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). Basal activation of both Epac1 and Epac2 has been confirmed in the experiments using selective Epac1 and Epac2 inhibitors. Specifically, inhibition of Epac1 by CE3F4 or Epac2 by HJC-0350 produced marked decrease in LCR parameters and increase in the LCR periods accompanied by an increase in the spontaneous SANC cycle lengths (confocal microscopy, Ca2+ indicator Fluo-3AM). Overall, 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. (2) We found that the main targets, regulated by basal Epac activation, include L-type Ca2+ current and phosphorlamban (PLB), i.e. PLB phosphorylation at CaMKII-dependent Thr17 site, which modulates kinetics of Ca2+ pumping into the SR. (3) To evaluate downstream targets of EPAC we, first, studied Epac-PLC-PKC-CaMKII pathway. 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. To regulate physiological responses PLC employs two downstream targets including inositol-1, 4, 5-trisphosphate (IP3) and diacylglycerol (DAG) dependent signaling. IP3 mediates the release of Ca2+ via intracellular Ca2+ stores, and DAG activates PKC. To verify role of IP3 receptor Ca2+ release we employed membrane-permeable antagonist of IP3 receptor Ca2+ release 2-APB, which produced no changes in the parameters of LCRs or amplitudes of AP-induced Ca2+ transients or spontaneous firing of rabbit SANC. Therefore, IP3 receptor-mediated Ca2+ release is likely 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. 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.