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

窦房结细胞（SANC）的正常自动性涉及耦合时钟系统内的细胞内Ca2+循环：周期性局部，肌浆网（Ca2+时钟）的肌下Ca2+释放（LCRs）激活向内的Na+-Ca2+交换电流，加速舒张去极化，促使表面膜离子通道（膜时钟）的集合产生下一个动作电位（AP）。细胞内Ca2+是否在搏动基础上调节SANC AP放电速率是有争议的。我们将单个分离的SANC装入笼状Ca2+缓冲液NP-EGTA，并同时记录膜电位和细胞内Ca2+。在引入笼式Ca2+缓冲液之前，舒张期去极化（DD）期间的自发性lcr与节律性APs紧密耦合（r2=0.9）。缓冲液明显延长AP诱导的Ca2+瞬态衰变时间（T50），部分降低SR负荷水平，抑制自发性舒张lcr，使其与AP产生分离，并导致AP放电明显减慢和节律紊乱。当Ca2+通过闪光光解从笼状化合物中急性释放时，细胞内Ca2+动力学被急性恢复，节律性ap立即以正常速率恢复。然而，经过几个有节奏的周期后，随着笼状缓冲液对Ca2+动力学的干扰被重新建立，这些闪光的影响减弱了。我们的研究结果直接支持了细胞内Ca2+时钟与表面膜电压时钟耦合的系统在节拍到节拍的基础上调节正常的SANC自动性的假设。

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.