The mechanisms underlying atrial fibrillation (AF) remain poorly understood. Our general hypothesis is that AF results from activity of a small number of relatively stable, high frequency reentrant sources (rotors) localized in one atrium, with fibrillatory conduction to the other atrium. We will use two well-established experimental models (sheep and goat) of acute and chronic AF with vastly different patterns of AF excitation to elucidate electrophysiological and molecular mechanisms in both acute and chronic AF, toward the following Specific Aims: 1. To compare the global mechanisms of acute AF, study in both species the effects of ACh on the activity of the high-frequency sources that maintain acute AF, and determine whether ACh increases complexity of AF and LA-RA gradients of local activation frequency in a dose-dependent manner. We achieve this aim using high-resolution optical mapping in combination with multiple electrode mapping in the isolated sheep heart in the presence of varying concentrations of ACh. 2. To investigate LA-RA differences in IK,(ACh) and IK1 density in the atria of sheep and goat hearts, determine the role of such differences in establishing frequency- and ACh concentration dependent changes in APD of atrial myocytes obtained from specific regions of the LA and RA, and compare the distribution of Kir3.x and Kir2.x channels. We will carry out patch-clamp experiments and RNAse protection assays, as well as western blot and immunolocalization studies. We will test the hypothesis that, in the sheep, the greater degree of frequency adaptation of normal LA myocytes during acute AF is due to the existence of a larger amount of Kir3.4 channels and greater density of IKACh than in the RA. In contrast, we expect to show that, in the goat, Kir3.x channels and IK,ACh density are equally distributed in both atria. 3. To determine the pathophysiological consequences of reduced functional expression of Kir3.x channels caused by pacing induced, chronic AF in sheep and goat hearts. ECG and Doppler echocardiography will be used in vivo to monitor LA-vs-RA frequency differences; optical mapping will be conducted ex vivo to determine the effects of chronic AF on LA vs RA frequencies and AF dynamics. In addition, RNAse protection assays, western blot and immunolocalization studies will be conducted to determine changes in Kir3.4, and Kir2.x channel expression. Finally, patch clamp studies in LA and RA myocytes obtained from chronic AF animals will be carried out to determine changes in action potential rate adaptation, and IK,ACh, ICa,L and IK1 densities. The proposed studies offer the opportunity of correlating for the first time propagation patterns and excitation frequency during AF with ion channel density distributions in two different species. Accomplishment of the proposed studies should lead to an increased understanding of the mechanisms of AF at the molecular, cellular and organ levels and may lead to therapeutic advances. ? ?
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