Modulation of Contraction-Induced Changes in Muscle mTOR Signaling by Alcohol

描述(由申请人提供)：酗酒和长期酗酒(即酗酒)是重要的公共卫生问题，因为这些问题与高危行为、多器官系统功能障碍、疾病恢复受阻，甚至过早死亡有关。习惯性酗酒会导致骨骼肌病，其特征是肌肉萎缩、虚弱和身体功能下降。这些症状与分子变化有关，包括骨骼肌中依赖mTOR的蛋白质合成受到抑制。无论是在酒精持续存在的情况下，还是在实现戒酒的情况下，都几乎没有治疗策略来帮助这种疾病的康复。然而，骨骼肌收缩激活了mTOR激酶活性，增加了蛋白质合成。重要的是，当间歇性收缩刺激持续几周时，它会诱导肌肉显著生长和肌肉功能的协调增加。因此，刺激肌肉收缩，无论是自愿的还是通过经皮电刺激，对于活动能力和功能能力有限的酒精性肌肉疾病患者来说，可能是一种潜在的治疗方式。我们的长期目标是确定酒精对骨骼肌蛋白质合成和因刺激肌肉收缩而增加的mTOR活性的影响，并确定潜在的信号变化和介导观察到的反应的新蛋白质。朝着这一目标的进展将通过以下具体目标来实现：(1)确定急性酒精中毒是否逆转由急性或慢性肌肉收缩引起的原有的mTOR活性、蛋白质合成和肌肉肥大的增加；(2)建立慢性酒精性肌病模型中急性和慢性肌肉收缩与肌肉mTOR活性、蛋白质合成和肌肉肥大变化的治疗相关性；(3)确定急性和慢性酒精中毒后TSC2和mTOR的内切酶/溶酶体靶向作用，以及与先前观察到的肥大、收缩和mTOR信号变化相关的肌肉收缩；以及(4)使用嘌呤霉素相关的新生链蛋白组学(PUCK-P)识别其合成速率被酒精和/或肌肉收缩改变的骨骼肌蛋白质。这些目标代表了一系列新颖和全面的实验，利用了几种创新的体内和体外技术，包括急性和慢性酒精滥用的啮齿动物模型，分离的肌肉力量和疲劳性测试，mTOR和TSC2分子运输的可视化，以及鉴定酒精和肌肉收缩后新产生的蛋白质的质谱学。从这些实验中获得的数据将提供临床相关知识，以显著推进治疗策略，并有助于从机制上理解急性和慢性酒精性肌肉疾病的病因。

DESCRIPTION (provided by applicant): Binge drinking and chronic alcohol abuse (i.e. alcoholism) represent important public health issues as these are associated with high risk behaviors, multiple organ system dysfunction, impaired recovery from illness and even premature death. Habitual alcohol abuse leads to skeletal muscle myopathy characterized by muscle loss, weakness, and decreased physical function. These symptoms are associated with molecular changes including a depressed rate of mTOR-dependent protein synthesis in skeletal muscle. Few treatment strategies exist to aid in the recovery from this disease both in the continued presence of alcohol and under circumstances where abstinence is achieved. However, skeletal muscle contraction activates mTOR kinase activity and increases protein synthesis. Importantly, when an intermittent contractile stimulus is sustained over several weeks it induces significant muscle growth and a coordinate increase in muscle function. Therefore, stimulated muscle contraction, either voluntarily or via transcutaneous electrical stimulation, may represent a potential therapeutic modality for individuals with alcoholic muscle disease who have limited mobility and functional capacity. Our long term goal is to determine the effects of alcohol on skeletal muscle protein synthesis and mTOR activity that has been increased by stimulated muscle contraction, and to identify potential signaling changes and new proteins that mediate the observed response. Progression towards this goal will be achieved through the following specific aims: (1) Determine whether acute alcohol intoxication reverses the pre-existing increase in mTOR activity, protein synthesis and muscle hypertrophy induced by acute or chronic muscle contraction; (2) Establish the therapeutic relevance of acute and chronic muscle contraction in a model of chronic alcoholic myopathy with regards to changes in muscle mTOR activity, protein synthesis and muscle hypertrophy; (3) Define the role of endosomal/lysosomal targeting of TSC2 and mTOR following acute and chronic alcohol, and muscle contraction relevant to previously observed changes in hypertrophy, contractility and mTOR signaling; and (4) Identify skeletal muscle proteins whose synthetic rate is altered by alcohol and/or muscle contraction using puromycin-associated nascent chain proteomics (PUNCH- P). These aims represent a set of novel and comprehensive experiments utilizing several innovative in vivo and ex vivo techniques including rodent models of acute and chronic alcohol abuse, isolated muscle strength and fatigability testing, visualization of the molecular trafficking of mTOR and TSC2, as well as mass spectrometry to identify newly made proteins following alcohol and muscle contraction. Data garnered from these experiments will provide clinically relevant knowledge to significantly advance treatment strategies and contribute to the mechanistic understanding of the etiology of acute and chronic alcoholic muscle disease.