Nebulin Alters Cross-bridge Cycling Kinetics and Increases Thin Filament Activation A NOVEL MECHANISM FOR INCREASING TENSION AND REDUCING TENSION COST
Nebulin Alters Cross-bridge Cycling Kinetics and Increases Thin Filament Activation A NOVEL MECHANISM FOR INCREASING TENSION AND REDUCING TENSION COST
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DOI：
10.1074/jbc.m109.049718
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发表时间：
2009-11-06
影响因子：
4.8
通讯作者：
Granzier, Henk
中科院分区：
文献类型：
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作者：
Chandra, Murali;Mamidi, Ranganath;Granzier, Henk
伴肌动蛋白是一种巨大的丝状F -肌动蛋白结合蛋白（约800 kDa），它沿着骨骼肌肌节的细肌丝结合。伴肌动蛋白是人们了解最少的主要肌肉蛋白之一。尽管伴肌动蛋白通常被视为一种结构蛋白，但在这里我们通过使用来自伴肌动蛋白敲除（NEB KO）小鼠模型的去皮肌纤维束，研究了伴肌动蛋白是否在肌肉收缩中起作用。我们测量了力 - pCa（-log[Ca²⁺]）和力 - ATP酶的关系，以及胫骨前肌纤维中张力再发展的速率（k₍ₜᵣ₎）。为了排除肌钙蛋白（Tn）异构体表达和/或Tn磷酸化状态的任何改变，我们研究了用细菌表达的快骨骼肌重组Tn重构的纤维束。我们还对肌球蛋白重链、肌球蛋白轻链以及肌球蛋白轻链2的磷酸化进行了详细分析，结果显示野生型和NEB KO之间没有显著差异。我们的力学研究表明，NEB KO纤维的张力消耗增加（5.9比4.4 pmol·mN⁻¹·mm⁻¹·s⁻¹），k₍ₜᵣ₎降低（4.7比7.3 s⁻¹），钙敏感性降低（pCa₅₀为5.74比5.90），激活的协同性降低（n₍ₕ₎为3.64比4.38）。我们的研究结果表明：1）在骨骼肌中，伴肌动蛋白增加细肌丝的激活；2）通过改变横桥循环动力学，伴肌动蛋白增加收缩力和收缩效率。伴肌动蛋白的这些新特性增加了对骨骼肌功能的新认识，并为基于伴肌动蛋白的线状体肌病患者的严重肌无力提供了一种机制。
Nebulin is a giant filamentous F-actin-binding protein (similar to 800 kDa) that binds along the thin filament of the skeletal muscle sarcomere. Nebulin is one of the least well understood major muscle proteins. Although nebulin is usually viewed as a structural protein, here we investigated whether nebulin plays a role in muscle contraction by using skinned muscle fiber bundles from a nebulin knock-out (NEB KO) mouse model. We measured force-pCa (-log[Ca2+]) and force-ATPase relations, as well as the rate of tension re-development (k(tr)) in tibialis cranialis muscle fibers. To rule out any alterations in troponin (Tn) isoform expression and/or status of Tn phosphorylation, we studied fiber bundles that had been reconstituted with bacterially expressed fast skeletal muscle recombinant Tn. We also performed a detailed analysis of myosin heavy chain, myosin light chain, and myosin light chain 2 phosphorylation, which showed no significant differences between wild type and NEB KO. Our mechanical studies revealed that NEB KO fibers had increased tension cost (5.9 versus 4.4 pmol millinewtons(-1) mm(-1) s(-1)) and reductions in ktr (4.7 versus 7.3 s(-1)), calcium sensitivity (pCa(50) 5.74 versus 5.90), and cooperativity of activation (n(H) 3.64 versus 4.38). Our findings indicate the following: 1) in skeletal muscle nebulin increases thin filament activation, and 2) through altering cross-bridge cycling kinetics, nebulin increases force and efficiency of contraction. These novel properties of nebulin add a new level of understanding of skeletal muscle function and provide a mechanism for the severe muscle weakness in patients with nebulin-based nemaline myopathy.