The influence of eccentric exercise on mRNA expression of skeletal muscle regulators

To evaluate change in myostatin, follistatin, MyoD and SGT mRNA gene expression using eccentric exercise to study mechanisms of skeletal muscle hypertrophy. Young (28 ± 5 years) and older (68 ± 6 years) men participated in a bout of maximal single-leg eccentric knee extension on an isokinetic dynamometer at 60°/s: six sets, 12–16 maximal eccentric repetitions. Muscle biopsies of the vastus lateralis were obtained from the dominant leg before exercise and 24 h after exercise. Paired t tests were used to compare change (pre versus post-exercise) for normalized gene expression in all variables. Independent t tests were performed to test group differences (young vs. older). A probability level of P ≤ 0.05 was used to determine statistical significance with Bonferroni adjustments. We observed no significant change in myostatin (−0.59 ± 2.1 arbitrary units (AU); P = 0.42), follistatin (0.22 ± 3.4; P = 0.85), MyoD (0.23 ± 3.1; P = 0.82), or SGT (1.2 ± 6.4; P = 0.58) mRNA expression in young subjects 24 h after eccentric exercise. Similarly, we did not observe significant change in myostatin (−3.83 ± 8.8; P = 0.23), follistatin (−2.66 ± 5.2; P = 0.17), MyoD (−0.13 ± 3.1; P = 0.90), or SGT (−1.6 ± 3.5; P = 0.19) mRNA expression in older subjects. Furthermore, the non-significant changes in mRNA expression were not different between young and older subjects, P > 0.23 for all variables. Our data suggests that a single bout of maximal eccentric exercise does not alter myostatin, follistatin, MyoD or SGT mRNA gene expression in young or older subjects. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

肌肉生长抑制因子Myostatin引起骨质疏松症的机制研究

肌肉生长抑制因子Myostatin由肌细胞分泌,是肌肉生长和再生的负调节因子。在本文中,我们探讨了Myostatin在骨骼代谢、骨骼生物力学和肌肉发展过程中,影响骨质疏松症的原因,以及在骨骼代谢过程中的复杂变化。例如在体外实验中,Myostatin抑制成骨细胞的分化并以剂量依赖的方式刺激破骨细胞的活性。缺少Myostatin的小鼠破骨细胞数量减少,皮质厚度增加,胫骨皮质组织矿物质密度增加。胫骨中的皮质骨密度增加,而椎体骨密度增加。虽然肌肉骨骼系统是最常见的增龄性退化的主要原因,但是骨质疏松症究其原因,在于骨重建的基本过程如骨形成和骨吸收的精密程序化过程中,成骨细胞与破骨细胞的分化或功能如何是骨代谢平衡的重要因素。另外,Myostatin对于人类骨质疏松症和肌少症中的转化应用的探索仍有相应复杂性和进一步需求。     

siRNA真核表达载体构建及对细胞MSTN基因抑制的研究

肌肉生长抑制素是肌肉生长的负调控因子。本研究为了得到有效抑制绵羊MSTN基因的siRNA分子,设计8对siRNA寡聚核苷酸引物,经退火与pSilencerTM4.1-CMV neo连接构建siRNA真核表达载体,转染C2C12细胞后,利用RT-PCR及real time PCR检测干扰效果。结果显示,8条siRNA分子中有3条对MSTN基因有显著的抑制作用,其中psi-mstn-717和psi-mstn-986分子对MSTN mRNA抑制效率分别达到61%和53%。本研究证实载体表达siRNA能有效抑制细胞MSTN mRNA的表达,下一步筛选的siRNA将用于体内实验。     

Myostatin基因疫苗动物免疫效果的初步研究

目的:检测Myostatin基因疫苗的免疫效果,观察其对免疫动物的影响,为Myostatin基因疫苗的应用提供实验研究.方法:以Myostatin基因疫苗pVAC-TT-Ms免疫小鼠,ELISA法测定其免疫小鼠血清的抗体滴度,通过全自动生化分析仪检测血清指标.以组织化学染色(HE)染色检测Myostatin基因疫苗对免疫小鼠骨骼肌的影响.利用Scion Image 4.02 分析Myostatin基因疫苗对免疫小鼠肌纤维横截面积的影响.结果:基因疫苗Myostatin能够诱导小鼠产生针对Myostatin的抗血清.与正常对照组比较,基因疫苗pVAC-TT-Ms免疫的动物平均体重增加了9.8%,股四头肌、腓肠肌和胸大肌分别增加了24.1%、10.9%和20.3%.结论:基因疫苗Myostatin能够诱导小鼠产生针对Myostatin的特异性中和抗体.Myostatin基因疫苗免疫的动物体重增加,肌细胞有肥大现象,肌肉质量明显增加.     

