E/C COUPLING AND CALCIUM REGULATION IN ATRIAL MYOCYTES

本研究的总体目标是探讨哺乳动物心房肌兴奋-收缩（e-c）偶联过程中[Ca 2 +]i调节和肌浆网（SR）Ca 2+释放机制。我们将测试的假设，在心房细胞正常的电-电耦合涉及Ca 2+释放从交界（j-SR）和非交界SR（nj-SR）。 然而，只有j-SR的释放直接依赖于膜电压（即通过电压门控Ca 2+通道进入触发Ca 2+诱导的Ca 2+释放（CICR）），而nj-SR的释放仅由Ca 2+和CICR的扩散触发（通过类似于心脏[Ca 2+]~波传播的机制）。在心房肌中，“局部控制”的电-电耦合模型严格地仅适用于从j-SR释放。SR Ca 2+释放受到磷酸化-去磷酸化循环的严格调节，其中ATP在SR释放通道的微区中糖酵解产生。 代谢诱导的电机械和[Ca 2 +]1瞬时交替是房性心律失常的主要危险因素，将提供一个模型系统来研究通过区室化糖酵解ATP形成对e-c偶联的动态调节。本研究的五个主要目的是：1。表征动作电位触发的哺乳动物心房细胞全细胞[Ca 2 +]1-瞬变的时空特性。2.验证心房肌细胞电-钙耦合的局部控制模型的有效性，并探讨电-钙耦合和j-SR和nj-SR钙释放的机制。定量定义心房细胞j-SR和nj-SR钙释放基本事件（钙火花）的性质。4.表征代谢诱导的[Ca 2 +]i交替及其细胞机制。5.明确糖酵解ATP产生在调节SR Ca 2+释放和CICR中的作用。为了实现这些目标，将使用多种实验技术，包括通过激光扫描共聚焦显微镜在单个心房肌细胞中进行的高分辨率[Ca 2 +]i成像，研究膜电流的全细胞电压钳技术，通过重构成平面脂质双层的心脏SR Ca 2+释放通道进行的单通道记录，通过双光子激发进行的笼状Ca 2+的亚细胞光解，以及药理学上对Ca 2+进入、释放和摄取的操纵。这项研究将为心房电-心耦合和正常和改变的房性心律失常相关条件下的Ca 2+释放提供基本的新信息。

The overall goal of the proposed study is to characterize [Ca2+]i regulation and sarcoplasmic reticulum (SR) Ca2+ release mechanisms during excitation-contraction (e-c) coupling in mammalian atrial muscle. We will test the hypothesis that in atrial cells normal e-c coupling involves Ca2+ release from both junctional (j-SR) and non-junctional SR (nj-SR). However only release from j-SR is directly dependent on membrane voltage (i.e. via Ca2+ entering through voltage-gated Ca2+ channels triggering Ca 2+-induced Ca2+-release (CICR)), whereas release from nj-SR is triggered solely by diffusion of Ca2+ and CICR (by a mechanism similar to cardiac [Ca 2+]~ wave propagation). In atrial muscle the model of 'local control' of e-c coupling strictly applies only to release from j-SR. SR Ca2+ release is tightly regulated by a phosphorylation-dephosphorylation cycle for which ATP is produced glycolytically in the microdomain of the SR release channel. Metabolically-induced electromechanical and [Ca2+]1 transient alternans, a major risk factor for atrial arrhythmias, will provide a model system to study the dynamic regulation of e- c coupling by compartmentalized glycolytic ATP formation. The five major specific aims of the proposed research are: 1. Characterize the spatio-temporal properties of whole-cell [Ca2+]1-transients in mammalian atrial cells triggered by action potentials. 2. Test the validity of the local control model for e-c coupling in atrial cells and characterize the mechanisms of e-c coupling and Ca2+ release from j-SR and nj-SR. 3. Define quantitatively the properties of elementary events of Ca2+ release (Ca2+ sparks) from j-SR and nj-SR in atrial cells. 4. Characterize metabolically-induced [Ca2+]i alternans and its cellular mechanisms. 5. Define the role of compartmentalized glycolytic ATP production in modulating SR Ca2+ release and CICR. To achieve these aims a multitude of experimental techniques will be used, including high resolution [Ca2+]i imaging by laser scanning confocal microscopy in single atrial myocytes, whole- cell voltage clamp techniques to study membrane currents, single channel recordings through cardiac SR Ca2+ release channels reconstituted into planar lipid bilayers, subcellular photolysis of caged Ca2+ by 2-photon excitation, and pharmacological manipulation of Ca2+ entry, release and uptake. The proposed research will provide fundamental new information on atrial e-c coupling and Ca2+ release under normal and altered conditions relevant to atrial arrhythmias.