Résumé
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 Ca 2+ signalling in cardiac myocytes. In this study, we analysed the effects of Epac activation on Ca 2+ handling by using confocal microscopy in isolated adult rat cardiomyocytes. [Ca 2+ ] i transients were evoked by electrical stimulation and Ca 2+ 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 (I Ca), and Western blot to evaluate phosphorylated ryanodine receptor (RyR). [Ca 2+ ] i transients were slightly reduced by 10 μM 8-CPT (F/F 0 : 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. I Ca was unaltered by Epac activation, so this cannot explain the decreased [Ca 2+ ] i transients. Instead, a decrease in the sarcoplasmic reticulum (SR) Ca 2+ load underlies the decrease in the [Ca 2+ ] i transients. This decrease in the SR Ca 2+ load was provoked by the increase in the SR Ca 2+ leak induced by Epac activation. 8-CPT significantly increased Ca 2+ spark frequency (Ca 2+ sparks s −1 (100 μm) −1 : from 2.4 ± 0.6 to 6.9 ± 1.5, P < 0.01) while reducing their amplitude (F/F 0 : 1.8 ± 0.02 versus 1.6 ± 0.01, P < 0.001) in a Ca 2+ /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 Ca 2+ release and signalling in cardiac myocytes.