Date of Award

4-1-1989

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Anatomy and Cell Biology

Abstract

Previous work has demonstrated that adult newt myocytes possess a proliferative ability in response to an experimentally-induced injury, in vivo. The work outlined in this study has been an effort to develop an additional in vitro model to study proliferative events in the adult cardiac myocyte.Ventricles were minced and then enzymatically dissociated in a Ca$\sp{++}$ and Mg$\sp{++}$-free salt solution containing 0.5% trypsin and 625 U/ml of CLS II collagenase for 8 to 10 hours at 25$\sp\circ$C. Enzyme digests were preplated and then cultured in either serum-free or serum-supplemented modified Liebovitz's medium for up to 30 days.Light and transmission electron microscopic characterization demonstrated that the myocytes underwent an initial period of disorganization which was characterized by a "rounding-up" of the cell and a loss of myofibrillar organization. Once the myocytes had attached to the culture substratum they began to spread out, underwent a reassembly of their contractile elements, resumed spontaneous contractions, and demonstrated ultrastructural evidence of protein synthesis.DNA synthetic ability was assessed by incubating the myocytes with $\sp3$H-thymidine. In the first study, myocytes cultured in serum-supplemented medium were incubated with $\sp3$H-thymidine for 24 hours at 10, 15, 20, and 30 days following isolation. The labeling indices were 20.5 $\pm$ 2.5, 26.5 $\pm$ 2.8, 10.5 $\pm$ 2.2, and 2.9 $\pm$ 0.6. In the second study, myocytes in serum-supplemented medium were incubated with $\sp3$H-thymidine continuously from 5 to 15 and 5 to 30 days following isolation. The labeling indices were 26.4 $\pm$ 4.9 and 34.5 $\pm$ 6.8 at 15 and 30 days following isolation, respectively. In the third study, myocytes were cultured in serum-free medium and incubated with $\sp3$H-thymidine continuously from 5 to 30 days. This study demonstrated that 8.3 $\pm$ 1.6% of the nuclei were labeled.Mitosis was observed in several of the myocytes 10 to 15 days following isolation. A significant number of the myocytes were observed to become binucleated or multinucleated at the later stages of culture.These results demonstrate that adult newt ventricular myocytes can be successfully placed into primary culture and are capable of resuming DNA synthesis and mitosis. This work may be considered as a foundation for future investigations which will focus on the mechanisms which control cardiac myocyte proliferation.

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