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降低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降低时骨骼肌会发生什么。拟议的实验将解决三个具体目标：1。测试以下假设：发育后肌肉生长抑制素耗尽将增加小鼠的肌肉质量，该效应将由肌纤维肥大介导，而每块肌肉的肌纤维数量或肌核数量没有增加，并且该效应将长期持续（在肌肉生长抑制素耗尽后测试长达一年）; 2.检查肌生长抑制素耗尽后肌肉质量的某些方面，包括产生力的能力、对牵张诱导的损伤的敏感性、疲劳性、通过肌球蛋白重链同种型的表达或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;以及，3.以检验以下假设：发育后肌肉生长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水解来诱导肌肉肥大，将上调参与mRNA翻译的基因的表达，并将通过激活Akt-mTOR-4 EBP 1/S6 K1途径来刺激mRNA翻译。肌肉生长抑制素基因的发育后破坏将通过在小鼠中激活由转基因组成型表达的他莫昔芬诱导型Cre重组酶来完成，其中肌肉生长抑制素基因的关键第三外显子侧接loxP序列（Cre重组酶的靶标）。在短期机制研究中，通过注射JA 16抗肌肉生长抑制因子抗体将抑制肌肉生长抑制因子活性。肌原纤维蛋白合成将通过稳定同位素示踪剂掺入来确定。将通过定量RT-PCR测定基因表达。将通过免疫印迹法测定选定蛋白质的浓度和磷酸化状态。免疫组织化学将用于评估肌纤维肥大、纤维类型、纤维数量和肌纤维结构域体积。力的产生、疲劳性和牵拉损伤将通过体外刺激离体肌肉来研究。与公共卫生的相关性：肌肉萎缩在各种情况下引起虚弱，包括肌肉营养不良、癌症、糖皮质激素施用、不活动、关节炎、营养不良、艾滋病和老年。在这些条件下降低肌生长抑制素活性可能会改善这种肌肉损失和虚弱。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.