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.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL033832-03
Application #
3346066
Study Section
Cardiovascular Study Section (CVA)
Project Start
1985-09-10
Project End
1990-04-30
Budget Start
1987-09-10
Budget End
1990-04-30
Support Year
3
Fiscal Year
1987
Total Cost
Indirect Cost
Name
University of California Los Angeles
Department
Type
Schools of Medicine
DUNS #
119132785
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
Lewis, W; Sadeghi, A M; Laks, H et al. (1991) Altered cytoskeletal protein synthesis in rat cardiac isografts. J Heart Lung Transplant 10:92-9
Lewis, W; Gonzalez, B (1990) Actin isoform mRNA alterations induced by doxorubicin in cultured heart cells. Lab Invest 62:69-76
Lewis, W (1989) AIDS: cardiac findings from 115 autopsies. Prog Cardiovasc Dis 32:207-15
Lewis, W; Perillo, N L; Gonzalez, B (1988) Alpha-actin synthesis changes in cultured cardiac myocytes: relationship to anthracycline structure. J Lab Clin Med 112:43-51
Lewis, W; Warner-Stevenson, L; MacAlpin, R et al. (1987) Polypeptide composition and histopathologic changes in endomyocardial biopsies from transplanted human hearts. J Heart Transplant 6:362-8
Lewis, W; Gonzalez, B (1987) Actin isoform synthesis by cultured cardiac myocytes. Effects of doxorubicin. Lab Invest 56:295-301
Lewis, W; Gonzalez, B (1987) Actin synthesis in cultured cardiac myocytes: comparative effects of doxorubicin, dactinomycin, and plicamycin. J Lab Clin Med 109:67-74
Lewis, W; Gonzalez, B (1986) Anthracycline effects on actin and actin-containing thin filaments in cultured neonatal rat myocardial cells. Lab Invest 54:416-23