the cAMP analogue 8-(4-chlorophenylthio)-2′-O- methyladenosine-3′,5′-cyclic monophosphate (8-CPT). Patch-clamp was used to record the L-type calcium current (ICa), and Western blot to evaluate phosphorylated ryanodine receptor (RyR). [Ca2+]i transients were slightly reduced by 10 μm 8-CPT (F/F0: decreased from 4.7 ± 0.5 to 3.8 ± 0.4, P < 0.05), an effect that was boosted when cells were previously infected with an adenovirus encoding human Epac. ICa was unaltered by Epac activation, so this cannot explain the decreased [Ca2+]i transients. Instead, a decrease in the sarcoplasmic reticulum (SR) Ca2+ load underlies the decrease in the [Ca2+]i transients. This decrease in the SR Ca2+ load was provoked by the increase in the SR Ca2+ leak induced by Epac activation. 8-CPT significantly increased Ca2+ spark frequency (Ca2+ sparks s-1 (100 μm)-1: from 2.4 ± 0.6 to 6.9 ± 1.5, P < 0.01) while reducing their amplitude ( F/F0: 1.8 ± 0.02 versus 1.6 ± 0.01, P < 0.001) in a Ca2+/calmodulin kinase II (CaMKII)-dependent and PKA-independent manner. Accordingly, we found that Epac increased RyR phosphorylation at the CaMKII site. Altogether, our data reveal a new signalling pathway by which cAMP governs Ca2+ release and signalling in cardiac myocytes. © 2007 The Authors. Journal compilation © 2007 The Physiological Society.