Sirenko Syevda G, Yang Dongmei, Maltseva Larissa A, Kim Mary S, Lakatta Edward G, Maltsev Victor A
Laboratory of Cardiovascular Science, National Institute on Aging, National Institutes of Health, Baltimore, Maryland, United States of America.
PLoS One. 2017 Sep 25;12(9):e0185222. doi: 10.1371/journal.pone.0185222. eCollection 2017.
Uptake and release calcium from the sarcoplasmic reticulum (SR) (dubbed "calcium clock"), in the form of spontaneous, rhythmic, local diastolic calcium releases (LCRs), together with voltage-sensitive ion channels (membrane clock) form a coupled system that regulates the action potential (AP) firing rate. LCRs activate Sodium/Calcium exchanger (NCX) that accelerates diastolic depolarization and thus participating in regulation of the time at which the next AP will occur. Previous studies in rabbit SA node cells (SANC) demonstrated that the basal AP cycle length (APCL) is tightly coupled to the basal LCR period (time from the prior AP-induced Ca2+ transient to the diastolic LCR occurrence), and that this coupling is further modulated by autonomic receptor stimulation. Although spontaneous LCRs during diastolic depolarization have been reported in SANC of various species (rabbit, cat, mouse, toad), prior studies have failed to detect LCRs in spontaneously beating SANC of guinea-pig, a species that has been traditionally used in studies of cardiac pacemaker cell function. We performed a detailed investigation of whether guinea-pig SANC generate LCRs and whether they play a similar key role in regulation of the AP firing rate. We used two different approaches, 2D high-speed camera and classical line-scan confocal imaging. Positioning the scan-line beneath sarcolemma, parallel to the long axis of the cell, we found that rhythmically beating guinea-pig SANC do, indeed, generate spontaneous, diastolic LCRs beneath the surface membrane. The average key LCR characteristics measured in confocal images in guinea-pig SANC were comparable to rabbit SANC, both in the basal state and in the presence of β-adrenergic receptor stimulation. Moreover, the relationship between the LCR period and APCL was subtended by the same linear function. Thus, LCRs in guinea-pig SANC contribute to the diastolic depolarization and APCL regulation. Our findings indicate that coupled-clock system regulation of APCL is a general, species-independent, mechanism of pacemaker cell normal automaticity. Lack of LCRs in prior studies is likely explained by technical issues, as individual LCRs are small stochastic events occurring mainly near the cell border.
肌浆网（SR）以自发、有节律、局部舒张期钙释放（LCRs）的形式摄取和释放钙（被称为“钙时钟”），与电压敏感离子通道（膜时钟）一起形成一个耦合系统，调节动作电位（AP）发放频率。LCRs激活钠/钙交换体（NCX），加速舒张期去极化，从而参与调节下一个AP出现的时间。先前对兔窦房结细胞（SANC）的研究表明，基础AP周期长度（APCL）与基础LCR周期（从前一个AP诱导的Ca2+瞬变到舒张期LCR出现的时间）紧密耦合，并且这种耦合会受到自主受体刺激的进一步调节。尽管在各种物种（兔、猫、小鼠、蟾蜍）的SANC中都报道了舒张期去极化期间的自发LCRs，但先前的研究未能在豚鼠的自发搏动SANC中检测到LCRs，豚鼠是一种传统上用于心脏起搏细胞功能研究的物种。我们对豚鼠SANC是否产生LCRs以及它们在调节AP发放频率中是否发挥类似的关键作用进行了详细研究。我们使用了两种不同的方法，二维高速摄像机和经典的线扫描共聚焦成像。将扫描线定位在肌膜下方，与细胞长轴平行，我们发现有节律搏动的豚鼠SANC确实在表面膜下方产生自发的舒张期LCRs。在豚鼠SANC的共聚焦图像中测量的平均关键LCR特征在基础状态和β-肾上腺素能受体刺激存在的情况下与兔SANC相当。此外，LCR周期与APCL之间的关系由相同的线性函数支撑。因此，豚鼠SANC中的LCRs有助于舒张期去极化和APCL调节。我们的研究结果表明，APCL的耦合时钟系统调节是起搏细胞正常自律性的一种普遍的、不依赖物种的机制。先前研究中未检测到LCRs可能是由技术问题导致的，因为单个LCRs是主要发生在细胞边界附近的小随机事件。
