Molecular Pathogenesis of Skeletal Muscle Atrophy

6.项目摘要/摘要 骨骼肌萎缩是一种知之甚少但几乎普遍存在的严重人类后果。 目前尚无治疗方法的疾病。我们假设，在本病的发病机制中有一个中心事件 骨骼肌萎缩是一种进化上古老的转录因子ATF4的表达增加 这是由压力引起的。这一假设是基于几条证据，这些证据来自我们的 初步研究。首先，我们发现萎缩的骨骼中ATF4的mrna水平升高。 来自患有脊髓损伤的受试者的肌肉。第二，我们发现小鼠的转基因 携带编码ATF4基因的小鼠骨骼肌可引起肌纤维萎缩。相反， 靶向ATF4或显性负性ATF4结构的人工microRNA的转染率降低 禁食条件下肌纤维萎缩。这些结果表明ATF4是一种新的转录因子 对骨骼肌萎缩既必要又充分的肌肉质量调节器。使用 为了确定治疗人类肌肉萎缩的新方法的长期目标，我们 提出三个目标。首先，为了确定ATF4是否对肌肉萎缩是必要的，我们将研究骨骼 肌肉特异性ATF4基因敲除小鼠，并确定它们是否抵抗禁食或去神经 导致肌肉萎缩。第二，确定ATF4升高的上游机制 表达，我们将在野生型小鼠中使用RNA干扰，并确定四种哺乳动物中的一种 应激激活的eIF2α激酶是ATF4表达和肌肉萎缩所必需的。第三，到 确定ATF4介导的萎缩的下游机制(S)，我们将转染骨骼 用腺病毒表达ATF4的肌管，并确定这是否会减少蛋白质合成和/或 增加蛋白质分解。我们还将研究骨骼肌特异性ATF4基因敲除小鼠以 确定ATF4是否是诱导抑制蛋白质合成的萎缩相关蛋白所必需的 (4E-BP1)，增加蛋白质合成(阿托金-1和MuRF1)，或者如果ATF4激活氨基酸 对肌肉萎缩至关重要的转运蛋白基因。

6. PROJECT SUMMARY/ABSTRACT Skeletal muscle atrophy is a poorly understood yet nearly universal consequence of severe human illness for which no therapy currently exists. We hypothesize that a central event in the pathogenesis of skeletal muscle atrophy is increased expression of ATF4, an evolutionarily ancient transcription factor that is induced by stress. This hypothesis is based on several lines of evidence derived from our preliminary studies. First, we found that ATF4 mRNA levels were increased in atrophied skeletal muscle from human subjects with spinal cord injuries. Second, we found that transfection of mouse skeletal muscle with plasmid DNA encoding mouse ATF4 induced myofiber atrophy. Conversely, transfection of an artificial microRNA targeting ATF4 or a dominant negative ATF4 construct reduced myofiber atrophy under fasting conditions. These results identify ATF4 as a novel transcriptional regulator of muscle mass that may be both necessary and sufficient for skeletal muscle atrophy. With the long-term goal of identifying new therapeutic approaches for muscle atrophy in human patients, we propose three aims. First, to determine if ATF4 is essential for muscle atrophy, we will study skeletal muscle-specific ATF4 gene knockout mice and determine if they are resistant to fasting- or denervation- induced muscle atrophy. Second, to determine the upstream mechanism of increased ATF4 expression, we will use RNA interference in wild-type mice and determine if one of the four mammalian stress-activated eIF2alpha kinases is required for ATF4 expression and muscle atrophy. Third, to determine the downstream mechanism(s) of ATF4-mediated atrophy, we will transfect skeletal myotubes with adenovirus expressing ATF4 and determine if this decreases protein synthesis and/or increases protein breakdown. We will also study skeletal muscle-specific ATF4 gene knockout mice to determine if ATF4 is required for induction of atrophy-associated proteins that inhibit protein synthesis (4E-BP1), increase protein synthesis (atrogin-1 and MuRF1), or if ATF4 activates amino acid transporter genes that are essential for muscle atrophy.