ROLE OF MYOFILAMENT BOUND CALCIUM IN MUSCLE CONTRACTION

描述（申请人的逐字描述）：长期目标

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.