Abstract
Spontaneous firing of sinoatrial node cells (SANC) is controlled by sarcoplasmic reticulum (SR) generated local subsarcolemmal Ca2+ releases (LCRs) which appear during diastolic depolarization and activate an inward Na+-Ca2+exchange current, regulating diastolic depolarization rate and SANC beating rate. Whether the ubiquitous enzyme, PLC that plays a key role in Ca2+ signaling of numerous cell types, controls spontaneous SANC firing is unknown. Here we show that PLC inhibitor U-73122 (6μmol/L, n=7), but not its inactive analog U-73343 (n=4), stops spontaneous firing (perforated patch-clamp recordings) of freshly isolated rabbit SANC, indicating important role of basal PLC activity for cardiac pacemaking. To determine whether signaling via the cAMP-sensor Epac elevates PLC activity in SANC and stimulates PLC-mediated IP3-dependent Ca2+ release, we employed Epac activator cpTOME or IP3 receptor inhibitor 2-APB, respectively. Neither cpTOME (10 μmol/L, n=3), nor 2-APB (2 μmol/L, n=4) altered the spontaneous SANC beating rate. PLC inhibition by U-73122, in a time-dependent manner, suppressed LCRs (confocal microscopy, Ca2+indicator Fluo-3), i.e. decreased LCR's size, amplitude and prolonged the LCR period (the interval between AP-induced Ca2+ transient and subsequent LCR); afterwards eliminated LCRs and stopped SANC firing. The time-dependent increase in the LCR period predicted the concomitant increase in the spontaneous cycle length, suggesting that Ca2+ cycling could be a major target of PLC-dependent control of SANC firing. LCRs are critically dependent upon amount of Ca2+ in SR, supplied by L-type Ca2+current (ICa,L). U-73122, but not U-73343, markedly suppressed ICa,L amplitude (from −11.8±4.8 to −5.9±2.4 pA/pF, n=6, P<0.001). We conclude that PLC-dependent control of spontaneous SANC firing, independent of either Epac signaling or IP3-dependent Ca2+ release, regulates Ca2+ influx through L-type Ca2+ channels and, thus, SR Ca2+ cycling and via these mechanisms, in part at least, is obligatory for SANC normal automaticity.
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