Cardiac Muscle: Thick Filament Structure and Proteins

心肌：粗丝结构和蛋白质

• 批准号: 7501359
• 负责人: ROBERT W KENSLER
• 金额: $ 23.38万
• 依托单位: UNIVERSITY OF PUERTO RICO MED SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7501359
• 关键词: Adrenergic Agents; Binding; Binding Sites; Biochemical; Calcium; Cardiac; Cardiac Muscle Contraction; Cellular biology; England; Excision; Familial Hypertrophic Cardiomyopathy; Filament; Genetic; Goals; Head; Heart; Heart Diseases; Inherited; Knockout Mice; Length; Life; Link; Location; Mediating; Modeling; Molecular; Mus; Muscle; Mutation; Myocardium; Myosin ATPase; Negative Staining; Numbers; Oryctolagus cuniculus; Patients; Phosphorylation; Physiological; Positioning Attribute; Procedures; Property; Proteins; Regulation; Relative (related person); Reporting; Resolution; Rest; Role; Sarcomeres; Skeletal Muscle; Staining method; Stains; Structure; Sturnus vulgaris; System; Techniques; Testing; Thick Filament; Thinking; Universities; Vertebral column; Wisconsin; adrenergic; bean; clinically relevant; connectin; myosin-binding protein C; particle; protein function; protein structure; reconstruction

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，但调节分子水平心脏肌肉收缩的因素尚不清楚。最近的证据表明，卡里达克厚细丝辅助蛋白肌球蛋白结合蛋白C（CMYBP-C）可能具有调节作用，并且在厚细丝中具有结构作用。了解该蛋白质功能的重要性是由25％的所有家族性肥大患者强调了 心肌病是一种遗传性心脏病，在CMYBP-C中有突变。拟议的研究的目的是确定CMYBP-C在稳定肌球蛋白头在厚细丝上的螺旋排列的重要性，以及巨型弹性蛋白滴定蛋白与厚细丝的结合。我们已经开发了一种隔离心肌厚细丝的程序，以保留肌球蛋白头和结构的有序排列。 最近，威斯康星大学已经开发了一种CMYBP-C的淘汰小鼠，其中肌肉中没有CMYBP-C。该鼠标提供了一个强大的系统，可用于检查CMYBP-C在厚细丝中的作用及其与其他蛋白质（例如Titin）的关系。该项目的具体目的是：1。）使用基因敲除小鼠进行CMYBP-C研究CMYBP-C丢失对孤立心脏厚细丝的详细结构的影响； 2.）使用免疫标记进行钛来确定它是否保持在孤立的心脏厚细丝上，并确定CMYBP-C是否与厚丝丝相关性是否需要；和3.）使用最近开发的冷冻阴性染色和单个颗粒重建技术来计算分离的小鼠和兔心脏厚细丝的3D重建，并具有足够的分辨率，以显示CMYBP-C相对于肌球蛋白头的精确排列。比较正常小鼠和CMYBP-C基因敲除鼠标的细丝重建，将用于确定识别CMYBP-C在重建中的位置。这些研究将允许测试CMYBP-C的假设，即稳定Crossbridge阵列的螺旋排列并结合Titin与厚细丝的结合是必要的。研究还将允许检验CMYBP-C位置的假设 相对于肌球蛋白头，它与肌球蛋白头的S2区域的结合一致，从而调节收缩。这些研究将提供有关CMYBP-C在正常心脏和患病心脏中的作用的重要信息。