Myostatin在小鼠腓肠肌失神经支配萎缩过程中的表达

目的:分析myostatin在腓肠肌失神经支配萎缩过程中的表达变化及其在肌萎缩过程中的作用。方法:采用坐骨神经横断术制备小鼠腓肠肌失神经支配模型,实时荧光定量PCR和Western印迹法分别检测失神经支配不同时段腓肠肌myostatin mRNA和蛋白表达水平,并对失神经前后肌肉湿重比、肌纤维横截面积进行比较。结果:失神经支配1 d时,腓肠肌myostatin mRNA迅速上升,3 d达到高峰,随后逐渐下降,而相应蛋白水平逐渐增高,7 d达到高峰继而逐渐下降,至56 d时mRNA和蛋白水平仍略高于正常水平。结论:腓肠肌失神经支配萎缩过程中myostatin的表达变化是一重要的分子事件。     

PPM1B and P-IKKβ expression levels correlated inversely with rat gastrocnemius atrophy after denervation

Activated inhibitor of nuclear factor-κB kinase β (IKKβ) is necessary and sufficient for denervated skeletal muscle atrophy. Although several studies have shown that Mg²⁺/Mn²⁺-dependent protein phosphatase 1B (PPM1B) inactivated IKKβ, few studies have investigated the role of PPM1B in denervated skeletal muscle. In this study, we aim to explore the expression and significance of PPM1B and phosphorylated IKKβ (P-IKKβ) during atrophy of the denervated gastrocnemius. Thirty young adult female Wistar rats were subjected to right sciatic nerve transection and were sacrificed at 0 (control), 2, 7, 14, and 28 days after denervation surgery. The gastrocnemius was removed from both the denervated and the contralateral limb. The muscle wet weight ratio was calculated as the ratio of the wet weight of the denervated gastrocnemius to that of the contralateral gastrocnemius. RT-PCR and Western blot analysis showed that mRNA and protein levels of PPM1B were significantly lower than those of the control group at different times after the initiation of denervation, while P-IKKβ showed the opposite trends. PPM1B protein expression persistently decreased while P-IKKβ expression persistently increased for 28 days after denervation. PPM1B expression correlated negatively with P-IKKβ expression by the Spearman test, whereas decreasing PPM1B expression correlated positively with the muscle wet weight ratio. The expression levels of PPM1B and P-IKKβ were closely associated with atrophy in skeletal denervated muscle. These results suggest that PPM1B and P-IKKβ could be markers in skeletal muscle atrophy.     

陈旧性坐骨神经缺损对大鼠腓肠肌组织蛋白含量及泛素表达的影响

目的：了解陈旧性坐骨神经缺损后肌肉萎缩过程中蛋白质降解的机制。方法：用SD大鼠建立坐骨神经缺损模型,切断大鼠右侧坐骨神经,形成10 mm缺损。测定术后1、2、3、4、6、9及12个月腓肠肌肌总蛋白的含量;免疫组化法观察组织中泛素的表达变化;Western印迹法测定组织中泛素蛋白的表达水平。结果：坐骨神经缺损后腓肠肌肌总蛋白含量随缺损时间延长呈进行性下降;正常腓肠肌组织中泛素呈低表达,随缺损时间延长泛素表达水平增强,持续到9个月,随后呈下调趋势。结论：陈旧性坐骨神经缺损后腓肠肌蛋白的降解、肌总蛋白量下降及肌萎缩可能和泛素-蛋白酶体途径有关。     

Beta adrenoceptor agonists, clenbuterol, and isoproterenol retard denervation atrophy in rat gastrocnemius muscle: use of 3-methylhistidine as a marker of myofibrillar degeneration

The effects of beta adrenergic agonists, clenbuterol (2 mg/kg body weight/d) and isoproterenol (12 mg/kg body weight/d), in normal innervated and denervated rat gastrocnemius muscle were investigated. The daily administration of beta adrenergic agonists to normal innervated rats for a short period (7 d) resulted in the hypertrophy of gastrocnemius as confirmed from the measurement of total tissue protein contents. The development of denervation atrophy witnessed a stimulation in the expression of acid and alkaline phosphatases, pointing to an enhanced myofibrillar degeneration. An administration of beta adrenergic agonists inhibited the expression of raised levels of these enzymes in denervated muscle. A measurement of 3-methylhistidine in muscle revealed a loss of amino acid with the progress in the development of denervation atrophy. Serum and urine samples from denervated rats showed a progressive accumulation of 3-methylhistidine. Clenbuterol and isoproterenol treatment to these rats resulted in an inhibition of 3-methylhistidine accumulation. When 3-methylhistidine was used as a marker of myofibrillar degeneration, the results seemed to suggest that the degeneration of cyto-contractile apparatus accompanying denervation atrophy is attenuated in the presence of beta adrenergic agonists, implying that these sympathomimetic drugs are capable of reversing denervation atrophy in rat gastrocnemius.     

