Beat to beat Ca2+-dependent regulation of pacemaker cell rate and rhythm

窦房结细胞(SANC)的正常自律性涉及细胞内钙离子在耦合时钟系统内的循环：周期性的肌浆网局部、肌膜下钙释放(钙时钟)激活内向的Na+-钙交换电流，加速舒张期去极化，促使表膜离子通道(膜时钟)产生下一个动作电位(AP)。细胞内钙离子是否在节拍的基础上调节SANC AP的放电率是有争议的。我们用笼式钙缓冲液NP-EGTA加载单个分离的SAEC，并同时记录膜电位和细胞内钙离子。在引入笼式钙缓冲液之前，舒张期去极化(DD)时自发的LCR与节律性AP紧密耦合(R2=0.9)。该缓冲剂显著延长AP诱发的钙瞬变的衰减时间(T50)，部分耗竭SR负荷水平，抑制自发舒张期LCR并使其与AP产生解偶联，并使AP放电明显减慢和节律紊乱。当通过闪光光解从笼子化合物中迅速释放出钙离子时，细胞内钙离子动力学迅速恢复，节律性AP立即以正常速度恢复。然而，在几个有节奏的循环之后，这些闪光的影响减弱，因为笼式缓冲器对钙动力学的干扰重新建立起来。我们的结果直接支持这样的假设，即细胞内钙离子时钟与表膜电压时钟耦合的系统在节拍的基础上调节正常的SANC自律性。

Normal automaticity in sinoatrial node cells (SANC) involves intracellular Ca2+ cycling within a coupled-clock system: periodic local, subsarcolemmal Ca2+ releases (LCRs) from sarcoplasmic reticulum (Ca2+ clock) activate an inward Na+-Ca2+ exchange current that accelerates the diastolic depolarization prompting the ensemble of surface membrane ion channels (membrane clock) to generate the next action potential (AP). Whether intracellular Ca2+ regulates SANC AP firing rate on a beat-to-beat basis is controversial. We loaded single isolated SANC with a caged Ca2+ buffer, NP-EGTA, and simultaneously recorded membrane potential and intracellular Ca2+. Prior to introduction of the caged Ca2+ buffer, spontaneous LCRs during diastolic depolarization (DD) were tightly coupled to rhythmic APs (r2=0.9). The buffer markedly prolonged the decay time (T50) of the AP-induced Ca2+ transient and partially depleted the SR load level, suppressed spontaneous diastolic LCRs and uncoupled them from AP generation, and caused AP firing to become markedly slow and dysrhythmic. When Ca2+ was acutely released from the caged compound by flash photolysis, intracellular Ca2+ dynamics were acutely restored and rhythmic APs resumed immediately at a normal rate. After a few rhythmic cycles, however, these effects of the flash waned as interference with Ca2+ dynamics by the caged buffer was reestablished. Our results directly support the hypothesis that a system of an intracellular Ca2+ clock coupled to a surface membrane voltage clock regulates normal SANC automaticity on a beat-to-beat basis.