Cardiac Muscle: Thick Filament Structure and Proteins

虽然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还不清楚。最近的证据表明，贲门粗丝辅助蛋白肌球蛋白结合蛋白C（cMyBP-C）可能具有调节作用，以及在粗丝的结构作用。了解这种蛋白质的功能的重要性被以下事实所强调，即25%的家族性肥厚性甲状腺肿患者， 心肌病是一种遗传性心脏病，具有cMyBP-C突变。所提出的研究的目标是确定cMyBP-C的重要性，稳定的螺旋有序排列的肌球蛋白头上的粗丝和巨弹性蛋白肌联蛋白的绑定到粗丝。我们已经开发了一种分离心肌粗丝的方法，该方法保留了肌球蛋白头部和结构的有序排列。 最近，威斯康星州大学已经开发了cMyBP-C敲除小鼠，其中cMyBP-C在肌肉中缺失。这种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细节结构的影响; 2.）使用肌联蛋白的免疫标记以确定其是否保持附着于分离的心脏粗丝，并确定cMyBP-C是否是其与粗丝缔合所必需的;和3.）使用最近开发的冷冻负染色和单颗粒重建技术，以足够的分辨率计算分离的小鼠和兔心脏粗丝的3D重建，以显示cMyBP-C相对于肌球蛋白头的精确排列。来自正常小鼠和cMyBP-C敲除小鼠的细丝的重建的比较将用于明确地鉴定cMyBP-C在重建中的位置。这些研究将允许测试cMyBP-C对于稳定横桥阵列的螺旋排列和肌联蛋白与粗丝的结合是必要的假设。这些研究还将允许检验cMyBP-C的位置与其在细胞中的表达有关的假设。 相对于肌球蛋白头部的收缩与其与肌球蛋白头部的S2区域结合从而调节收缩一致。这些研究将为cMyBP-C在正常心脏和病变心脏中的作用提供重要信息。

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.