Date of Award

9-25-1992

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

First Advisor

John O. Oberpriller

Abstract

To better understand the mechanisms governing the proliferation of cardiac myocytes it is important to identify the factors controlling this phenomenon, and to characterize their actions. The ventricular myocyte from the adult newt, Notophthalmus viridescens, is well suited to this type of study, due to its large chromosomes, ease of culture maintenance, and ability to undergo mitosis with cytokinesis. DNA synthesis was quantified in vitro in myocytes treated with platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF-$\beta$), fibroblast growth factors (FGFs), 12-0-tetradecanoylphorbol-13-acetate (TPA), heparin, or conditioned medium from ventricular myocytes or nonmyocytes (primarily endothelial cells). Ultrastructure of TPA-treated myocytes was analyzed.Ventricles were enzymatically separated for 10 hours. A cell population consisting primarily of myocytes was collected in the last 5 hours, suspended in modified Leibovitz L-15 medium and 10% fetal bovine serum, and preplated onto plastic (day zero). By day two many nonmyocytes had attached, leaving a relatively pure solution of myocytes, which was plated onto laminin-coated dishes. On days 5, 7, 9, and 11, the cells were fed with modified L-15 medium with 10% fetal bovine serum with and without the addition of experimental agents. On day 11 cells used in DNA-synthetic studies were given 1$\mu$Ci/ml of tritiated thymidine, and 24 hours later were fixed and stained. Dishes were coated with photographic emulsion, exposed, and developed. The percent of cells with labeled nuclei was determined. On day 11 myocytes used in electron microscopic studies were fixed, and subsequently embedded and thin-sectioned en face.Experimental media significantly increasing DNA synthesis included those containing TPA (137% above control), PDGF (27% above control), or conditioned medium from ventricular myocytes (115% above control) or nonmyocytes (28% above control). Media significantly inhibiting DNA synthesis were those containing heparin (70% below control) or TGF-$\beta$ (63% below control). FGFs did not alter DNA synthesis. TPA did not alter ultrastructure.These results indicate that the newt ventricular myocyte can have its DNA synthetic activity altered by certain factors. This information may be added to the body of knowledge concerning the cardiac myocyte and its microenvironment, and may aid in the eventual understanding of cardiac myocyte proliferation.

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