In mammals, several outward K+ and Cl- currents have been described that play important roles in repolarization of cardiac action potentials. However, there are considerable species, tissue, and regional differences within a single tissue (e.g., epi- vs endocardium of ventricle) in the specific types and magnitudes of these currents. Thus, while studies of myocytes isolated from non-human mammals have elucidated the biophysical properties and physiological roles for specific ion channels, they do not necessarily provide relevant models of human cardiac myocytes. Project 1 is a characterization of the currents that are responsible for repolarization of action potentials in normal human ventricles. The project is divided into four projects: 1) characterization of repolarizing K+ (transient outward, delayed rectifier, inward rectifier) and Cl- currents, 2) mechanisms of action of antiarrhythmic drugs, 3) characterization of Na-Ca exchange current, and 4) expression cloning of a delayed rectifier K+ channel ((l/Kr) using Xenopus oocytes. Most of these currents, including K+, Cl- and Na-Ca exchange currents will be studied using standard whole cell voltage clamp techniques with freshly dissociated cells obtained from epicardial and endocardial biopsies. Characterization of repolarizing currents from nondiseased tissue represents essential baseline data for comparison to currents with cells isolated from diseased human tissue. To that end we propose to record the same currents in myocytes isolated from endomyocardial biopsy samples of patients with long QT syndrome, in an attempt to determine the ionic basis of this genetic disease (Project 2). Several antiarrhythmic drugs act by prolonging action potential duration of myocardial cells and thereby lengthen refractory period. The mechanism of action of three representative drugs [dofetilide and tedisamil (class III), and quinidine (class 1a)] on repolarizing currents will be studied in isolated human ventricular cells. Additionally, we will define the mechanisms of induction of early afterdepolarizations by these agents, and determine the cellular mechanism of rate-dependent prolongation of action potentials by dofetilide.
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