TROPHIC EFFECTS IN ADULT HEART CELL CULTURES

点击翻译按钮获取中文摘要

The overall goal of this project is to develop and characterize reproducible cultures of adult rat ventricular myocytes. The following hypothesis will be critically evaluated: that catecholamines have major trophic effects on adult cultured heart cells similar to those found by us in neonatal cells, specifically (1) stimulation of hypertrophic growth through an alpha1-adrenergic receptor and (2) development of contractility through an alpha-and beta-adrenergic receptor interaction. Growth will be monitored as cell attachment and survival in culture, and subsequent change in size (hypertrophy, as quantitated by cell protein, RNA, area, and volume) and indices of mitotic activity (nuclear counts and DNA synthesis). Both inotropic and chronotropic aspects of spontaneous beating will be evaluated using a videomotion detector. Several factors that subserve the ability to grow and to beat will be sampled, asking which participate in the trophic effects. These include electrophysiologic properties by intracellular recording and patch clamp; alpha- and beta-adrenergic receptor number and affinity by radioligand binding techniques; adenylate cyclase activity; radiolabeled hexose and calcium transport; myosin accumulation and myosin isoenzyme distribution by gel electrophoresis; and sarcomere formation, assembly, and alignment into myofibrils by light and electron microscopy. Frames of reference for comparison of these parameters will be published data on the heart in vivo, and data obtained from freshly isolated neonatal and adult cells and from cultured neonatal cells. Thus our work seeks to define new and important trophic systems involving catecholamines in normal adult cells. Abnormal hypertrophy and/or contractility in myocardial disease could reflect aberrancies in these regulatory systems, and future work could test this possibility. All analytic methods are in use our laboratories. In preliminary work we have cultured adult rat heart myocytes without nonmyocardial cell overgrowth and have found stimulation of protein synthesis by norepinephrine. Two critical techniques in our strategy for developing the culture system are the extracellular matrix secreted by cultured endothelial cells and serum-free medium with transferrin and insulin.