Costamere Defects in Muscular Dystrophies

 描述（由申请人提供）：虽然丰富的肌节肌动蛋白同种型因其在横纹肌收缩中的重要作用而闻名，但低丰度的非肌肉“细胞质”肌动蛋白同种型（肌细胞-肌动蛋白和肌细胞-肌动蛋白）在维持正常和患病骨骼肌中的特化结构中也是重要的。在这个项目中，我们产生和表征肌肉特异性的小鼠品系，它们缺乏肌动蛋白，或肌动蛋白，或过表达肌动蛋白，以了解它们在肌营养不良蛋白缺陷型肌营养不良症中的内源性功能和作用。有趣的是，每一个β-肌动蛋白或β-肌动蛋白单敲除产生了定性相似的表型，其特征在于具有显著肌纤维变性/再生和肌无力的进行性肌病。我们已经证明，在肌营养不良蛋白缺陷型mdx小鼠中，2000倍的肌肉特异性过表达的肌细胞肌动蛋白提供了显著的保护，免受离心收缩诱导的力下降。我们的新数据表明，离心收缩驱动了一种快速可逆的活性氧（ROS）介导的肌节收缩性抑制，这种抑制可能起到保护营养不良肌肉免受重复高力收缩引起的肌原纤维损伤的作用。最后，我们已经获得了新的数据表明，线粒体和肌浆网之间的相互作用，线粒体和肌浆网肌动蛋白的合作，以维持功能。展望未来，我们将利用我们独特的动物模型，亚型特异性试剂以及生化和生理方法来解决有关正常骨骼肌功能和肌营养不良蛋白缺陷型肌营养不良症中细胞质肌动蛋白的基本问题。在目标1中，我们将研究一个关键的氧化还原缓冲蛋白的损失如何有助于营养不良mdx骨骼肌离心收缩引起的力下降。在目标2中，我们将检验以下假设：牵张诱导的ROS可能通过肌节肌动蛋白或对收缩功能至关重要的其他肌原纤维蛋白的可逆氧化修饰引起mdx肌肉中离心收缩诱导的力下降。在目标3中，将通过小鼠品系的表征来研究线粒体和肌浆网之间界面处的胞浆细胞肌动蛋白和胞浆细胞肌动蛋白的作用，在所述小鼠品系中，胞浆细胞肌动蛋白和胞浆细胞肌动蛋白已经在骨骼肌中单独地或组合地被敲除。拟议的研究结果将明确解决细胞质肌动蛋白亚型的功能正常和患病骨骼肌的独特和重要的贡献。

 DESCRIPTION (provided by applicant): While abundant sarcomeric actin isoforms are famous for their essential role in striated muscle contraction, low abundance non-muscle "cytoplasmic" actin isoforms (cyto- and cyto-actin) are also emerging as important in the maintenance of specialized structures in normal and diseased skeletal muscle. During this project, we generated and characterized muscle-specific mouse lines lacking either cyto-actin, or cyto-actin, or overexpressing cyto-actin to understand their endogenous functions and role(s) in dystrophin-deficient muscular dystrophy. Interestingly, each cyto-actin or cyto-acin single knockout develops a qualitatively similar phenotype characterized by a progressive myopathy with significant myofiber degeneration/regeneration and muscle weakness. We have shown that 2000-fold muscle-specific overexpression of cyto-actin in dystrophin- deficient mdx mice affords significant protection from eccentric contraction-induced force drop. Our new data suggest that eccentric contraction drives a rapidly-reversible, reactive oxygen species (ROS)-mediated inhibition of sarcomeric contractility that may function to protect dystrophic muscles from myofibrillar damage caused by repeated, high force contractions. Finally, we have obtained new data suggesting that cyto- and cyto-actins collaborate to maintain the functional interaction between mitochondria and sarcoplasmic reticulum. Going forward, we will make use of our unique animal models, isoform-specific reagents and biochemical and physiological methodologies to address fundamental questions about cytoplasmic actins in normal skeletal muscle function and in dystrophin-deficient muscular dystrophy. In aim 1, we will investigate how loss of a key redox buffering protein contributes to eccentric contraction-induced force drop in dystrophic mdx skeletal muscle. In aim 2, we will test the hypothesis that stretch-induced ROS may cause eccentric contraction induced force drop in mdx muscle via reversible oxidative modification of sarcomeric actin or other myofibrillar proteins critical for contractile function. n aim 3, the roles of cyto- and cyto-actins at the interface between mitochondria and the sarcoplasmic reticulum will be investigated through characterization of mouse lines in which cyto-actin and cyto-actins have been knocked out in skeletal muscle individually, or in combination. The results of the proposed studies will definitively address the unique and important contributions of cytoplasmic actin isoforms to the function of normal and diseased skeletal muscle.