Cardiac Muscle: Thick Filament Structure and Proteins

心肌：粗丝结构和蛋白质

• 批准号: 6766620
• 负责人: ROBERT W KENSLER
• 金额: $ 14.3万
• 依托单位: UNIVERSITY OF PUERTO RICO MED SCIENCES
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-08-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6766620
• 关键词: SDS polyacrylamide gel electrophoresis; actins; binding proteins; electron microscopy; genetically modified animals; immunocytochemistry; laboratory mouse; laboratory rabbit; microfilaments; muscle contraction; muscle function; muscle proteins; myocardium; myosin light chain kinase; myosins; phosphorylation; protein isoforms; protein protein interaction; protein structure function; troponin

Although a beating heart is vital for life, the factors which regulate contraction of cardiac muscle at the molecular level are not well understood. Recent evidence has shown that the caridac thick filament accessory protein myosin binding protein C (cMyBP-C) may have a regulatory role as well as a structural role in the thick filament. The importance of understanding the function of this protein is underscored by the fact that 25% of all patients with familial hypertrophic cardiomyopathy, an inherited cardiac disease, have a mutation in cMyBP-C. The goal of the proposed studies is to determine the importance of cMyBP-C for stabilizing the helically ordered arrangement of the myosin heads on the thick filament and for the binding of the giant elastic protein titin to the thick filament. We have developed a procedure for isolating cardiac muscle thick filaments that preserves the ordered arrangement of the myosin heads and structure. Recently, a knockout mouse for cMyBP-C in which cMyBP-C is absent from the muscle has been developed at the University of Wisconsin. This mouse provides a powerful system for examining the role of cMyBP-C in the thick filament and its relation to other proteins such as titin. The specific Aims of the project are to: 1 .) use the knockout mouse for cMyBP-C to study the effect of loss of cMyBP-C on the detailed structure of the isolated cardiac thick filament; 2.) use immunolabeling for titin to determine if it remains attached to the isolated cardiac thick filaments and to determine if cMyBP-C is necessary for its association with the thick filament; and 3.) use the recently developed technique of cryo-negative staining and single particle reconstruction to compute 3D-reconstructions of isolated mouse and rabbit cardiac thick filaments with sufficient resolution to show the precise arrangement of cMyBP-C relative to the myosin heads. Comparison of the reconstructions of the filaments from the normal mouse and the cMyBP-C knockout mouse will be used to definitively identify the location of cMyBP-C in the reconstructions. These studies will allow testing of the hypotheses that cMyBP-C is necessary to stabilize the helical arrangement of the crossbridge array and for binding of titin to the thick filament. The studies will also allow testing of the hypothesis that the location of cMyBP-C relative to the myosin head is consistent with its binding to the S2 region of the myosin head thus regulating contraction. These studies will provide significant information on the role of cMyBP-C in both the normal heart and in the diseased heart.

尽管跳动的心脏对生命至关重要，但在分子水平上调节心肌收缩的因素尚不清楚。最近的证据表明，心粗丝辅助蛋白肌球蛋白结合蛋白 C (cMyBP-C) 可能在粗丝中具有调节作用和结构作用。 25% 的家族性肥厚患者中有 25% 患有家族性肥厚症，这一事实强调了了解这种蛋白质功能的重要性。 心肌病是一种遗传性心脏病，cMyBP-C 存在突变。拟议研究的目的是确定 cMyBP-C 对于稳定粗丝上肌球蛋白头的螺旋有序排列以及巨型弹性蛋白肌联与粗丝结合的重要性。我们开发了一种分离心肌粗丝的程序，可以保留肌球蛋白头部和结构的有序排列。 最近，威斯康星大学开发出了一种 cMyBP-C 基因敲除小鼠，其中肌肉中不存在 cMyBP-C。该小鼠提供了一个强大的系统，用于检查 cMyBP-C 在粗丝中的作用及其与其他蛋白质（如肌联蛋白）的关系。该项目的具体目标是： 1）利用cMyBP-C基因敲除小鼠，研究cMyBP-C缺失对离体心脏粗丝详细结构的影响； 2.) 使用肌联蛋白的免疫标记来确定它是否仍然附着在分离的心脏粗丝上，并确定 cMyBP-C 是否是其与粗丝关联所必需的； 3.) 使用最近开发的冷冻阴性染色和单粒子重建技术来计算离体小鼠和兔子心脏粗丝的 3D 重建，具有足够的分辨率，以显示 cMyBP-C 相对于肌球蛋白头的精确排列。正常小鼠和 cMyBP-C 敲除小鼠的肌丝重建的比较将用于明确识别重建中 cMyBP-C 的位置。这些研究将允许测试以下假设：cMyBP-C 对于稳定横桥阵列的螺旋排列以及肌联蛋白与粗丝的结合是必需的。这些研究还将检验 cMyBP-C 位置的假设 与肌球蛋白头的相对关系与其与肌球蛋白头的 S2 区域的结合一致，从而调节收缩。这些研究将提供关于 cMyBP-C 在正常心脏和患病心脏中的作用的重要信息。