Costamere Defects in Muscular Dystrophies

 描述(申请人提供)：虽然丰富的肌节肌动蛋白异构体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”肌动蛋白异构体(细胞和细胞肌动蛋白)在维持正常和疾病骨骼肌的特殊结构方面也越来越重要。在这个项目中，我们建立并鉴定了缺失细胞肌动蛋白或细胞肌动蛋白或过表达细胞肌动蛋白的肌肉特异性小鼠系，以了解它们在营养不良蛋白缺乏性肌营养不良中的内源性功能和作用(S)。有趣的是，每个细胞肌动蛋白或细胞肌动蛋白单基因敲除都发展出一种定性相似的表型，以进行性肌病为特征，伴有显著的肌纤维变性/再生和肌肉无力。我们已经证明，在肌营养不良蛋白缺乏的mdx小鼠中，肌肉特异的细胞肌动蛋白高表达2000倍，可以显著保护小鼠免受偏心收缩诱导的力量下降。我们的新数据表明，偏心收缩导致快速可逆的活性氧(ROS)介导的肌节收缩抑制，这可能起到保护营养不良肌肉免受反复、高强度收缩引起的肌纤维损伤的作用。最后，我们获得了新的数据，表明细胞和细胞肌动蛋白协同维持线粒体和肌浆网之间的功能相互作用。展望未来，我们将利用我们独特的动物模型、异构体特异性试剂以及生化和生理学方法来解决细胞质肌动蛋白在正常骨骼肌功能和肌营养不良症中的基本问题。在目标1中，我们将研究一个关键的氧化还原缓冲蛋白的丢失是如何在营养不良的MDX骨骼肌中导致离心性收缩诱导的力量下降的。在目标2中，我们将验证这一假说，即拉伸诱导的ROS可能通过肌节肌动蛋白或其他对收缩功能至关重要的肌原纤维蛋白的可逆氧化修饰而导致MDX肌肉的偏心收缩诱导的力量下降。在目标3中，将通过在骨骼肌中单独或联合敲除细胞肌动蛋白和细胞肌动蛋白的小鼠品系的特征来研究细胞肌动蛋白和细胞肌动蛋白在线粒体和肌浆网之间的界面上的作用。拟议的研究结果将明确阐述细胞质肌动蛋白亚型对正常和疾病骨骼肌功能的独特和重要贡献。

 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.