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。这些死亡中的许多是由心律失常引起的，心律失常可能是由弱化组织中拉伸机械敏感通道（MSC）引起的异常兴奋引起的。该建议特别关注心脏中的机械转导机制，MSC的一般特性及其在影响细胞Ca 2+中的作用。拟议的实验涉及完整组织中的细胞、分离的细胞、单个离子通道和重建。这些方法涉及电生理学、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。为了确定体内细胞如何响应拉伸，我们在双蔗糖间隙中电压钳住小梁。组织将被拉伸，我们将测量膜电位转导电流，药理学中的细胞Ca2+作为拉伸参数和电压的函数。这对于理解分离的细胞和通道的哪些特性与原位功能相关至关重要。孤立的细胞该制备提供了比用于体内研究的细胞更高的光学和电学分辨率。我们将研究机械诱导的Ca 2+释放和波在离体心肌细胞的性质。机械敏感性离子通道的活性及其药理学研究：单通道和全细胞膜片钳和荧光成像。MSC的药理学。我们将在通道，细胞和组织水平上检查对MSC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MSC和细胞Ca 2+的影响的初步数据。我们将研究两亲物激活和抑制MSC作为影响拉伸诱导效应的工具的效果。真核MSC的应力分布的定义。使用膜片钳和成像的补丁，我们将测量膜片的膜力学和激活的内源性和克隆的MSC作为细胞骨架和细胞外基质的状态的函数。

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.