PROTEIN TURNOVER IN CULTURED ADULT CARDIAC MYOCYTES

培养的成年心肌细胞中的蛋白质周转率

• 批准号: 3345668
• 负责人: ROBERT S DECKER
• 金额: $ 16.73万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-09-30 至 1996-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3345668
• 关键词: cyclic AMP; cytoskeletal proteins; fluorescence microscopy; genetic transcription; heart cell; heart metabolism; hormone regulation /control mechanism; immunoelectron microscopy; immunofluorescence technique; inositol phosphates; laboratory rabbit; membrane proteins; messenger RNA; myofibrils; myosins; nucleic acid probes; protein degradation; proteolysis; second messengers; tissue /cell culture; ventricular hypertrophy

Fluctuations in circulating hormones, activation of the autonomic nervous system and altered mechanical work load are implicated as potential mediators of evolving hypertrophic heart disease. How the transduction of neurohumoral and load dependent signals modulate adult myocyte growth has been difficult to pursue in whole heart preparations; however, adult feline myocytes maintained in long term culture provide a unique approach to directly explore the cellular mechanisms that regulate myocyte growth. A fine balance must be struck between the rates of synthesis and degradation of cardiac proteins if the heart is to retain its normal size and function. Although much is known about the synthetic side of this equation, virtually nothing is known about rates of contractile protein degradation or the messengers and mechanisms that regulate their breakdown. The principal objective of this proposal is to determine the rates of total, contractile and cytoskeletal protein degradation in the cultured adult myocyte where experimental conditions can be precisely defined and controlled. The long-term stability of this preparation facilitates the study of protein breakdown because the half-lives of adult contractile proteins are measured in days rather than hours. To measure the degradation of long-lived and short-lived proteins, myocytes will be biosynthetically labeled with [14C] and [3H] amino acids and the radiolabeled heart cells then will be exposed to neurohumoral factors and/or mechanical stretch. Rates of degradation will be measured for total and purified proteins. Parallel immunofluorescence and immunoelectron microscopic approaches will be employed to correlate alterations in proteolysis with any changes in the reorganization of the contractile apparatus. Four proteolytic pathways will be investigated to determine whether they participate in the degradation of specific-classes of myocyte proteins. In addition, cAMP production, inositol phosphate turnover and [Ca2+]i will be monitored to assess whether these second messengers directly activate specific proteolytic pathways. Since adult heart cells synthesize proteins at rates identical to those documented in the whole animal, this stable myocyte culture model should divulge fundamental new observations on the mechanisms that regulate protein turnover in the adult heart. As such, the experiments outlined in this proposal will provide valuable insight into assessing whether any alteration in the rate of protein degradation may limit myocyte growth and, ultimately, pathophysiologic changes that develop during cardiac hypertrophy.

循环激素波动，自主神经激活 系统和改变机械工作负荷被暗示为潜在的 肥大性心脏病的介质。 传导是如何 神经体液和负荷依赖性信号调节成年心肌细胞生长 很难在全心的准备中追求;然而，成年人 长期培养的猫肌细胞提供了一种独特的方法， 直接探索调节肌细胞生长的细胞机制。 必须在合成速率和 如果心脏要保持其正常大小， 和功能 尽管我们对这种物质的合成方面了解很多， 方程，几乎没有什么是已知的收缩蛋白的速率 降解或信使和机制，调节他们的 崩溃 本建议的主要目的是确定 细胞总蛋白、收缩蛋白和细胞骨架蛋白降解率 培养的成年心肌细胞，实验条件可以精确地 定义和控制。 该制剂的长期稳定性 有助于研究蛋白质分解，因为 成体收缩蛋白以天而不是小时来测量。 到 测量长寿命和短寿命蛋白质、肌细胞 将用[14 C]和[3 H]氨基酸生物合成标记， 然后放射性标记的心脏细胞将暴露于神经体液因子 和/或机械拉伸。 将测量降解速率， 总蛋白和纯化蛋白。 平行免疫荧光和 免疫电镜方法将被用来关联 蛋白质水解的改变， 收缩器 将研究四种蛋白水解途径， 确定它们是否参与特定类别的退化 肌细胞蛋白质。 此外，cAMP的产生，磷酸肌醇 将监测周转率和[Ca 2 +]i，以评估这些第二次 信使直接激活特定的蛋白水解途径。 从成人开始 心脏细胞合成蛋白质的速率与 整个动物，这个稳定的心肌细胞培养模型应该泄露 对蛋白质调节机制的基本新观察 成人心脏的周转。 因此，本文中概述的实验 建议将提供宝贵的见解，以评估是否有任何 蛋白质降解速率的改变可能限制肌细胞的生长 最终，在心脏病发作期间发生的病理生理变化 肥厚