PROTEIN TURNOVER IN CULTURED ADULT CARDIAC MYOCYTES

循环激素的波动，自主神经的激活 系统和改变的机械工作负载被认为是潜在的 发展中的肥厚性心脏病的介质。 如何转导 神经体液和负荷依赖性信号调节成体肌细胞生长 全心准备已难以追求；然而，成人 长期培养的猫肌细胞提供了一种独特的方法 直接探索调节心肌细胞生长的细胞机制。 必须在合成速率和合成速率之间取得良好的平衡 如果心脏要保持其正常大小，则需要降解心脏蛋白质 和功能。 尽管人们对这种物质的合成方面了解很多 方程，实际上对收缩蛋白的速率一无所知 退化或调节其的信使和机制 崩溃。 该提案的主要目标是确定 总蛋白、收缩蛋白和细胞骨架蛋白降解率 培养的成体肌细胞，其中实验条件可以精确 定义和控制。 该制剂的长期稳定性 促进蛋白质分解的研究，因为其半衰期 成人收缩蛋白以天而不是小时为单位进行测量。 至 测量长寿命和短寿命蛋白质、肌细胞的降解 将被生物合成标记为[14C]和[3H]氨基酸并且 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。 将测量降解率 总蛋白和纯化蛋白。 平行免疫荧光和 将采用免疫电子显微镜方法来关联 蛋白质水解的改变与重组的任何变化 收缩装置。 将研究四种蛋白水解途径 确定它们是否参与特定类的降级 肌细胞蛋白质。 此外，cAMP 的产生、磷酸肌醇 将监测周转率和 [Ca2 ]i，以评估这些第二 信使直接激活特定的蛋白水解途径。 自成年以来 心脏细胞合成蛋白质的速度与文献中记载的相同 整个动物，这种稳定的肌细胞培养模型应该揭示 关于调节蛋白质机制的基本新观察 成年人心里的周转。 因此，本节中概述的实验 该提案将为评估是否存在任何问题提供宝贵的见解 蛋白质降解率的改变可能会限制心肌细胞的生长 最终，心脏过程中发生的病理生理变化 肥大。

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.