TROPHIC EFFECTS IN ADULT HEART CELL CULTURES

该项目的总体目标是开发和表征 成年大鼠心室肌细胞的可重复培养物。 以下 假设将被严格评估：儿茶酚胺具有主要 对成人培养心脏细胞的营养作用与我们发现的相似 在新生细胞中，特别是（1）刺激肥大生长 通过α 1-肾上腺素能受体和（2）收缩性的发展 通过α和β肾上腺素能受体的相互作用。 生长将作为培养物中的细胞附着和存活进行监测，并且 随后大小变化（肥大，如通过细胞蛋白定量， RNA、面积和体积）和有丝分裂活性指数（核计数和 DNA合成）。 自发性的变力性和变时性方面 将使用视频运动检测器来评估搏动。 有几个因素有助于增长和击败的能力， 取样，询问哪些参与营养效应。 这些包括 细胞内记录和膜片钳的电生理特性; 放射性配体法测定α和β肾上腺素能受体数量和亲和力 结合技术;腺苷酸环化酶活性;放射性标记的己糖和 肌球蛋白的积累和肌球蛋白同工酶的分布 凝胶电泳;和肌节形成，组装和排列成 光镜和电镜观察肌原纤维。 参考框架 这些参数的比较将是关于体内心脏的公开数据， 以及从新鲜分离的新生儿和成人细胞以及从 培养的新生细胞 因此，我们的工作旨在定义新的和重要的营养系统， 正常成人细胞中的儿茶酚胺。 异常肥大和/或 心肌疾病的收缩性可以反映这些异常， 监管系统和未来的工作可以测试这种可能性。 所有的分析方法都在我们的实验室使用。 在前期工作中， 培养成年大鼠心肌细胞，不含非心肌细胞 过度生长，并发现刺激蛋白质合成， 去甲肾上腺素 在我们的发展战略中， 培养系统是由培养的细胞分泌的细胞外基质， 内皮细胞和含转铁蛋白和胰岛素的无血清培养基。

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.