The aim of this project is to explore the biochemical and subcellular events in congestive cardiomyopathy (COCM) by examining cardiac actin in vitro. This project will test hypotheses about COCM by using Adriamycin (ADR, an anthracycline antineoplastic known to cause COCM) as a probe. Cell biologic, biochemical and biophysical methods will be used to test the following hypotheses: (1) ADR alters normal compartmentalization of F- and G-actin by decreasing the amount of actin synthesized. (2) ADR causes cardiac cells to synthesize non-cardiac actin isomers which may not be properly incorporated into the cardiac thin filament. (3) Changes induced by ADR in actin synthesis lead to structurally altered thin filaments in heart cells. (4) Biochemical and biophysical events in the polymerization of purified cardiac actin in vitro caused by ADR are different from polymerization event of cardiac actin caused by cations. (5) The effect of ADR on cardiac actin polymerization in vitro is unique to actin from this tissue course. The characterization of ADR COCM will be undertaken by examining the influence of ADR on the ultrastructural assembly and biochemical properties of cardiac actin and thin filaments in myocardial cells. With cultured cardiac myocytes (CMC) as a model, actin monomer and polymer compartments in the cell will be analyzed by complimentary techniques: (a) one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE and quantitative densitometry); (b) DNAase I inhibition assays of CMC extracts. CMC actin synthesis and isoactin pool distribution will be monitored by SDS PAGE autoradiography and two-dimensional electrophoresis. ADR treated CMC will be examined by electron microscopy to evaluate thin filament structure. In parallel biochemical and biophysical studies, actins will be extracted and purified from cardiac and non-cardiac sources and will be interacted with ADR to assess actin polymerization comparatively. Methods employed will include: (a) gel filtration to separate actin polymers from monomers, (b) ultracentrifugal pelleting of ADR-actin polymers, (c) viscometric studies of ADR-actin, (d) negative staining electron microscopy of actin filaments. From these studies, insight into basic mechanisms of myocardial processing of actin and the disturbance of these mechanisms in ADR COCM is anticipated.
