MECHANICAL TRANSDUCTION BY CARDIOCYTES

根据世界卫生组织的数据，截至1997年，世界上最常见的死亡原因是心血管疾病。这些死亡中许多是由心律失常引起的，心律失常可能是由在衰弱组织中拉伸机械敏感通道（MSCs）引起的异常兴奋引起的。该建议特别关注心脏机械转导的机制，MSCs的一般特性，以及它们在影响细胞Ca2+中的作用。所提出的实验涉及完整组织中的细胞、分离细胞、单个离子通道和重构。方法涉及电生理学、生物力学、荧光和明场显微镜、分子生物学、肽化学、药理学和数学建模。完整的组织。为了确定细胞在体内对拉伸的反应，我们在双蔗糖间隙中电压夹紧小梁。组织将被拉伸，我们将测量膜电位转导电流，细胞Ca2+在药理学上作为拉伸参数和电压的函数。这对于理解分离细胞和通道的哪些特性与原位功能相关至关重要。孤立的细胞。这种制备提供了比体内研究中使用的细胞更高的光学和电分辨率。我们将研究机械诱导Ca2+释放的性质和波在分离的心脏细胞。利用单通道膜片钳、全细胞膜片钳和荧光成像技术研究机械敏感离子通道的活性及其药理作用。msc的药理学。我们将在通道，细胞和组织水平上检查对MSCs有活性的药物，研究电生理学和Ca2+调节。我们将继续合成阻断或增强MSC活性的肽。我们还将扩展内皮素和相关肽对间充质干细胞和细胞Ca2+影响的初步数据。我们将研究两亲细胞的作用，它们可以激活和灭活间充质干细胞，作为影响拉伸诱导效应的工具。真核间充质干细胞应力分布的定义。利用膜片钳和膜片成像，我们将测量膜片的膜力学以及内源性和克隆MSCs的激活作为细胞骨架和细胞外基质状态的功能。

According toe the World Health Organization, as of 1997 the most common cause of death in the world is cardiovascular disease. Many of these deaths are caused by arrhythmias that may arise by abnormal excitation caused by stretching mechanosensitive channels (MSCs) in weakened tissue. This proposal concerns specifically the mechanism of mechanical transduction in the heart, the general properties of MSCs, and their role in affecting cell Ca2+. The proposed experiments deal with cells in intact tissue, isolated cells, individual ion channels and reconstitution. The methods involve electrophysiology, biomechanics, fluorescence and bright field microscopy, molecular biology, peptide chemistry, pharmacology and mathematical modeling. Intact tissue. To determine how cells in vivo respond to stretch, we voltage clamp trabeculae in a double sucrose gap. The tissue will be stretched and we will measure the membrane potentials transduction currents, cell Ca2+ in a pharmacology as a function of stretching parameters and voltage. This is critical to understanding which properties of isolated cells and channels are relevant to in situ function. Isolated cells. This preparation provides higher optical and electrical resolution than the cells used for in vivo studies. We will study the properties of mechanically induced Ca2+ release and waves in isolated cardiac cells. The activity of mechanistically sensitive ion channels and their pharmacology using patch clamp of single channels and whole cells and fluorescent imaging. Pharmacology of MSCs. We'll examine drugs active on MSCs at the level of channels, cells and tissue, studying electrophysiology and Ca2+ regulation. We will continue to synthesize peptides that block or potentiate MSC activity. We will also extend preliminary data on the effects of endothelin and related peptides on MSCs and cell Ca2+ We will examine the effect of amphiphiles that activate and inactivate MSCs as tools for affecting stretch induced effects. Definition of stress distribution for eukaryotic MSCs. Using the patch clamp and imaging of the patch we will measure the membrane mechanics of patches and the activation of endogenous and cloned MSCs as a function of the status of the cytoskeleton and extracellular matrix.