Muscle protein synthesis and gene expression in myostatin-deficient mice

描述(由申请人提供)：在发育过程中抑制或丧失肌肉生长抑制素活性会导致肌肉过度生长，但对肌肉停止生长后肌肉抑制素活性降低的后果知之甚少。这项研究的主要目标是了解当肌肉生长抑素活性在发育后减少时，骨骼肌会发生什么。拟议的实验将针对三个具体目标：1.检验发育后肌肉抑制素耗竭会增加小鼠肌肉质量的假设，即这种效应将由肌肉纤维肥大介导，而肌肉纤维数量或每块肌肉的肌核数量不会增加，并且这种影响将长期持续(测试到肌肉生长抑素耗竭后一年)；2.检测肌肉生长抑制素耗竭后肌肉质量的某些方面，包括力量生成能力、对牵拉损伤的易感性、疲劳性、不同类型纤维的比例(根据肌球蛋白重链亚型或SDH活性的表达、胶原含量和前胶原基因的表达来分类)；3.验证发育后肌抑制素缺乏将通过增加肌原纤维蛋白合成而不是抑制蛋白分解而导致肌肉肥大的假说，将上调与mRNA翻译有关的基因的表达，并将通过激活Akt-mTOR-4EBP1/S6K1途径来刺激mRNA翻译。在肌肉生长抑素基因关键的第三外显子与loxP序列(Cre重组酶的靶标)相邻的小鼠中，肌肉生长抑素基因的发育后破坏将通过激活三苯氧胺诱导的Cre重组酶来完成，该重组酶由转基因组成表达。在短期机制研究中，通过注射JA16抗肌肉生长抑素抗体将抑制肌肉生长抑制素的活性。肌原纤维蛋白的合成将通过稳定的同位素示踪剂掺入来确定。基因的表达将通过定量RT-PCR来确定。选定蛋白质的浓度和磷酸化状态将通过免疫印迹来确定。免疫组织化学将用于评估肌肉纤维肥大、纤维类型、纤维数量和肌核结构域体积。将通过刺激体外分离的肌肉来研究力量的产生、疲劳性和拉伸损伤。与公共卫生相关：肌肉萎缩导致各种情况下的虚弱，包括肌肉营养不良、癌症、糖皮质激素应用、缺乏活动、关节炎、营养不良、艾滋病和老年。在这些情况下减少肌肉抑制素的活性可能会改善肌肉的损失和虚弱。了解降低成熟肌肉中肌肉生长抑素活性的生理和分子效应将揭示抗肌肉生长抑制素治疗的潜在益处和局限性。

DESCRIPTION (provided by applicant): Inhibition or loss of myostatin activity during development causes excessive muscle growth, but little is known about the consequences of reducing myostatin activity after muscle has stopped growing. The broad goal of this research is to understand what happens to skeletal muscle when myostatin activity is reduced post-developmentally. The proposed experiments will address three specific aims: 1. to test the hypothesis that post-developmental myostatin depletion will increase muscle mass in mice, that this effect will be mediated by muscle fiber hypertrophy with no increase in the number of muscle fibers or the number of myonuclei per muscle, and that the effect will persist long term (tested up to one year after myostatin depletion); 2. to examine certain aspects of muscle quality after myostatin depletion, including capacity for force generation, susceptibility to stretch-induced injury, fatigability, proportion of different fiber types as classified by expression of myosin heavy chain isoforms or SDH activity, collagen content, and expression of procollagen genes; and, 3. to test the hypothesis that post-developmental myostatin deficiency will induce muscle hypertrophy via increased myofibrillar protein synthesis rather than inhibition of proteolysis, will upregulate expression of genes involved in mRNA translation, and will stimulate mRNA translation through activation of the Akt-mTOR-4EBP1/S6K1 pathway. Post-developmental disruption of the myostatin gene will be accomplished by activation of a tamoxifen-inducible Cre recombinase, constitutively expressed by a transgene, in mice in which the critical third exon of the myostatin gene is flanked with loxP sequences (the targets of Cre recombinase). Myostatin activity will be inhibited in short-term mechanistic studies by injecting the JA16 anti-myostatin antibody. Myofibrillar protein synthesis will be determined by stable isotope tracer incorporation. Gene expression will be determined by quantitative RT-PCR. Concentrations and phosphorylation status of selected proteins will be determined by immunoblotting. Immunohistochemistry will be used to assess muscle fiber hypertrophy, fiber types, numbers of fibers, and myonuclear domain volume. Force generation, fatigability, and stretch injury will be studied by stimulation of isolated muscles in vitro. Relevance to public health: Muscle atrophy causes weakness in various conditions, including muscular dystrophies, cancer, glucocorticoid administration, inactivity, arthritis, malnutrition, AIDS, and old age. Reducing myostatin activity in these conditions might ameliorate this muscle loss and weakness. Knowledge of the physiological and molecular effects of reducing myostatin activity in mature muscle will reveal potential benefits and limitations of anti-myostatin therapy.