E/C COUPLING AND CALCIUM REGULATION IN ATRIAL MYOCYTES

本研究的总体目标是研究哺乳动物心房肌兴奋-收缩(e-c)偶联过程中[Ca~(2+)]i的调节和肌浆网(SR)的钙释放机制。我们将检验这一假设，即在心房细胞中，正常的e-c偶联涉及交界性(j-SR)和非交界性SR(NJ-SR)的钙释放。然而，只有j-SR的释放直接依赖于膜电压(即通过电压门控的钙通道进入触发钙诱导的钙释放(CICR))，而NJ-SR的释放仅由钙和CICR的扩散触发(其机制类似于心脏的[Ca2+]波传播)。在心房肌中，e-c偶联的“局部控制”模式严格适用于j-SR的释放。肌浆网钙离子的释放受到一个磷酸化-去磷酸化循环的严格调控，在这个循环中，ATP在SR释放通道的微区中以糖酵解的方式产生。作为房性心律失常的主要危险因素，代谢诱导的机电和[Ca~(2+)]_1瞬时交替为研究房性心律失常的动态调节提供了一个模型系统。本研究的五个主要目标是：1.研究动作电位触发的哺乳动物心房细胞内全细胞[Ca~(2+)]_1-瞬变的时空特性。2.验证心房细胞e-c偶联的局部控制模型的有效性，并研究j-SR和Nj-SR的e-c偶联和钙释放机制。3.定量确定心房细胞j-SR和Nj-SR钙释放的基本事件(钙火花)的性质。4.研究代谢诱导的[Ca~(2+)]_i交替及其细胞机制。5.明确糖酵解ATP产物在调节肌浆网钙释放和CICR中的作用。为了实现这些目标，将使用多种实验技术，包括激光扫描共聚焦显微镜在单个心房肌细胞上的高分辨率[Ca~(2+)]i成像，研究膜电流的全细胞电压钳技术，通过重构为平面脂质双层的心脏SR Ca~(2+)释放通道的单通道记录，双光子激发对笼中Ca~(2+)的亚细胞光解，以及对Ca~(2+)进入、释放和摄取的药理学操作。这项研究将为房性心律失常相关的正常和异常状态下的心房e-c偶联和钙释放提供新的基础信息。

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.