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荷被认为是潜在的 演变中的肥厚性心脏病的中介物。转导是如何 神经体液和负荷依赖信号调节成人心肌细胞生长 很难在全心全意的准备中进行；然而，成年人 长期培养的猫科动物心肌细胞提供了一种独特的方法 直接探索调控心肌细胞生长的细胞机制。 必须在合成速度和合成速度之间取得微妙的平衡 如果心脏要保持正常大小，心脏蛋白质的降解 和功能。尽管人们对这一现象的合成方面已有很多了解 方程式中，关于收缩蛋白的速率几乎一无所知 退化或信使和调节它们的机制 崩溃了。这项建议的主要目标是确定 总蛋白、收缩蛋白和细胞骨架蛋白降解率 培养的成年心肌细胞的实验条件可以精确地 定义和控制。该制剂的长期稳定性 有助于蛋白质分解的研究，因为蛋白质的半衰期 成人收缩蛋白是以天而不是小时来测量的。至 测量长寿命和短寿命蛋白、心肌细胞的降解 将被生物合成地标记为[14C]和[3H]氨基酸，以及 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 将使用免疫电子显微镜方法来关联 蛋白分解的改变与重组的任何变化 收缩装置。四条蛋白质分解途径将被研究以 确定他们是否参与了特定班级的退化 肌细胞蛋白。此外，cAMP生产、肌醇磷酸 将监测成交量和[Ca~(2+)]i以评估这些秒 信使直接激活特定的蛋白分解途径。从成年开始 心脏细胞合成蛋白质的速度与文献中记录的相同 整个动物，这个稳定的心肌细胞培养模型应该泄露 蛋白质调控机制的新发现 成人心的周转。因此，本文中概述的实验 建议书将为评估是否有任何 蛋白质降解率的改变可能会限制心肌细胞的生长 最终，心脏疾病期间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.