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 (I_Ca), 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. I_Ca 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.