Cytokine Regulation of Muscle Protein Synthesis During Infection

描述（由申请人提供）：肌肉萎缩仍然是创伤，烧伤和感染后发病和死亡的原因。尽管萎缩反应的机制是多因素的且定义不清，但我们的工作证明了炎症调节剂（特别是TNF和NO）的上调与肌肉蛋白合成的翻译控制下降之间的因果关系。败血症引起的缺陷不仅表现在基础条件下，而且表现为对作为合成代谢营养信号的亮氨酸的反应性降低。我们的数据表明，这些败血症引起的变化是由骨骼肌中mTOR激酶信号的抑制介导的。我们的长期目标是阐明脓毒症损害肌肉蛋白平衡导致肌肉萎缩的细胞和分子机制。为了解决这一目标中隐含的问题，我们提出的研究有以下具体目标：(1)通过确定脓毒症是否改变4E-BP1和S6K1与支架蛋白raptor的相互作用、eIF3支架复合物的结合活性和/或PDCD4与eIF4A的结合从而改变解旋酶活性，阐明脓毒症在基础条件下和对赖氨酸的反应中损害肌肉中mTOR激酶活性和帽盖依赖性翻译起始的机制。(2)描述脓毒症通过改变翻译装置组件的细胞内定位来损害蛋白质合成拓扑结构的机制。具体来说，我们将评估Vps34（一种营养调节的脂质激酶）和Rab5在调节细胞内mTOR定位中的作用。(3)确定脓毒症是否通过改变Akt底物PRAS40的磷酸化和/或结合来降低mTOR激酶活性。(4)确定脓毒症诱导的microrna，特别是miR133的变化，降低全局翻译起始的机制。(5)阐明全身与局部产生的TNF"和NO在破坏肌肉mTOR信号中的相对作用。通过使用从TNFR1缺失小鼠中分离的肌细胞的原代培养物或补充的体内研究，将TNF特异性shRNA局部电穿孔到肌肉中，最终目的直接解决了脓毒症诱导的肌肉萎缩中炎症介质自分泌/旁分泌产生的重要性。总的来说，这些研究是独特的，因为它们结合了体外和体内方法，允许定义详细的效应机制，并确认观察结果的生理相关性。这些研究将为这一关键的调控系统和关键营养信号所发挥的综合控制机制提供新的见解。他们将导致不仅彻底了解败血症引起的肌病，而且其他健康相关的条件下，特征是肌肉萎缩和营养抵抗。提出的研究具有转化基础，因为它们将刺激新的临床干预措施的发展，旨在消除脓毒症和创伤性肌病的长期后果，这些后果阻碍了恢复和康复。公共卫生相关性：感染或外伤性损伤后患者的肌肉丧失会延迟恢复，并可能降低生存率。发生这种浪费的原因尚不完全清楚，但不能通过提供足够的营养来完全纠正。我们的研究将确定这种肌肉萎缩的机制，这将最终导致新的治疗方法的发展，并改善患者的预后。

DESCRIPTION (provided by applicant): Muscle wasting remains a cause of morbidity and mortality after trauma, burns and infection. Although the mechanisms for the atrophic response are multifactorial and poorly defined, our work documents the causal relationship between the up regulation of inflammatory modulators, particularly TNF" and NO, and the decreased translational control of muscle protein synthesis. The sepsis-induced defect is manifested not only under basal conditions but also as a reduced responsiveness to leucine which acts as an anabolic nutrient signal. Our data suggest these sepsis-induced changes are mediated by inhibition of mTOR kinase signaling in skeletal muscle. Our long-term goal is to elucidate the cellular and molecular mechanisms by which sepsis impairs muscle protein balance leading to the development of muscle atrophy. To address the questions implicit in this goal our proposed research has the following specific aims: (1) Elucidate the mechanism by which sepsis impairs mTOR kinase activity and cap-dependent translation initiation in muscle under basal conditions and in response to leucine by determining whether sepsis alters the interaction of 4E-BP1 and S6K1 with the scaffold protein raptor, the binding activity of the eIF3 scaffolding complex, and/or the binding of PDCD4 to eIF4A and thereby altering helicase activity. (2) Delineate the mechanism by which sepsis impairs the topology of protein synthesis by altering the intracellular localization of components of the translational apparatus. Specifically, we will assess the role of Vps34, a nutrient-regulated lipid kinase, and Rab5 in regulating intracellular mTOR localization. (3) Determine whether sepsis decreases mTOR kinase activity by altering the phosphorylation and/or binding of the Akt substrate PRAS40. (4) Determine the mechanism by which sepsis-induced changes in microRNAs, particularly miR133, decrease global translation initiation. (5) Elucidate the relative contribution of systemic versus locally produced TNF" and NO in disrupting mTOR signaling in muscle. This final aim directly addresses the importance of autocrine/paracrine production of inflammatory mediators in sepsis-induced muscle wasting by using primary cultures of myocytes isolated from TNFR1 null mice or in complementary in vivo studies where shRNA specific for TNF" is electroporated locally into muscle. Collectively, these studies are unique as they integrate in vitro and in vivo approaches permitting detailed effector mechanisms to be defined and the physiological relevance of the observations to be confirmed. These studies will provide new insights into this critical regulatory system and the integrative control mechanisms exerted by a key nutrient signal. They will lead not only to a thorough understanding of sepsis- induced myopathy but also of other health-related conditions characterized by muscle wasting and nutrient resistance. The proposed studies have a translational underpinning in that they will stimulate the development of novel clinical interventions designed to abrogate the long-term consequences of sepsis- and trauma-induced myopathy which impedes recovery and rehabilitation. PUBLIC HEALTH RELEVANCE: The loss of muscle in patients after infection or traumatic injury delays recovery and may decrease survival. The reason why this wasting occurs is not fully understood, but it cannot be fully corrected by providing adequate nutrition. Our research will determine the mechanism for this muscle atrophy which will ultimately lead to development of novel therapeutic treatment and improve patient outcome.