LOCAL CONTROL OF CARDIAC EXCITATION/CONTRACTION COUPLING

肌浆网钙离子释放的调控 (Sr)是正常心脏功能的基础，并通过 荷尔蒙、神经递质和电刺激。的总体目标 本研究旨在验证关于肌质网钙释放的两个假说：1)局部 控制：SR释放通道在空间上离散的群或群 首先被钙离子通过共同结合的L型钙离子进入激活。 钙离子通道。在本地控制中，集群的激活发生 而不依赖于任何其他这样的集群的激活。2)损失 “局部控制”会导致某些心脏功能异常，例如 Ca~(2+)波和触发的心律失常。失去对当地的控制意味着 团簇最初不再被附近的钙离子内流激活，但可能 被从其他团簇到达它们的钙离子激活。这是假设的 此外，这种局部控制的丧失可以被细胞内拮抗 镁离子(Mg2+)。当地控制被认为是由一种 单个L型钙通道与α-钙离子通道的微观“耦合” 位于SR和横向交界处的SR释放通道的“簇” 小管。据推测，引起钙释放的可能性 由L类钙离子通道首次开放的潜伏期决定， 并由单位L型电流的大小决定(而不是由 全细胞钙电流、Na+/Ca~(2+)交换或已释放的钙离子 来自其他集群)。根据当地控制，‘耦合器’([Ca2+]) 在RyR的局部亚细胞区域与 从全细胞钙离子的常规测量中可以预测到 电流和空间平均钙瞬变。相应地，共聚焦激光器 提供更高空间分辨率的扫描显微镜将是 用于观察电压钳引起的局部[钙]i瞬变(LCT) 豚鼠和大鼠单个心肌细胞的去极化 内置荧光钙指示器。图像处理 将用于确定诱发局部的(空间)特征 [Ca~(2+)]i-瞬变和自发的Ca~(2+)火花，这是评估 严格的局部控制假说。全细胞和膜片钳 记录将用于确定局部[Ca~(2+)]i瞬变 诱发的方式与L类钙离子的门控特性一致- 频道。SR负荷和细胞内镁离子对细胞大小的影响 诱发局部[Ca~(2+)]i-瞬变发生的概率和位置， 将研究自发的钙离子火花和钙离子波。这项研究 将提供有关正常兴奋-收缩(E-C)的新信息 耦合，它将检验出现异常E-C耦合的假设 由于失去了对肌质网钙离子释放的正常局部控制。

Control of the release o calcium ions (Ca2+) from sarcoplasmic reticulum (SR) is fundamental to normal cardiac function and its modulation by hormones, neurotransmitters, and electrical excitation. The overall aim of this research is to test two hypotheses on SR calcium release; 1) 'Local control': Spatially discrete groups or 'clusters" of SR release channels are first activated exclusively by Ca2+ entering via co-associated L-type. Ca2+ channels. In local control activation of a cluster occurs independently of the activation of any other such clusters. 2) Loss of 'local control" leads to certain abnormalities of cardiac function such as Ca2+ waves and triggered arrhythmias. Loss of local control means that clusters are no longer activated initially by nearby Ca2+ influx, but may be activated by Ca2+ reaching them from other clusters. It is hypothesized also that loss of local control can be antagonized by intracellular magnesium ions (Mg 2+). Local control is thought to result from a microscopic 'coupling' between individual L-type Ca2+-channels and a 'cluster' of SR release channels at the junction between SR and transverse tubule. It is hypothesized that the probability of evoking Ca2+ release there is determined by latency to first opening of the L-type Ca2+ channel, and by the magnitude of the unitary L-type current (rather than by the whole-cell Ca2+-current, Na+/Ca2+_ exchange, or by Ca2+ already released from other clusters). According to local control, the 'coupler' ([Ca2+]) in the local subcellular region of the RyR is very different from that which would be predicted from conventional measurements of whole-cell Ca2+- current and spatial-average Ca2+ transient. Accordingly, confocal laser scanning microscopy, which provides increased spatial resolution, will be used to observe local [Ca2+]i transients (LCTs) evoked by voltage clamp depolarization in single guinea-pig and rat cardiac ventricular cells perfused internally with fluorescent Ca2+ indicators. Image processing will be used to determine (spatial) characteristics of evoked local [Ca2+]i-transients and spontaneous Ca2+-sparks, as required to evaluate the hypothesis of local control rigorously. Whole-cell and patch clamp recording will be used to determine whether local [Ca2+]i-transients are evoked in a manner consistent with gating properties of L-type Ca2+ - channels. The effect of SR loading and intracellular Mg2+ on the size, probability, and location of occurrence of evoked local [Ca2+]i-transients, spontaneous Ca2+-sparks, and Ca2+ -waves will be studied. The research will provide new information on normal excitation-contraction (E-C) coupling, and it will test the hypothesis that abnormal E-C coupling arises from loss of the normal 'local control of SR Ca2+ release.