PROPERTIES OF CULTURED ADULT MYOCYTES

使心脏蛋白体外研究复杂化的两个主要问题 代谢是可用制剂中细胞类型的异质性 (i.e.,成纤维细胞、内皮细胞）和存活时间短 从成年动物获得的制剂（例如，灌注的心脏， 乳头肌）。 前者导致解释上的歧义 结果（即，是由于肌细胞Tenon肌细胞而观察到的结果） 而后者阻碍了对重要的长寿心脏蛋白的研究 感兴趣的（例如肌球蛋白）。 由于这些原因， 建议是a）开发必要的技术和方法， 维持耐钙成年兔心室肌细胞的纯培养 在培养物中培养肌细胞长达7-14天; B）全面 将该制剂中蛋白质和氨基酸代谢表征为 与目前在其他国家获得的这些过程的可用数据相比， 常用的体外兔心脏制备物;和c）仅在 已经进行了充分的表征研究，以启动研究 的蛋白质周转和溶酶体蛋白酶代谢。 成年兔心肌细胞培养的初步方法， 由PI定义。 这种方法允许稳定培养 相对纯的成人肌细胞制备物4天。 技术来 消除培养物中的所有成纤维细胞污染，延长培养时间， 存活7-14天，并表征蛋白质和氨基酸代谢 在本提案中提出了这一准备工作。 我们认为， 新开发的制剂必须在 启动当前感兴趣的关于蛋白质特定方面的实验 新陈代谢. 因此，尽管提出了一些这样的实验，但大部分实验都是在实验中进行的。 该提案确定了改进养殖技术的方法， 记录制备中关于蛋白质的“稳态”， 氨基酸代谢

Two major problems which complicate all in vitro studies of cardiac protein metabolism are the heterogeneity of cell type in available preparations (i.e., fibroblasts, endothelial cells) and the short duration of viability of preparations obtained from adult animals (e.g., perfused hearts, papillary muscles). The former results in an ambiguity of interpretation of results (i.e., is the observed result due to myocytenon myocyte cells) while the latter prevents studies on important long lived cardiac proteins of interest (e.g. myosin). For these reasons the specific goals of the proposal are a) to develop techniques and the methodology requisite to maintain pure cultures of calcium tolerant adult rabbit-ventricular myocytes in culture for periods upto 7-14 days; b) to comprehensively characterize protein and amino acid metabolism in this preparation as compared to presently available data of these processes obtained in other commonly used in vitro rabbit cardiac preparations; and c) only after adequate characterization studies have been performed, to initiate studies of protein turnover and lysosomal protease metabolism in this preparation. Preliminary methodology for culture of adult rabbit myocytes has been defined by the P.I. This methodology allows for stable culture of a relatively pure adult myocyte preparation for 4 days. Techniques to eliminate all fibroblast contamination in the culture, prolong culture viability to 7-14 days and characterize protein and amino acid-metabolism in this preparation are presented in this proposal. We believe such a newly developed preparation must be comprehensively characterized prior to initiating experiments of current interest on specific aspects of protein metabolism. Thus, although some such experiments are proposed, the bulk of this proposal defines methodology for improving culture technique and documenting a "steady state" in the preparation as regards protein and amino acid metabolism.