Mechanotransduction and the Regulation of Skeletal Muscle Mass

描述（由申请人提供）：机械刺激在骨骼肌质量的调节中起主要作用，肌肉质量的维持对疾病预防和生活质量有显著贡献。虽然机械刺激和肌肉质量调节之间的联系已经被认识了几十年，但这一过程背后的分子机制尚不清楚。因此，我们研究的长期目标是确定机械刺激调节骨骼肌质量的分子事件。该项目的主要目的是确定哺乳动物雷帕霉素靶蛋白（mTOR）在调节骨骼肌质量中的作用，并确定机械刺激如何激活mTOR信号。我们关注mTOR的基本原理来自我们的初步研究，其表明：i）mTOR的激活在机械诱导的生长中起关键作用，和ii）机械刺激通过涉及磷脂酸（PA）的独特的PI 3 K/PKB非依赖性机制激活mTOR信号传导。鉴于机械刺激激活mTOR，因此存在将机械信号转化为mTOR激活的分子机制（即机械转导途径）。因此，我们的计划是确定这一途径中的关键事件，并确定模仿这些事件是否可以诱导肌肉生长和减轻废用性萎缩。我们目前的假设是，机械刺激促进磷脂酸（PA）的增加，随后激活mTOR信号传导并最终生长。为了检验这一假设，我们将追求以下四个具体目标：1）确定mTOR的激活是否足以诱导生长和减弱废用性萎缩; 2）确定mTOR在机械诱导生长中的作用; 3）确定[PA]的增加是否足以诱导生长和减弱废用性萎缩;以及4）鉴定调节mTOR的机械激活的上游分子。在第一个目标中，Rheb的过表达将用于诱导小鼠骨骼肌中mTOR的PI 3 K/PKB非依赖性活化，并将确定在正常使用和停用期间对肌肉质量的影响。在第二个目标中，表达mTOR的各种突变体的转基因小鼠将用于确定mTOR的肌肉特异性作用，和mTOR激酶活性，在机械诱导的生长。在第三个目标中，PA合成酶的过表达将用于确定[PA]的增加是否足以诱导生长和减弱废用性萎缩。在第四个目标中，活性测定将用于鉴定调节PA中机械诱导的变化的酶，然后药理学和分子干预将用于进一步确定这些酶在mTOR的机械激活中发挥的作用。这些研究具有重要意义，因为这些结果将填补我们目前对机械刺激如何调节mTOR信号传导和骨骼肌质量的认识中的主要空白。此外，这些结果可能导致识别模拟机械刺激效果的治疗目标，从而防止在卧床休息，固定和衰老等废用期间的萎缩。

DESCRIPTION (provided by applicant): Mechanical stimuli play a major role in the regulation of skeletal muscle mass, and the maintenance of muscle mass contributes significantly to disease prevention and the quality of life. Although the link between mechanical stimulation and the regulation of muscle mass has been recognized for decades, the molecular mechanisms underlying this process are not known. Hence, the long-term goal of our research is to define the molecular events through which mechanical stimuli regulate skeletal muscle mass. The primary objective of this project is to define the role of the mammalian target of rapamycin (mTOR) in regulating skeletal muscle mass, and determine how mechanical stimuli activate mTOR signaling. Our rationale for focusing on mTOR comes from our preliminary studies which suggest that: i) the activation of mTOR plays a critical role in mechanically-induced growth, and ii) mechanical stimuli activate mTOR signaling through a unique PI3K/PKB- independent mechanism involving phosphatidic acid (PA). Given that mechanical stimuli activate mTOR, it follows that a molecular mechanism (i.e. mechanotransduction pathway) exists for converting mechanical signals into mTOR activation. Thus, our plan is to identify the critical events in this pathway and determine if mimicking these events can induce muscle growth and attenuate disuse atrophy. Our current hypothesis is that mechanical stimuli promote an increase in phosphatidic acid (PA) which subsequently activates mTOR signaling and ultimately growth. To test this hypothesis we will pursue the following four specific aims: 1) Determine if the activation of mTOR is sufficient to induce growth and attenuate disuse atrophy; 2) Define the role of mTOR in mechanically-induced growth; 3) Determine if an increase in [PA] is sufficient to induce growth and attenuate disuse atrophy and 4) Identify the upstream molecules that regulate the mechanical activation of mTOR. In the first aim, over-expression of Rheb will be used to induce a PI3K/PKB-independent activation of mTOR in mouse skeletal muscles, and the resulting effect on muscle mass during normal use and disuse will be determined. In the second aim, transgenic mice expressing various mutants of mTOR will be used to define the muscle specific role of mTOR, and mTOR kinase activity, in mechanically-induced growth. In the third aim, over-expression of PA synthesizing enzymes will be used to determine if an increase in [PA] is sufficient to induce growth and attenuate disuse atrophy. In the fourth aim, activity assays will be used to identify the enzymes that regulate mechanically-induced changes in PA, and then pharmacological and molecular interventions will be used to further define the role that these enzymes play in the mechanical activation of mTOR. The proposed studies are significant because the outcomes will fill major gaps in our current knowledge of how mechanical stimuli regulate mTOR signaling and skeletal muscle mass. Furthermore, the outcomes could lead to the identification of targets for therapies that mimic the effects of mechanical stimuli and, in-turn, prevent atrophy during periods of disuse such as bedrest, immobilization and aging.