ELECTOPHYSIOLOGY OF NEONATAL AND ADULT HEART

这项研究计划的目的是检查基本的分子 在哺乳动物中， 心 先前的研究表明，心脏信号发生在 在二氢吡啶/兰尼定受体周围的微区中 复杂的，通过交换的Ca 2+信号之间的纵横交错，这些 两种蛋白质和Na+-Ca 2+交换剂。 这些研究还表明， ICa门控释放的增益是电压依赖性的，这表明 Ca 2+释放复合物受一种以上的机制调节， 有不同的Ca 2+释放单位群体， 不同的门控模式。 我们建议测试的中心思想是， 心脏中收缩的信号可以是多模式门控的。 对这一假设的评估不仅需要精确的识别 持续时间，幅度，以及这种释放部位的细胞位置， 而且能够同时监控许多这样的站点， 以区分它们的门控、电压依赖性和 代谢因素的调节。 使用一种方法， Ca 2+扩散到约50 nm，我们建议监测50-300 Ca 2 + 同时在约20 x 50 mum 2的区域释放位点， 心肌细胞采用快速二维共焦成像。我们 应特别：a）表征局灶性Ca 2+释放的动力学 作为电压、伊卡、SR的Ca 2+负载的函数的位点， 磷酸化和药理学干预; B）测量 与超微结构相关的局灶性Ca 2+释放位点分布 决定因素在心室和心房细胞（即细胞与 无t-小管）; c）确定病灶释放位点的分布 相对于电压门控，伊卡，INaCa，INa; d）测量 作为氧化还原状态的函数的焦点释放位点的性质，以及 e）探索Ca 2+通道的C-末端尾部是否是 Ca 2+和电压信号的通道。 我们的论点是 可能需要能够监测数百个释放点的技术， 为了研究钙离子信号转导机制的多样性， 心拟议的研究可能会更好地了解 调节信号传导的基本分子机制 收缩在正常和患病的心脏。

This research proposal aims to examine the fundamental molecular mechanisms that underlie the signaling of contraction in the mammalian heart. Previous studies have shown that cardiac signaling takes place in microdomains surrounding the dihydropyridine/ryanodine receptor complex, via interchange of Ca2+ signals criss-crossing between these two proteins and the Na+-Ca2+ exchanger. Such studies also show that the gain of ICa-gated release was voltage-dependent, suggesting that the Ca2+ release complex is either regulated by more than one mechanism or that there are different populations of Ca2+ release units with different gating modes. The central idea we propose to test is that the signaling of contraction in the heart might be multi-modally gated. Evaluation of this hypothesis requires not only precise identification of the duration, magnitude, and cellular location of such release sites, but also the ability to monitor many such sites simultaneously in order to differentiate between their gating, voltage dependence, and regulation by metabolic factors. Using a method that limits the diffusion of Ca2+ to about 50 nm, we propose to monitor 50-300 Ca2+ release sites simultaneously in areas of approximately 20 x 50 mum2 of a cardiac myocyte employing rapid two-dimensional confocal imaging. We shall specifically: a) characterize the kinetics of focal Ca2+ release sites as a function of voltage, ICa, Ca2+ load of the SR, phosphorylation, and pharmacological interventions; b) measure the distribution of focal Ca2+ release sites relative to ultrastructural determinants in both ventricular and atrial cells (i.e. cells with and without t-tubules); c) identify distribution of focal release sites relative to gating by voltage, ICa, INaCa, INa; d) measure the properties of focal release sites as a function of redox state, and temperature; e) explore whether the C-terminal tail of Ca2+ channel is the conduit to both Ca2+ and voltage signaling. It is our contention that techniques that can monitor 100s of release sites may be required to examine the multiplicity in the mechanisms of Ca2+ signaling in the heart. The proposed studies may provide a better insight into fundamental molecular mechanisms that regulate the signaling of contraction in normal and diseased heart.