Cardiac Muscle: Functional Role of Myosin - Binding Protein C in Contraction

描述(申请人提供)：粗丝相关蛋白cMyBP-C的突变是家族性肥厚型心肌病的主要原因。心肌肌球蛋白结合蛋白-C突变的病理效应与越来越多的证据一致，即它在心肌收缩中既起结构作用又起调节作用。尽管表明cMyBP-C在心肌收缩中的调节作用的证据似乎令人信服，但其作用机制还远未明确。大多数关于cMyBPC的功能作用及其磷酸化影响的研究都是在主要含有α-肌球蛋白重链的大鼠或小鼠心肌中进行的，尽管有证据表明，含有(3-肌球蛋白重链)的粗丝(如在人类中)在cMyBP-C磷酸化时所显示的变化可能不同。这表明，更详细地研究cMyBP-C的磷酸化对大鼠以外物种心肌粗丝结构的影响是很重要的；包括具有3-肌球蛋白重链的物种，如兔。此外，越来越多的证据表明，cMyBP-C的N末端片段可能不依赖于cMyBP-C将肌球蛋白S2区域拴在肌丝骨架上而影响心肌的收缩能力，也可能能够直接结合肌动蛋白。这些研究强调需要观察这些N末端片段的结合对分离的心脏粗丝的跨桥排列和结构的影响；并通过电子显微镜进一步评估cMyBP-C的这些末端片段与肌动蛋白的结合潜力。此外，为了充分了解cMyBP-C在粗丝中的功能，拥有比目前可用的更高分辨率的心脏粗丝30重建是至关重要的。本研究的具体目的是：1)确定cMyBP-C的磷酸化对小鼠、大鼠和兔心脏粗丝交叉桥排列的影响；2)确定cMyBP-C的C_1-C2和C_1 N端片段对兔心脏、野生型和cMyBP-C“‘”敲除小鼠心脏的交叉桥排列的影响；3)评估c MyBP-C的C_1-C2和C_2 N端片段与肌动蛋白的结合；4)利用单粒子分析方法对小鼠和兔的心脏粗丝进行高分辨率三维重建，精确定位肌球蛋白头部和cMyBP-C。

DESCRIPTION (provided by applicant): Mutations in the thick filament associated protein cMyBP-C are a major cause of familial hypertrophic cardiomyopathy. The pathological effects of mutations in cardiac myosin binding protein-C are consistent with increasing evidence that it plays both a structural and a regulatory role in myocardial contraction. Although the evidence suggesting a regulatory role for cMyBP-C in myocardial contraction appears compelling, the mechanism of action is far from clear. Most of the research on the functional role of cMyBPC and the effect of its phosphorylation has been done in rat or mouse myocardium which contains primarily the a-myosin heavy chain, despite evidence that thick filaments containing the (3-myosin heavy chain (such as in humans) may differ in the changes shown upon phosphorylation of cMyBP-C. This suggests the importance of investigating in more detail the effect of phosphorylation of cMyBP-C on the structure of the myocardial thick filament in species other than the rat; including species with the (3-myosin heavy chain such as the rabbit. In addition, increasing evidence suggests that N-terminus fragments of cMyBP-C may be able effect myocardial contractility independent of a tethering of the myosin S2 region to the backbone of the filament by cMyBP-C, and may also be able to directly bind actin. These studies emphasize the need to look at the effects of binding of these N-terminus fragments on the cross bridge arrangement and structure of the isolated cardiac thick filament; and further assess by electron microscopy the binding potential of these Nterminus fragments of cMyBP-C to actin. In addition in order to fully understand how cMyBP-C functions in the thick filament, it is vital to have higher resolution 30-reconstructions of the cardiac thick filaments than are currently available. The specfic aims of the research are to: 1) Determine the effect of phosphorylation of cMyBP-C on the crossbridge arrangement of isolated mouse, rat, and rabbit cardiac thick filaments; 2) Determine the effect of the C1-C2 and CO-C1 N-terminus fragments of cMyBP-C on the crossbridge arrangement of the isolated thick filaments from rabbit heart, and both wildtype and cMyBP-C"'" knockout mouse hearts; 3) Assess the of binding of C1-C2 and CO-C2 N-terminus fragments of cMyBP-C to actin; and 4) Compute higher resolution 3D-reconstructions of the cardiac thick filaments from both mouse and rabbit thick filaments by single particle analysis to precisely locate the myosin heads and cMyBP-C.