Date of Award

8-1-1995

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Anatomy and Cell Biology

Abstract

The cultured adult newt ventricular myocyte has been shown to undergo mitosis and cytokinesis in a fully differentiated state. A more complete insight into its proliferation and cellular changes in regeneration involves a better understanding of its morphology and cytoskeletal changes during mitotic events. The major advantage of cell culture of the newt cardiac myocyte is that it exhibits large chromosomes, making mitosis in myocytes easily observed by phase-contrast, epifluorescence and electron microscopy.In the present study, ventricles were enzymatically dissociated and myocardial cells were cultured on laminin-coated dishes or glass coverslips. Myocyte cultures fed modified L-15/10% FBS every other day showed a small percentage (3.5% at day 5, 10.3% at day 20) of nonmyocytes, some of which were endothelial cells.From days eight through nineteen in culture, the process of mitosis in mononucleated and binucleated newt ventricular myocytes was recorded and timed using time-lapse video microscopy. Mitotic mononucleated myocytes produced mononucleated daughter cells 80% of the time, while binucleated myocytes were more variable with only 32% undergoing complete cytokinesis resulting in binucleated daughter cells. In binucleated myocytes the period of anaphase to midbody formation was significantly shorter than in mononucleated myocytes.At day 12, ventricular myocyte cultures were fixed and stained with either antibodies or direct fluorescent stains for cytoskeletal proteins, or were prepared for transmission electron microscopy. During mitosis, myosin, actin and $\alpha$-actinin immunolocalization and the ultrastructural morphology of the myocytes indicated that myofibrillar disassembly occurred centrally, while myofibrillogenesis occurred peripherally. During mitosis, desmin and vimentin lost their filamentous patterns, indicating that a breakdown of cytoskeletal elements was necessary for the accommodation of the mitotic and cytokinetic processes. Tubulin was observed in or near Z-band regions which may indicate that it provided structural support for myofibrillae or was involved in organelle transport necessary for myofibrillogenesis. The results obtained in this study demonstrated the dynamic nature of the cytoskeleton and the synthetic processes in its mitotic process, and will aid in future investigations of the mechanisms involved in the proliferative and regenerative capabilities of this important cardiac cell type.

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