Molecular Pathogenesis of Skeletal Muscle Atrophy

6.项目总结/摘要 骨骼肌萎缩是一种知之甚少，但几乎普遍的后果，严重的人类 目前没有治疗方法的疾病。我们假设，在发病机制中的一个中心事件， 骨骼肌萎缩是ATF 4（一种进化上古老的转录因子）表达增加 是由压力引起的。这一假设是基于几个线的证据来自我们的 初步研究。首先，我们发现ATF 4 mRNA水平在萎缩的骨骼肌中增加， 脊髓损伤患者的肌肉。第二，我们发现转染的小鼠 用编码小鼠ATF 4的质粒DNA处理骨骼肌诱导肌纤维萎缩。相反地， 靶向ATF 4或显性失活ATF 4构建体的人工微小RNA的转染减少 禁食条件下的肌纤维萎缩。这些结果表明ATF 4是一种新的转录因子， 肌肉质量的调节剂，可能是骨骼肌萎缩的必要和充分的。与 为了确定人类患者肌肉萎缩的新治疗方法的长期目标，我们 提出三个目标。首先，为了确定ATF 4是否是肌肉萎缩所必需的，我们将研究骨骼肌 肌肉特异性ATF 4基因敲除小鼠，并确定它们是否对禁食或去神经支配有抵抗力， 导致肌肉萎缩。第二，确定ATF 4增加的上游机制 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是否有一种 应激激活的eIF 2 α激酶是ATF 4表达和肌肉萎缩所必需的。三是 为了确定ATF 4介导的萎缩的下游机制，我们将检测骨骼肌萎缩。 用表达ATF 4的腺病毒感染肌管，并确定这是否减少蛋白质合成和/或 增加蛋白质分解。我们还将研究骨骼肌特异性ATF 4基因敲除小鼠， 确定是否需要ATF 4来诱导抑制蛋白质合成的萎缩相关蛋白 （4 E-BP 1），增加蛋白质合成（atrogin-1和MuRF 1），或者如果ATF 4激活氨基酸 肌肉萎缩所必需的转运基因。

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.