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失去肌肉生长抑制素活性会导致肌肉过度生长，但在肌肉停止生长后减少肌肉生长抑制素活性的后果知之甚少。这项研究的广泛目标是了解当肌肉生长抑制素活性在发育后减少时骨骼肌会发生什么。拟议的实验将解决三个具体目标：1。为了验证发育后肌生长抑制素耗竭会增加小鼠肌肉质量的假设，这种影响将通过肌纤维肥大介导，而肌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（在肌生长抑制素耗竭后测试长达一年）；2. 检查肌肉生长抑制素消耗后肌肉质量的某些方面，包括产生力的能力、对拉伸损伤的易感性、疲劳程度、根据肌球蛋白重链异构体的表达或SDH活性分类的不同纤维类型的比例、胶原含量和前胶原基因的表达；, 3。为了验证发育后肌生长抑制素缺乏会通过增加肌原纤维蛋白合成而不是抑制蛋白质水解来诱导肌肉肥大，会上调mRNA翻译相关基因的表达，并会通过激活Akt-mTOR-4EBP1/S6K1通路刺激mRNA翻译的假设。肌生长抑制素基因的发育后破坏将通过激活他莫昔芬诱导的Cre重组酶来完成，该重组酶由转基因组成，在小鼠中，肌生长抑制素基因的关键第三外显子两侧是loxP序列（Cre重组酶的靶标）。在短期机制研究中，注射JA16抗肌生长抑制素抗体可抑制肌生长抑制素活性。肌原纤维蛋白的合成将通过稳定同位素示踪剂掺入来测定。通过定量RT-PCR检测基因表达。所选蛋白的浓度和磷酸化状态将通过免疫印迹测定。免疫组织化学将用于评估肌纤维肥大、纤维类型、纤维数量和肌核结构域体积。力的产生、疲劳和拉伸损伤将通过体外孤立肌肉的刺激进行研究。与公共卫生的相关性：肌肉萎缩导致各种情况下的虚弱，包括肌肉萎缩症、癌症、糖皮质激素管理、不活动、关节炎、营养不良、艾滋病和老年。在这些情况下，减少肌肉生长抑制素的活性可能会改善这种肌肉损失和无力。了解降低成熟肌肉中肌肉生长抑制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.