Department of Diabetes and Clinical Nutrition, Faculty of Medicine, Kyoto University, Kyoto, Japan.
Am J Physiol Heart Circ Physiol. 2011 Jan;300(1):H251-61. doi: 10.1152/ajpheart.00764.2010. Epub 2010 Oct 15.
The question of the extent to which cytosolic Ca(2+) affects sinoatrial node pacemaker activity has been discussed for decades. We examined this issue by analyzing two mathematical pacemaker models, based on the "Ca(2+) 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(+)/Ca(2+) exchange current via spontaneous Ca(2+) 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 Ca(2+), 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 Ca(2+) dynamics were acutely disrupted using high doses of SR blockers. These experimental results suggested that rapid disruption of normal Ca(2+) dynamics would not markedly affect spontaneous activity. Experimental prolongation of the action potentials, as well as slowing of the Ca(2+)-mediated inactivation of the L-type Ca(2+) currents induced by BAPTA, were well explained by assuming Ca(2+) 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 Ca(2+) current rundown induced secondarily through inhibition of Ca(2+)/calmodulin kinase II and/or Ca(2+)-stimulated adenylyl cyclase was discussed as underlying the disruption of spontaneous activity after prolonged intracellular Ca(2+) concentration reduction for >5 min.
几十年来，人们一直在讨论细胞质 Ca(2+) 对窦房结起搏活动的影响程度。我们通过分析基于“Ca(2+) 钟”（C）和“膜钟”（M）假说的两个数学起搏模型，以及在分离的豚鼠窦房结细胞中的膜片钳实验来研究这个问题。通过对模型进行铅电位分析，发现 C 机制与 M 机制重叠，C 机制依赖于舒张期间来自肌浆网（SR）的自发性 Ca(2+) 释放对 Na(+)/Ca(2+) 交换电流的增强。通过螯合细胞内 Ca(2+)，在 C 模型中观察到起搏节律的快速抑制，而 M 模型不受影响。然而，当用 10 mM BAPTA 灌流液迅速平衡胞质溶胶时，实验性地破坏穿孔膜却未能在大约 30 秒内降低自发性动作电位的频率，而收缩则在大约 3 秒内停止。当使用高剂量的 SR 阻断剂急性破坏 SR Ca(2+) 动力学时，自发节律在几分钟内仍然保持完整。这些实验结果表明，快速破坏正常的 Ca(2+) 动力学不会显著影响自发性活动。实验延长动作电位的时间，以及 BAPTA 诱导的 L 型 Ca(2+) 电流的 Ca(2+) 介导失活的减慢，通过假设 Ca(2+) 螯合得到了很好的解释，即使在 M 模型中，除了胞质溶胶的主体之外，还在通道孔附近。综上所述，实验和模型研究结果强烈表明，C 模型明确描述的 C 机制很难应用于豚鼠窦房结细胞。可能涉及 L 型 Ca(2+) 电流衰竭的继发性激活，这是通过抑制 Ca(2+)/钙调蛋白激酶 II 和/或 Ca(2+)-刺激的腺苷酸环化酶引起的，被认为是在细胞内 Ca(2+) 浓度降低超过 5 分钟后自发活动中断的原因。
