THE N-TERMINAL MYOSIN-ELC REGULATION OF CARDIAC MUSCLE CONTRACTION

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 人们已经知道，在肌肉收缩过程中，心脏ELC的长N末端与肌动蛋白直接接触。 为了研究这可能如何调节收缩性，我们产生了表达ELC N末端的43个氨基酸残基截短（Tg-D43）的转基因小鼠（Kazmierczak等人，JMB 387，706-725，2009）。我们假设N-末端ELC延伸通过其与肌动蛋白的直接接触和/或通过影响肌球蛋白重链（MHC）的构象及其与肌动蛋白的相互作用来调节心肌中的力发展。另一种可能性是ELC的N-末端充当预先定位肌球蛋白头以附着于肌动蛋白并促进肌动蛋白-肌球蛋白相互作用的系链。因此，缺乏N-末端ELC延伸将导致最大力降低和肌丝协同性降低，这是我们在已发表的Tg-D43小鼠初步数据中观察到的表型。该项目将有助于确定ELC的N-末端的遗传消融是否可能导致Tg-D43横桥的径向和/或方位角位移，使得它们可以重新定位远离细丝的表面并且更靠近肌球蛋白粗丝。为了测试这一点，我们将使用低角X射线衍射比较晶格间距和赤道强度比（I1，1/I1，0），以评估肌球蛋白跨桥质量相对于肌动蛋白在Tg-D43小鼠心肌相比，Tg-WT小鼠含有全长ELC N-末端的接近。 这将阐明N-末端ELC延伸在肌肉收缩中的调节作用。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. It has been known for some time that during muscle contraction the long N terminus of cardiac ELC makes direct contacts with actin. To study how this might regulate contractility, we generated transgenic mice expressing a 43 amino acid residue truncation of the ELC N terminus (Tg-D43) (Kazmierczak et al. JMB 387, 706-725, 2009). We hypothesize that the N-terminal ELC extension regulates force development in cardiac muscle via its direct contacts with actin and/or through influencing the conformation of the myosin heavy chain(MHC) and its interaction with actin,. Another possibility is that the N-terminus of ELC acts as a tether pre-positioning the myosin heads for attachment to actin and facilitating Acto-myosin interaction. The lack of the N-terminal ELC extension would therefore result in a decreased maximal force and lower myofilament cooperativity, the phenotypes that we observe in our published preliminary data with Tg-D43 mice. This project will help determine whether the genetic ablation of the N-terminus of ELC may result in radial and/or azimuthal displacement of Tg-D43 crossbridges, such that they may re-locate further from the surface of the thin filaments and closer to the myosin thick filaments. To test this, we will use low-angle X-ray diffraction to compare lattice spacings and equatorial intensity ratios (I1,1/I1,0) to assess the proximity of myosin cross-bridge mass relative to actin in Tg-D43 mouse myocardium compared to Tg-WT mice containing a full length ELC N-terminus. This will elucidate the regulatory role of the N-terminal ELC extension in muscle contraction.