ROLE OF MYOFILAMENT BOUND CALCIUM IN MUSCLE CONTRACTION

描述(申请人的逐字描述)：长期目标 本研究旨在了解肌浆网升高的机制。 游离钙调节脊椎动物横纹肌的收缩。在……里面 特别是，目的是阐明钙与肌丝结合的方式 对收缩事件进行调制并被其调制。都是跨桥 肌动蛋白的附着和肌钙蛋白C(TNC)的钙结合现在被认为是 调节细丝的激活状态，但相对 每一项的作用和重要性都很难确定，因为许多 实验条件对跨桥结合和钙结合都有影响。 拟议的实验不仅提供了对 钙结合量，以及肌节内钙结合的空间分布。 心肌和骨骼肌处于不同的激活水平。建议 实验将扩展之前的工作，以调查骑自行车 交叉桥影响骨骼肌和心脏肌丝与钙的结合 肌肉，以及这种作用是否依赖于TNC亚型。他们会 也要检查肌球蛋白与钙的结合是否被肌球蛋白增强 相互作用，以及肌丝蛋白Titin是否在 激活条件。钙沿肌节的分布将是 使用定量电子探针X射线显微分析(EPXMA)和 兔腰大肌甘油化冷冻干燥切片的元素成像 比目鱼肌和兔、蛙心肌。样品将被冷冻在 含或不含镁三磷酸腺苷的溶液，以及不同水平的游离钙。 对肌原纤维蛋白成分的修改将包括提取 TNC、肌球蛋白轻链和肌动蛋白，以及TNC亚型的交换(骨骼， 心脏或具有修饰的钙结合特性的突变体)。跨桥 附件将由BDM、正钒酸酯、 N-苯基马来酰亚胺。这些操作将确定两个病例的位置 绑定，以及此绑定受交互影响的程度 肌丝蛋白之间。交叉桥效应对TNC钙结合的影响 提出为Frank-Starling关系提供分子基础，以及 可能是横纹肌收缩事件的其他特征。这些 研究将提供对调节的基本机制的洞察 骨骼肌和心肌力的激活。

DESCRIPTION (the applicant's description verbatim): The long term objective of this research is to understand the mechanisms by which elevation in myoplasmic free calcium regulates contraction of vertebrate striated muscle. In particular, the aim is to clarify the way calcium binding to myofilaments modulates and is modulated by the contractile event. Both cross-bridge attachment to actin and calcium binding to troponin C (TnC) are now believed to regulate the state of activation of the thin filament, however the relative role and importance of each has been difficult to determine because many experimental conditions affect both cross-bridge binding and calcium binding. The experiments proposed provide independent measurements not only of the amount, but also of the spatial distribution of Ca binding within sarcomeres of cardiac and skeletal muscle at different levels of activation. Proposed experiments will extend previous work to investigate whether cycling cross-bridges affect Ca binding to myofilaments in both skeletal and cardiac muscle, and whether such effects are dependent on the TnC isoform. They will also examine whether calcium binding to myosin is enhanced by actomyosin interaction, and whether the myofilament protein, titin, binds calcium under activating conditions. Distributions of calcium along the sarcomere will be determined using quantitative electron probe X-ray microanalysis (EPXMA) and elemental imaging of freeze dried cryosections of glycerinated rabbit psoas and soleus muscles and rabbit and frog cardiac muscle. Samples will be frozen in solutions with or without MgATP and with varying levels of free calcium. Modifications of myofibrillar protein composition will include extraction of TnC, myosin light chains and actin, and exchange of TnC isoforms (skeletal, cardiac or a mutant with modified Ca binding properties). Cross-bridge attachments will be modified by agents such as BDM, orthovanidate, N-phenylmaleimide. These manipulations will identify both the sites of Ca binding, and the degree to which this binding is affected by interactions between myofilament proteins. Crossbridge effects on TnC Ca binding are proposed to provide the molecular basis for the Frank-Starling relation, and may underlie other features of the contractile event in striated muscle. These studies will provide insight into the fundamental mechanisms which modulate activation of force in skeletal and cardiac muscle.