EPAC, PKC and CaMKII regulate local Ca2+ releases and cardiac pacemaker firing

National Institute On Aging

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Abstract

(1) We discovered that EPAC1 and EPAC2 are expressed in rabbit SANC (RT-qPCR). Selective inhibitors of EPAC1 (CE3F4) or EPAC2 (HJC-0350 or ESI-05) suppressed basal CaMKII activity (reflected in CaMKII autophosphorylation at Thr286/287 site, western blotting) and CaMKII-dependent phosphorylation of Ca2+-handling protein PLB (Thr17 site, western blotting) and RyR (Ser2815 site, immunostaining) and decreased the amplitude of L-type Ca2+ current (whole-cell patch-clamp technique). (2) Consistent with reduction of basal CaMKII-dependent phosphorylation, inhibition of EPAC1 or EPAC2 markedly decreased LCR characteristics, extended the LCR period (interval between action potential-induced Ca2+ transient and subsequent LCR) and increased the spontaneous SANC cycle length (confocal microscopy, Ca2+ indicator Fluo-3AM). Overall, the inhibition of either EPAC1 or EPAC2 produced ~30% decrease in the spontaneous SANC firing rate each, indicating that both EPAC1 and EPAC2 are activated and accelerate spontaneous SANC firing in the basal state. In contrast, activation of Epac by 8-pCPT-2’-O-Me-cAMP augmented LCR parameters, shortened the LCR period and, consistent with the coupled-clock pacemaker theory, accelerated the spontaneous beating rate of SANC by ~20%. (3) To assess whether EPAC employes Epac-PLC-PKC-CaMKII pathway we, first, used RT-qPCR to confirm expression of PLC in the rabbit SA node. Inhibition of PLC by U-73122, but not its inactive analog U-73343, stopped spontaneous firing of intact rabbit SANC, indicating a key role of basal PLC activation for cardiac pacemaker function. PLC regulates physiological responses via two downstream targets including inositol-1, 4, 5-trisphosphate (IP3), which mediates the release of Ca2+ via intracellular Ca2+ stores, and diacylglycerol (DAG), which activates protein kinase C (PKC). Membrane-permeable IP3 receptor antagonist 2-APB produced no changes in LCR parameters or spontaneous firing of rabbit SANC, suggesting that IP3 receptor-mediated Ca2+ release is not a target of EPAC-PLC pathway. In contrast, PKC inhibition by broad-spectrum PKC inhibitors Bis-I or calphostin-C suppressed or even arrested spontaneous firing of rabbit SANC, indicating essential role of PKC for cardiac pacemaker function. To establish whether in SANC EPAC-PLC-mediated PKC signaling operated via modulation of CaMKII activity we studied effects of Bis-I on CaMKII activity and CaMKII-dependent phosphorylation of Ca2+ cycling proteins (PLB and RyR). Bis-I significantly (P<0.05) reduced basal CaMKII activity (reflected in autophosphorylation of CaMKII) by ~20%, and this was accompanied by marked reduction of CaMKII-dependent phosphorylation of both PLB at Thr17 site and RyR at Ser2815 site. Therefore, EPAC-PLC pathway employs PKC as a downstream target of EPAC-PLC-PKC pathway to modulate CaMKII activation and CaMKII-dependent protein phosphorylation to regulate normal spontaneous firing of SANC. Next, we studied whether EPAC-PI3K-Akt-NOS1-CaMKII pathway also participates in the regulation of spontaneous SANC firing. We employed a potent, cell-permeable, multi-targeted PI3K inhibitor, PI-103, which inhibits all PI3K isoforms expressed in the heart. Prolonged ~10-minute superfusion with PI-103, however, produced no statistically significant changes either in LCR characteristics or LCR period or spontaneous AP firing rate, suggesting that PI3K is likely not a downstream target of EPAC in SANC. The removal of PI3K from the signaling cascade linking basal EPAC activation to CaMKII-dependent modulation of spontaneous SANC firing also disqualified activation of CaMKII via EPAC-PI3K-Akt-NOS1 pathway.