Differentiation of human rhabdomyosarcoma RD cells is regulated by reciprocal, functional interactions between myostatin, p38 and extracellular regulated kinase signalling pathways

Rhabdomyosarcoma (RMS) includes heterogeneous tumours of mesenchymal derivation which are genetically committed to the myogenic lineage, but fail to complete terminal differentiation. Previous works have reported on deregulated myostatin, p38 and extracellular regulated kinase (ERK) signalling in RMS cell lines; however, the functional link between these pathways and their relative contribution to RMS pathogenesis and/or maintenance of the transformed phenotype in vitro are unclear. Herein we show that the constitutive expression of a dominant-negative form of activin receptor type IIb (dnACTRIIb), which inhibits myostatin signalling, decreased proliferation and promoted differentiation of the human RMS RD cell line. DnACTRIIb-dependent differentiation of RD cells correlated with a reduced SMAD2/3 (small mother against decapentaplegic) and ERK signalling and the activation of p38 pathway. Conversely, the expression of a constitutively activated ALK5 (activin receptor-like kinase) (caALK5) form, activating SMAD3 and ERK pathways, led to further impairment of RD differentiation. Pharmacological blockade of ERK pathway in RD cells was sufficient to replicate the biological phenotype observed in dnACTRIIb-expressing RD cells, and also recovered the differentiation of caALK5-expressing RD cells. Conversely, deliberate activation of p38 signalling mimics the effect of dnActRIIb and overcame the differentiation block in RD cells. These data indicate the existence of a network formed by myostatin/SMAD2/3, ERK and p38 pathways that, when deregulated, might contribute to the pathogenesis of RMS. The components of this network might, therefore, be a valuable target for interventions towards correcting the malignant phenotype of RMS.     

多发性肌炎患者肌肉组织Myostatin mRNA的表达

目的 探讨肌肉生长抑制素Myostatin基因mRNA在多发性肌炎患者肌肉组织中的表达。方法 采用半定量RT-PCR方法检测5例多发性肌炎肌肉组织和配对非肌肉病对照者肌肉组织Myostatin mRNA表达及其与临床病理特征的关系。结果 两组肌肉组织均有Myostatin mRNA表达;多发性肌炎肌肉组织Myostatin mRNA的表达指数为0.62±0.393,对照组肌肉组织0.34±0.150,二者差异无统计学意义( P = 0.199)。结论: myostatin基因mRNA表达水平在多发性肌炎肌肉组织中与对照组无明显差异。     

Role of insulin, insulin-like growth factors, and muscle regulatory factors in the compensatory growth of the trout (Oncorhynchus mykiss)

To examine the various mechanisms involved in compensatory growth in Oncorhynchus mykiss, an experimental protocol involving 1, 2 or 4 weeks of fasting followed by a single ad libitum re-feeding period of 4 weeks was designed for alevins. Morphological parameters including body weight, specific growth rates (SGR), and coefficient factor decreased significantly during fasting. Re-feeding accelerated growth and restored final body weight in groups previously fasted. Plasma insulin and glucose decreased in fasting, while normal levels were restored in all re-fed groups. The expression profile of insulin-like growth factors (IGFs) in liver and of the main muscle growth regulators in white muscle was examined using real-time quantitative RT-PCR. Fasting decreased the expression of IGF-I mRNA in both tissues, while re-feeding restored expression to control values. In contrast, IGF-II expression was not affected by any treatment in either tissue. Insulin- and IGF-I-binding assays in partial semi-purifications (of soluble proteins) in white skeletal muscle showed that insulin binding was not affected by either fasting or re-feeding, whereas fasting up-regulated IGF-I binding. The expression of IGFRIb mRNA in white skeletal muscle also increased with fasting, while IGFRIa increased with re-feeding, indicating that the two receptor isoforms are differentially regulated. The mRNA expression of myogenic regulator factors and fibroblast growth factors (FGFs) was not affected throughout the experiment, except for myogenin, which first decreased and then showed a rebound effect after 4 weeks of fasting. Myostatin mRNA expression did not change during fasting, although re-feeding caused a significant decrease. In conclusion, re-feeding of previously fasted trout induced compensatory growth. The differential regulation in muscle expression of IGF-I, IGF-I receptors, and myostatin indicates their contribution to this compensatory mechanism.     

