ELECTOPHYSIOLOGY OF NEONATAL AND ADULT HEART

这项研究计划旨在研究基础分子。 哺乳动物收缩信号的基础机制 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域中 复杂的，通过钙信号的交换在它们之间交叉 两种蛋白质和Na+-钙离子交换器。这类研究还表明， ICA门控释放的增益依赖于电压，这表明 钙离子释放复合体要么受多种机制调节，要么受多种机制调节 有不同群体的钙离子释放单位 不同的选通模式。我们建议测试的中心思想是 心脏收缩的信号可能是多模式选通的。 对这一假说的评估不仅需要准确的识别 这些释放地点的持续时间、大小和蜂窝位置， 而且能够同时监控多个这样的站点，以便 以区分它们的门控、电压依赖和 代谢因素的调节。使用一种方法限制 钙离子扩散到50 nm左右，我们建议监测50-300个钙离子 同时在大约20×50平方米的区域内释放 使用快速二维共聚焦成像的心肌细胞。我们 应具体：a)表征局部钙离子释放的动力学 作为电压、ICA、SR的钙负荷的函数的位置， 磷酸化和药物干预；b)测量 局部钙释放部位相对于超微结构的分布 心室细胞和心房细胞中的决定因素(即有和 无T形小管)；c)确定局部释放部位的分布 相对于按电压选通，ICa、InaCa、Ina；d)测量 作为氧化还原状态的函数的焦点释放位置的性质，以及 温度；e)探索钙通道的C末端尾巴是否 钙离子和电压信号的通道。这是我们的论点 可能需要能够监控数百个投放点的技术 探讨钙离子信号转导机制的多样性。 心。拟议的研究可能会提供更好的洞察力 调节细胞外信号转导的基本分子机制 正常心脏和疾病心脏的收缩。

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.