Costamere Defects in Muscular Dystrophies

项目总结/摘要 虽然丰富的骨骼（ASK-肌动蛋白）和心脏（ACA-肌动蛋白）肌动蛋白同工型因其在细胞内的重要作用而闻名， 横纹肌收缩时，低丰度的非肌肉“细胞质”肌动蛋白同种型（bcyto-和gcyto-actin）是 在维持正常和不正常的专业结构（和职能）方面也越来越重要， 患病的骨骼肌在这个项目中，我们产生和特点的肌肉特异性小鼠线 缺乏或过表达bcyto-actin或gcyto-actin，以了解它们在 肌营养不良蛋白缺乏型肌营养不良症。有趣的是，每一个bcyto-actin或gcyto-actin单敲除都会产生一个 性质相似的表型，特征为进行性肌病伴显著肌纤维 退化/再生和肌肉无力。我们已经证明，骨骼肌特异性 抗肌萎缩蛋白缺陷型mdx小鼠中bcyto-actin或gcyto-actin的过表达提供了显著的保护， 离心收缩引起的力下降，而gcyto-actin的C272 A突变体的过表达不提供 保护这些和其他数据表明，离心收缩驱动一个快速可逆的，反应性的， 氧自由基（ROS）介导的抑制肌节收缩，可能起到保护营养不良的作用。 肌肉因反复的高强度收缩而受损。我们新的初步数据显示， 从野生型肌肉特异性切除bcyto-actin或gcyto-actin导致离心收缩诱导的 力下降，被非特异性抗氧化剂N-乙酰半胱氨酸逆转。最后，我们获得了新的 数据表明G细胞肌动蛋白对于膜损伤的修复是重要的。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， 解决了关于正常骨骼肌功能和细胞质肌动蛋白的新的基本问题， 肌营养不良蛋白缺乏型肌营养不良症。在目标1中，我们将确定ROS的来源， 在营养不良mdx骨骼肌以及ROS下游靶点中收缩诱导的力下降 最终抑制力的产生。在目标2中，我们将研究氧化应激在驱动细胞凋亡中的作用。 与bcyto-或gcyto-actin基因消融相关的肌病和离心收缩诱导的力下降 骨骼肌。在目标3中，细胞质肌动蛋白亚型和ROS在膜修复中的相互作用将 使用最先进的成像方法进行研究，以分析来自相同小鼠品系的肌肉 用于目标1和2。拟议研究的结果将进一步阐明 细胞质肌动蛋白亚型对正常和营养不良骨骼肌功能的贡献。

PROJECT SUMMARY/ABSTRACT While abundant skeletal (ask-actin) and cardiac (aca-actin) actin isoforms are famous for their essential role in striated muscle contraction, low abundance non-muscle “cytoplasmic” actin isoforms (bcyto- and gcyto-actin) are also emerging as important in the maintenance of specialized structures (and functions) in normal and diseased skeletal muscle. During this project, we generated and characterized muscle-specific mouse lines either lacking or overexpressing bcyto-actin or gcyto-actin to understand their endogenous functions and role(s) in dystrophin-deficient muscular dystrophy. Interestingly, each bcyto-actin or gcyto-actin single knockout develops a qualitatively similar phenotype characterized by a progressive myopathy with significant myofiber degeneration/regeneration and muscle weakness. We have shown that skeletal muscle-specific overexpression of bcyto-actin or gcyto-actin in dystrophin-deficient mdx mice affords significant protection from eccentric contraction-induced force drop while overexpression of a C272A mutant of gcyto-actin affords no protection. These and other 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 damage caused by repeated, high force contractions. Our new preliminary data show that muscle-specific ablation of bcyto-actin or gcyto-actin from wildtype muscle results in eccentric contraction-induced force drop that is reversed by the nonspecific antioxidant N-acetylcysteine. Finally, we have obtained new data suggesting that gcyto-actin is important for repair of membrane damage. Going forward, we will make use of our unique animal models, isoform-specific reagents, and biochemical and physiological methodologies to address new fundamental questions about cytoplasmic actins in normal skeletal muscle function and in dystrophin-deficient muscular dystrophy. In aim 1, we will identify the sources of ROS contributing to eccentric contraction-induced force drop in dystrophic mdx skeletal muscle as well as the downstream targets of ROS that ultimately inhibit force production. In aim 2, we will investigate the role of oxidative stress in driving the myopathy and eccentric contraction-induced force drop associated with genetic ablation of bcyto- or gcyto-actin in skeletal muscle. In aim 3, the interplay between cytoplasmic actin isoforms and ROS in membrane repair will be investigated using state-of-the-art imaging approaches to analyze muscles from the same mouse lines used in aims 1 and 2. The results of the proposed studies will further delineate the unique and important contributions of cytoplasmic actin isoforms to the function of normal and dystrophic skeletal muscle.