ROLE OF MYOFILAMENT BOUND CALCIUM IN MUSCLE CONTRACTION

描述（申请人的逐字描述）：长期目标 这项研究旨在了解肌质升高的机制 游离钙调节脊椎动物横纹肌的收缩。在 特别是，目的是阐明钙与肌丝结合的方式 调节收缩事件并被收缩事件调节。两者都过桥 现在认为，肌动蛋白的附着和钙与肌钙蛋白 C (TnC) 的结合 调节细丝的激活状态，但是相对 每个的作用和重要性很难确定，因为许多 实验条件影响跨桥结合和钙结合。 所提出的实验不仅提供了独立的测量 量，还包括肌节内 Ca 结合的空间分布 心肌和骨骼肌处于不同的激活水平。提议 实验将扩展之前的工作，以调查骑自行车是否 跨桥影响骨骼和心脏中 Ca 与肌丝的结合 肌肉，以及这种作用是否依赖于 TnC 同工型。他们会 还检查肌动球蛋白是否增强钙与肌球蛋白的结合 相互作用，以及肌丝蛋白肌联蛋白是否与钙结合 激活条件。钙沿肌节的分布是 使用定量电子探针 X 射线微量分析 (EPXMA) 确定 甘油化兔腰肌冻干冷冻切片的元素成像 比目鱼肌以及兔子和青蛙的心肌。样品将被冷冻在 含有或不含 MgATP 以及不同水平游离钙的溶液。 肌原纤维蛋白组成的修饰将包括提取 TnC、肌球蛋白轻链和肌动蛋白，以及 TnC 异构体的交换（骨骼、 心脏或具有修饰的 Ca 结合特性的突变体）。过桥 附件将被诸如 BDM、orthovanidate、 N-苯基马来酰亚胺。这些操作将识别 Ca 的两个位点 结合，以及这种结合受相互作用影响的程度 肌丝蛋白之间。跨桥效应对 TnC Ca 结合的影响是 提议为 Frank-Starling 关系提供分子基础，并且 可能是横纹肌收缩事件的其他特征的基础。这些 研究将深入了解调节的基本机制 激活骨骼肌和心肌的力量。

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.