Costamere Defects in Muscular Dystrophies

项目摘要/摘要 而丰富的骨骼肌动蛋白(ASK-肌动蛋白)和心脏肌动蛋白(AcA-肌动蛋白)亚型因其在 横纹肌收缩，低丰度的非肌肉“细胞质”肌动蛋白亚型(bcyto和gcyto-actin)是 在维护正常和非正常状态下的特殊结构(和功能)方面也变得重要 患病的骨骼肌。在这个项目中，我们培育并鉴定了肌肉特异的小鼠品系 缺乏或过表达细胞肌动蛋白或细胞肌动蛋白以了解其内源性功能和作用(S) 营养不良蛋白缺乏性肌营养不良症。有趣的是，每一个bcyto-actin或gcyto-actin单基因敲除都会产生一种 以具有明显肌纤维的进行性肌病为特征 退化/再生和肌肉无力。我们已经证明了骨骼肌特有的 在dystrophin缺陷型mdx小鼠中过表达bcyto-actin或gcyto-actin可提供显著的保护作用 偏心收缩诱导的力量下降，而过表达Gcyto-actin的C272a突变体不提供 保护。这些和其他数据表明，偏心收缩导致快速可逆的、反应性的 氧物种(ROS)介导的肌节收缩抑制可能起到保护营养不良的作用 肌肉因反复的、高强度的收缩而受损。我们新的初步数据显示， 肌肉特异性消融野生型肌肉的bcyto-actin或gcyto-actin可引起偏心收缩 被非特异性抗氧化剂N-乙酰半胱氨酸逆转的力量下降。最后，我们获得了新的 数据表明，细胞肌动蛋白在膜损伤的修复中起重要作用。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 解决有关细胞质肌动蛋白在正常骨骼肌功能和 营养不良蛋白缺乏性肌营养不良症。在目标1中，我们将确定导致古怪的ROS的来源 营养不良MDX骨骼肌及ROS下游靶点收缩诱导的力量下降 这最终会抑制武力的生产。在目标2中，我们将研究氧化应激在推动 肌病和离心性收缩引起的肌力下降与基因消融双细胞或双细胞肌动蛋白相关 骨骼肌。在目标3中，细胞质肌动蛋白亚型和膜修复中的ROS之间的相互作用将 使用最先进的成像方法来分析同一小鼠品系的肌肉 在目标1和目标2中使用。拟议研究的结果将进一步描绘独特和重要的 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.