PROTEIN TURNOVER IN CULTURED ADULT CARDIAC MYOCYTES

本研究计划的主要目标是评估 体液和机械因素如何调节 成年家兔心肌肌原纤维总蛋白和特异性蛋白 维持在细胞培养物中的肌细胞。 这些不扩散的 将用示踪剂氨基脉冲标记肌细胞制备物 酸来测量蛋白质合成的分数率， 在改变胰岛素、甲状腺激素和胰岛素后的特定时间间隔内 激素和去甲肾上腺素水平 同样，这些静止的 将刺激肌细胞搏动或周期性伸展， 确定这些物理参数如何改变蛋白质周转。 蛋白质合成分数率的变化也将是 相比于总poly A信使RNA含量的改变， 为了确定是否消息转录和速率 蛋白质的合成也会同步波动。 一个少校的表情 肌原纤维蛋白，肌球蛋白重链（MHC），将被 通过使用cDNA探针进行研究，以确定是否 MHC基因的转录响应于改变而改变， 培养条件 肌球蛋白的亚细胞分布 也可以通过免疫荧光和免疫电子 使用抗肌球蛋白单克隆抗体的显微术 尤其是MHC α的结构组织 收缩器将在显微镜下进行形态学检查， 试图描述这些复杂的蛋白质是如何组装的 并在改变分数的条件下分解 特异性myobifrillar蛋白的合成和降解速率。 溶酶体空泡器在介导细胞凋亡中的作用 总的和特定的心脏蛋白质的降解也将是 追求。 本实验旨在估计 激素调节、收缩活动和 被动牵张对心肌细胞合成和降解的影响 蛋白 这些研究将提供基本信息， 培养的心肌细胞调节它们的蛋白质组成， 推进我们对控制心脏的机制的理解 在不同的生理和病理生理状态下的肿块。

The principal objective of the present research plan is to evaluate how humoral and mechanical factors regulate the turnover of total and specific myofibrillar proteins in adult rabbit cardiac myocytes maintained in cell culture. These nonproliferating myocyte preparations will be pulse labeled with tracer amino acids to measure the fractional rates of protein synthesis and degradation at specific intervals after altering insulin, thyroid hormone and norepinephrine levels. Similarly, these quiescent myocytes will be stimulated to beat or periodically stretched to determine how these physical parameters alter protein turnover. Changes in the fractional rate of protein synthesis will also be compared to alterations in total poly A messenger RNA content in order to determine whether message transcription and rate of protein synthesis fluctuate in parallel. The expression of a major myofibrillar protein, myosin heavy chain (MHC), will be investigated by employing a cDNA probe to determine whether the transcription of MHC genes is altered in response to changing culture conditions. The subcellular distribution of myosin will also be monitored by immunofluorescent and immunoelectron microscopy using monoclonal antibodies directed against myosin and, in particular, the MHC alpha. The structural organization of the contractile apparatus will be examined morphometically in an attempt to describe how this complex set of proteins is assembled and broken down under conditions that alter the fractional synthesis and degradation rates of specific myobifrillar proteins. The role of the lysosomal vacuolar apparatus in mediating the degradation of total and specific cardiac proteins will also be pursued. The present experiments are designed to estimate the contribution of hormonal modulation, contractile activity and passive stretch on the synthesis and degradation of myocytic protein. These studies will provide basic information on how cultured cardiac myocytes regulate their protein composition and advance our understanding of the mechanisms that control cardiac mass under differing physiologic and pathophysiologic states.