Primary cell culture and morphological characterization of ventricular myocytes from the adult newt, notophthalmus viridescens

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Abstract
Previous work has demonstrated that adult newt cardiac myocytes possess a proliferative ability in response to an experimentally induced in jury, in vivo. This study describes an in vitro model in which the proliferative events of the adult cardiac myoctye may be studied. Ventricles were minced and then enzymatically dissociated in a Ca++- and MG++-free salt solution containing 0.5% trypsin and 625 U/ml of CLS II collagenase for 8 to 10 hours at 25°C. Enzyme digests were preplated and then cultured on bovine corneal endothelial-derived basement membrane “carpets” in either serum-free or serumsupplemented modified Leibovitz's medium for up to 30 days. Light and transmission electron microscopic characterization demonstrated that a majority of the myocytes underwent an initial period of disorganization 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. Mitosis was observed in several myocytes 8 to 15 days following isolation. In 15-day serum-supplemented and serum-free cultures, 6.5% ± 0.9% and 8.1% ± 1.4% of the myocytes were binucleated, respectively. These results demonstrate that adult newt ventricular myocytes can be successfully placed into primary culture and are capable of undergoing mitosis. This work may be considered as a foundation for future investigations which will focus on the mechanisms which control cardiac myocyte proliferation.