CONICET | Buscador de Institutos y Recursos Humanos

In the myocardium, the role of the exchange protein directly activated by cAMP (EPAC) in the regulation of Ca2+ homeostasis and contractility is controversial. With the aim to elucidate the reason for the discrepancies, we studied the effects of the selective activator of EPAC, 8-(-4-chlorophenilthio)-2-O-methyladenisine-3-5-cyclic monophosphate (8-CPT), in isolated adult rat myocytes, loaded with Fura-2AM and stimulated at 1 Hz, at different [Ca2+]o and in perfused rat hearts. After 10 min exposure to 10 μM 8-CPT, fractional sarcomere shortening increased at 0.5 mM [Ca2+]o, did not change at 1 mM [Ca2+]o, and decreased at higher [Ca2+]o (1.8 mM). These effects were associated with similar changes in steady-state twitch [Ca2+]i transient amplitudes and SR Ca2+ contents, measured by caffeine pulses, suggesting no alteration in myofilament Ca2+ sensitivity. 8-CPT-induced a decrease in half relaxation time at 0.5 and 1 mM [Ca2+]o, with no changes at 1.8 mM [Ca2+]o. Diastolic [Ca2+]i rose in a time dependent manner after 10 min exposure to 8-CPT. This effect, prevented by PKC inhibition with calphostin C, could be the consequence of the increased Ca2+ spark frequency, determined in Fluo-4-AM-loaded myocytes, observed at the 3 different [Ca2+]o. In line, with the myocyte results, experiments in perfused rat heart revealed an 8-CPT-induced increase in end diastolic pressure. The effects of 8-CPT were associated with an enhancement of the phosphorylation of CaMKII and its substrates: Ser2815 of RyR2 and Thr17 of phospholamban, which were prevented by the CaMKII inhibitor KN-93 and calphostin C. The results suggest that the effects of EPAC activation, via a PKC/CaMKII pathway, depend on the balance between SR Ca2+ reuptake and release which is modified by the Ca2+ supply to the cell.