PROTEIN TURNOVER IN CULTURED ADULT CARDIAC MYOCYTES

本研究计划的主要目标是评价 体液和机械因素是如何调节人的更替的 成年兔心脏肌原纤维蛋白的总蛋白和特异性蛋白 在细胞培养中维持肌细胞。这些非增殖性 肌细胞制剂将用示踪剂氨基脉冲标记 酸来测量蛋白质合成和蛋白质合成的分数 改变胰岛素、甲状腺后特定时间间隔的降解 荷尔蒙和去甲肾上腺素水平。同样，这些静止的 肌细胞会被刺激跳动或周期性地伸展到 确定这些物理参数如何改变蛋白质周转。 蛋白质合成分数的变化也将是 与总Poly A信使RNA含量的变化相比 以确定报文转录和速率是否 蛋白质的合成是平行波动的。一位少校的表达 肌原纤维蛋白，肌球蛋白重链(MHC)，将是 通过使用cdna探针来确定 MHC基因的转录会因改变而改变 培养条件。肌球蛋白Will的亚细胞分布 也可以通过免疫荧光和免疫电子技术进行监测 使用针对肌球蛋白的单抗进行显微镜检查 尤其是MHC阿尔法。的结构组织 将对收缩装置进行形态计量学检查 试图描述这组复杂的蛋白质是如何组装的 并在改变分数的条件下被分解 特定肌管蛋白的合成和降解速率。 溶酶体空泡器在调节血管紧张素转换酶活性中的作用 总的和特定的心脏蛋白的降解也将 被追捕。目前的实验是为了估计 荷尔蒙调节、收缩活动和 被动拉伸对肌细胞合成和降解的影响 蛋白。这些研究将提供有关如何 培养的心肌细胞调节其蛋白质组成和 促进我们对心脏控制机制的理解 不同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.