Minor contribution of cytosolic Ca2+ transients to the pacemaker rhythm in guinea pig sinoatrial node cells

Abstract

The question of the extent to which cytosolic Ca2+ affects sinoatrial node pacemaker activity has been discussed for decades. We examined this issue by analyzing two mathematical pacemaker models, based on the “Ca2+ clock” (C) and “membrane clock” (M) hypotheses, together with patch-clamp experiments in isolated guinea pig sinoatrial node cells. By applying lead potential analysis to the models, the C mechanism, which is dependent on potentiation of Na+/Ca2+ exchange current via spontaneous Ca2+ release from the sarcoplasmic reticulum (SR) during diastole, was found to overlap M mechanisms in the C model. Rapid suppression of pacemaker rhythm was observed in the C model by chelating intracellular Ca2+, whereas the M model was unaffected. Experimental rupturing of the perforated-patch membrane to allow rapid equilibration of the cytosol with 10 mM BAPTA pipette solution, however, failed to decrease the rate of spontaneous action potential within ∼30 s, whereas contraction ceased within ∼3 s. The spontaneous rhythm also remained intact within a few minutes when SR Ca2+ dynamics were acutely disrupted using high doses of SR blockers. These experimental results suggested that rapid disruption of normal Ca2+ dynamics would not markedly affect spontaneous activity. Experimental prolongation of the action potentials, as well as slowing of the Ca2+-mediated inactivation of the L-type Ca2+ currents induced by BAPTA, were well explained by assuming Ca2+ chelation, even in the proximity of the channel pore in addition to the bulk cytosol in the M model. Taken together, the experimental and model findings strongly suggest that the C mechanism explicitly described by the C model can hardly be applied to guinea pig sinoatrial node cells. The possible involvement of L-type Ca2+ current rundown induced secondarily through inhibition of Ca2+/calmodulin kinase II and/or Ca2+-stimulated adenylyl cyclase was discussed as underlying the disruption of spontaneous activity after prolonged intracellular Ca2+ concentration reduction for >5 min.