cAMP-Activated EPAC Signaling Is an Integral Component of Cardiac Pacemaker Cell Automaticity

BACKGROUND: Spontaneous automaticity of sinoatrial node cells (SANCs) is driven by a system that couples ion channels, membrane clock and Ca 2+ clock, the sarcoplasmic reticulum generated LCRs (local subsarcolemmal Ca 2+ releases). Although LCRs are critically dependent on high basal cAMP and both PKA (protein kinase A)- and CaMKII (Ca2+/calmodulin dependent protein kinase II)-dependent protein phosphorylation, the link between cAMP and CaMKII remains unclear. Here, we tested a hypothesis that high cAMP activates EPAC (exchange protein directly activated by cAMP) which increases basal CaMKII activity, reinforcing the coupled clock pacemaker system, to boost LCRs and accelerate spontaneous SANC firing. METHODS: Real-time quantitative polymerase chain reaction, Western blot, immunostaining, whole-cell patch clamp, and line-scan confocal microscopy were employed to study EPAC-dependent regulation of rabbit SANC firing. RESULTS: Both EPAC isoforms were expressed and active in SANC. Selective inhibition of EPAC1 (CE3F4) or EPAC2 (HJC0350) similarly suppressed basal CaMKII activity, CaMKII-dependent phosphorylation of Ca 2+ -cycling proteins (PLB [phospholamban] and RyR [ryanodine receptors]) and reduced the amplitude of L-type Ca 2+ current. EPAC1 and EPAC2 inhibitors significantly decreased LCR number, size, and prolonged the LCR period (interval between prior AP-induced Ca 2+ transient and LCR) reducing spontaneous SANC firing by ≈30%. In contrast, EPAC activator (8-pCPT) increased LCR number and size, shortened the LCR period and accelerated spontaneous firing by≈18%. EPAC-mediated effects were implemented in PKC-dependent manner via EPAC-PLC-PKC-CaMKII signaling pathway, since PKC inhibitor reproduced effects of EPAC inhibition on CaMKII activity, CaMKII-dependent phosphorylation of Ca 2+ -cycling proteins, LCR parameters, and spontaneous SANC firing. CONCLUSIONS: EPAC is an essential component of basal cardiac pacemaker function, which accelerates spontaneous automaticity of SANC via an increase in basal CaMKII-dependent phosphorylation of Ca 2+ -cycling proteins (PLB, RyR, L-type Ca 2+ channels, and likely others), leading to amplification of LCR parameters, shortening of LCR timing and resultant spontaneous cycle length. Consequently, EPAC might represent a novel therapeutic target to regulate resting heart rate and treat sinoatrial node dysfunction.