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.
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