verbatim): This project addresses the composition and function of contractile proteins in myocardium and thus assess the role of the contractile apparatus in the evolution of ischemic cardiomyopathy. Human myocardial biopsy samples will be characterized microscopically and by protein composition in both ischemic and nonischemic remodeled zones with direct comparison to samples from control myocardium which demonstrate no evidence of regional coronary artery disease or global contractile deficit. The biopsies will be characterized with respect to changes in proportions of isoforms, alterations of normal stoichiometric ratios (reflecting loss of protein or excessive degradation), and alterations in phosphorylation of proteins. Native thin filaments and myosin will be isolated and purified from selected zones (ischemic, non-ischemic remodeled, and apparently normal zones), and characterized with respect to calcium sensitivity, unloaded shortening, and average force using the in vitro motility assay. Sarcomeric proteins will be isolated from explanted human hearts. The thin filament will be reconstituted and characterized with respect to contractile function, including average cross-bridge force, calcium activation, ATPase, and maximal unloaded shortening. The use of explanted tissue will allow characterization of specific contractile proteins and thus permit delineation of casual links between specific native thin filament changes and mechanical performance. The overall hypothesis to be tested is that alterations in contractile machinery within ischemic zones and nonischemic remodeled zones underlie changes in mechanical performance that contribute to progressive diminution in regional and global myocardial performance. The proposed research is designed to determine whether specific changes within the sarcomere (for example altered thin filament activation or changes in mechanical behavior of myosin) underlie the progression evident in ischemic myopathy. Such data could ultimately be used to identify specific targets for interventions designed to confer protection.
