Regulation and Maintenance of Cardiac Muscle Sarcomere Integrity

描述（由申请人提供）：心肌肌节是一个复杂且高度有序的收缩和调节蛋白集合，旨在产生力。为了维持肌节的功能，需要精确的蛋白质周转，以平衡新蛋白质的合成和结合到肌节中，同时去除和降解磨损或受损的蛋白质。鉴于蛋白质周转率的异质性，个体肌节成分在保持肌节结构功能完整性的同时发生独立周转率的机制尚不清楚。几项研究表明，包括肌动蛋白和肌球蛋白在内的许多肌丝蛋白与细胞质前体池处于动力学平衡状态，这表明磨损的肌丝蛋白不断替代其前体进入其他完整的肌瘤。然而，随着蛋白质大小的增加，出现了一些问题，使简单的肌节蛋白质交换变得不可能。首先，维持高分子量蛋白质的前体池需要肌细胞付出越来越多的能量代价。另一个问题是，与在现有肌节中不断循环的较小的肌丝蛋白不同，肌纤维大分子复合物的周转可能需要部分或完全分解肌节。这些考虑将我们的注意力集中在调节巨肌丝蛋白titin周转的分子事件上。肌凝蛋白是肌节复合体的组成部分，作为(1)肌凝蛋白和其他结构和信号蛋白聚集的分子模板，以及(2)赋予心脏肌原纤维硬度的分子弹簧。因此，我们假设titin的降解会引发局部肌节的分解。基于初步数据，我们提出titin的氧化损伤触发钙蛋白酶和泛素蛋白酶体系统的顺序降解。我们认为肌节机械传感器在titin降解过程中被激活，并转运到细胞核以激活titin基因转录。我们认为titin mRNA是针对肌节的，局部的titin合成发生在肌节重组的同时。我们最后提出心脏肥厚调节titin转录和翻译途径，导致净肌节增加。提出的实验目的将使我们能够评估和完善该模型，并促进我们对肌丝肌节转换的复杂生理和时间方面的理解。

DESCRIPTION (provided by applicant): The cardiac sarcomere is a complex and highly ordered ensemble of contractile and regulatory proteins designed to generate force. To maintain functional sarcomeres, precise turnover of proteins is required that balances new protein synthesis and incorporation into the sarcomere with removal and degradation of worn out or damaged proteins. Given the heterogeneity in protein turnover rates, the mechanisms by which independent turnover of the individual sarcomere components occurs while maintaining the functional integrity of the sarcomere structure, is not well understood. Several studies have shown that a number of myofilament proteins, including actin and myosin, are in kinetic equilibrium with a cytoplasmic precursor pool, suggesting continual replacement of worn out myofilament proteins for their precursors into an otherwise intact sarcomere. As the size of the protein increases, however, several problems arise that make simple sarcomere protein exchange improbable. For one, the maintenance of a precursor pool of high molecular weight proteins comes at an increasing energetic cost to the myocyte. Another problem is that unlike smaller myofilament proteins that are continually recycled in the existing sarcomere, turnover of myofibrillar macromolecular complexes likely requires either partial or complete disassembly of the sarcomere. These considerations have focused our attention on the molecular events regulating the turnover of the giant myofilament protein titin. Titin is an integral part of the sarcomere complex, serving as (1) a molecular template around which the myosins and other structural and signaling proteins assemble, and (2) a molecular spring to impart myofibrillar stiffness to the heart. We therefore postulate that the degradation of titin will trigger local disassembly of the sarcomere. Based on preliminary data, we propose that oxidative damage to titin triggers sequential degradation by the calpains and ubiquitin-proteasome system. We propose that sarcomere mechanosensors are activated during the process of titin degradation, and translocate to the nucleus to activate titin gene transcription. We propose that titin mRNA is targeted to the sarcomere and that localized titin synthesis occurs with concurrent reassembly of the sarcomere. We finally propose that cardiac hypertrophy modulates titin transcription and translation pathways leading to net sarcomere addition. The proposed experimental aims will allow us to evaluate and refine this model, and advance our understanding of the complex physiological and temporal aspects of myofilament sarcomere turnover. PUBLIC HEALTH RELEVANCE: The cardiac sarcomere is the basic contractile unit of the heart and is composed of an ensemble of myofilament and regulatory proteins designed to generate force. It is generally assumed that maintenance of functioning sarcomeres requires precise control of synthesis, assembly, and degradation of myofilament proteins, however, the mechanisms that mediate turnover of large integrated myofilament proteins, such as titin, have not been looked at. Understanding the molecular mechanisms of titin protein turnover will provide insight into the regulatory processes of sarcomere maintenance, and may ultimately lead to novel therapeutic approaches for heart failure cardiomyopathies.