Regulation of body mass growth through activin type IIB receptor in teleost fish

Myostatin is a TGF-β family member that plays a key role in regulating skeletal muscle growth. Previous studies in mammals have demonstrated that myostatin is capable of binding the two activin type II receptors. Additionally, activin type II receptors have been shown to be capable of binding a number of other TGF-β family members besides myostatin. An injection of a soluble form of activin type IIB receptor obtained from CHO cells into wild-type mice generated up to a 60% increase in muscle mass in 2 weeks. The knowledge on the role of activin receptors in fish is limited. In the present study, we examined the growth effect of administering a recombinant, soluble form of goldfish activin type IIB receptor extracellular domain to juvenile and larval goldfish (Carassius auratus), African catfish (Clarias gariepinus) larvae and tilapia (Oreochromis aureus) larvae. We have expressed the goldfish activin type IIB receptor extracellular domain in the yeast Pichia pastoris and we have demonstrated for the first time that this recombinant molecule stimulates growth in teleost fish in a dose-dependent manner. We provide evidence that this body weight increase is achieved by an increase in muscle mass and protein content. Histological analysis of the goldfish muscle revealed that treated fish exhibited hyperplasia as compared to controls. These findings contribute to the understanding of the mechanisms that regulate growth in non-mammalian vertebrates and suggest a powerful biotechnology approach to improving fish growth in aquaculture.     

Myostatin inhibition slows muscle atrophy in rodent models of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease leading to motor neuron cell death, but recent studies suggest that non-neuronal cells may contribute to the pathological mechanisms involved. Myostatin is a negative regulator of muscle growth whose function can be inhibited using neutralizing antibodies. In this study, we used transgenic mouse and rat models of ALS to test whether treatment with anti-myostatin antibody slows muscle atrophy, motor neuron loss, or disease onset and progression. Significant increases in muscle mass and strength were observed in myostatin-antibody-treated SOD1(G93A) mice and rats prior to disease onset and during early-stage disease. By late stage disease, only diaphragm muscle remained significantly different in treated animals in comparison to untreated controls. Myostatin inhibition did not delay disease onset nor extend survival in either the SOD1(G93A) mouse or rat. Together, these results indicate that inhibition of myostatin does not protect against the onset and progression of motor neuron degenerative disease. However, the preservation of skeletal muscle during early-stage disease and improved diaphragm morphology and function maintained through late stage disease suggest that anti-myostatin therapy may promote some improved muscle function in ALS.     

Increased myostatin activity and decreased myocyte proliferation in chronic alcoholic cardiomyopathy

Background: Apoptosis mediates in alcohol‐induced heart damage leading to cardiomyopathy (CMP). Myocyte proliferation may compensate for myocyte loss. Myostatin is upregulated after cardiac damage and by alcohol consumption thereby decreasing myocyte renewal. We assess the potential role of alcohol in inducing myocyte apoptosis as well as in inhibiting myocyte proliferation. Methods: Heart samples were obtained from organ donors, including 22 high alcohol consumers, 22 with hypertension, 8 with other causes of CMP, and 10 healthy donors. Evaluation included medical record with data on daily, recent and lifetime ethanol consumption, chest X‐ray, left ventricular (LV) function assessed by two‐dimensional echocardiography, and LV histology and immunohistochemistry. Apoptosis was evaluated by TUNEL, BAX, and BCL‐2 assays. Myocyte proliferation was evaluated with Ki‐67 assay. Myostatin activity was measured with a specific immunohistochemical assay. CMP was assessed by functional and histological criteria. Results: Alcoholic and hypertensive donors with CMP showed higher apoptotic indices than did their partners without CMP. Myostatin activity was higher in alcoholics than in controls, mainly in those with CMP. The increase in myostatin expression in alcoholic CMP was higher than in other groups. The Ki‐67 proliferation index increased in all groups with CMP compared to those without CMP, with alcoholics showing a lower increase in this proliferation response. Conclusions: Alcohol produces cardiac myocyte loss through apoptosis but also partially inhibits myocyte proliferation through myostatin up‐regulation. The final result may suppose an imbalance in myocyte homeostasis, with a net loss in total ventricular myocyte mass and progressive ventricular dysfunction.