黄芪丹参联合应用可延缓大鼠失神经骨骼肌萎缩

目的 探讨黄芪丹参联合应用对大鼠失神经骨骼肌萎缩的治疗效果。方法 取雄性健康8周龄SD大鼠20只,随机分为对照组、治疗组。两组均切断右侧坐骨神经约1cm,建立右下肢失神经腓肠肌萎缩模型。治疗组术后每日采用黄芪与丹参各1.5ml混合液,腹腔注射给药;对照组不做任何处理。于给药后的第2周和第3周时间点,各取两组大鼠双侧腓肠肌,测量肌湿重,计算肌湿重比值;另取两组右侧腓肠肌测量肌纤维横截面积、总蛋白含量、细胞凋亡率、叉头蛋白（FoXO3a）、肌萎缩F-box蛋白（MAFbx）mRNA及蛋白表达水平。 结果 第2周对照组、治疗组肌湿重比值：0.62±0.07、0.76±0.05;肌纤维横截面积：300.24±17.87、365.49±24.19;总蛋白量：63.32±5.30、80.28±3.12;细胞凋亡率：20.34±2.51、12.62±1.20;FoXO3a蛋白及mRNA表达：0.828±0.090、0.643±0.090、24.45±7.21、19.38±5.55;MAFbx蛋白及mRNA表达：1.224±0.090、0.956±0.040、27.45±8.50、19.44±5.64;第3周对照组、治疗组肌湿重比值：0.35±0.03、0.59±0.07;肌纤维横截面积：253.38±13.32、314.50±16.74;总蛋白量：50.34±4.12、67.70±5.42;细胞凋亡率：33.45±1.71、17.53±1.26;FoXO3a蛋白及mRNA表达：1.963±0.150、1.236±0.060、35.62±6.24、27.91±7.71;MAFbx蛋白及mRNA表达1.487±0.050、1.240±0.123、43.63±8.22、35.39±7.96,两组相比,差异具有统计学意义（P<0.05)。 结论 黄芪丹参联合应用可延缓大鼠失神经骨骼肌萎缩,其可能的机制为抑制细胞凋亡、延缓总蛋白降解速度;抑制蛋白降解通路FoXO3a、MAFbX mRNA和蛋白表达水平。     

氯化胆碱与制动性骨骼肌萎缩肌肉生成抑制素mRNA的表达

背景：研究发现类胆碱物质可增加乙酰胆碱的弥散及终板电流的幅度,对神经肌肉接点功能退化有一定的对抗作用。 目的：观察氯化胆碱对制动性骨骼肌萎缩的防治作用及对骨骼肌萎缩大鼠肌肉生成抑制素mRNA表达的影响。 方法：将30只雄性SD大鼠随机分为对照组、模型组和治疗组,每组10只。采用可塑性石膏固定模型组和治疗组大鼠右后肢制备肌萎缩模型。治疗组每日灌胃氯化胆碱(150 mg/kg),对照组和模型组灌胃等体积蒸馏水。4周后解剖右后肢腓肠肌,检测腓肠肌收缩张力、肌湿质量、蛋白质水平及肌肉生成抑制素mRNA的表达。 结果与结论：与对照组比较,模型组大鼠腓肠肌的收缩张力、肌湿质量、蛋白质水平均显著降低(P < 0.05或P < 0.01),肌肉生成抑制素mRNA表达显著增高(P < 0.01)。与模型组比较,治疗组大鼠腓肠肌的收缩张力、肌湿质量、蛋白质水平均显著升高(P < 0.05),肌肉生成抑制素mRNA表达显著降低(P < 0.05)。说明氯化胆碱能够显著提高制动性萎缩骨骼肌的收缩张力、肌湿质量、蛋白质水平,减少肌肉生成抑制素mRNA的表达,从而有效抑制骨骼肌制动性萎缩的发生。     

Stretching and electrical stimulation reduce the accumulation of MyoD,myostatin and atrogin-1 in denervated rat skeletal muscle

Denervation causes muscle atrophy and incapacity in humans. Although electrical stimulation (ES) and stretching (St) are commonly used in rehabilitation, it is still unclear whether they stimulate or impair muscle recovery and reinnervation. The purpose of this study was to evaluate the effects of ES and St, alone and combined (ES + St), on the expression of genes that regulate muscle mass (MyoD, Runx1, atrogin-1, MuRF1 and myostatin), on muscle fibre cross-sectional area and excitability, and on the expression of the neural cell adhesion molecule (N-CAM) in denervated rat muscle. ES, St and ES + St reduced the accumulation of MyoD, atrogin-1 and MuRF1 and maintained Runx1 and myostatin expressions at normal levels in denervated muscles. None of the physical interventions prevented muscle fibre atrophy or N-CAM expression in denervated muscles. In conclusion, although ES, St and ES + St changed gene expression, they were insufficient to avoid muscle fibre atrophy due to denervation.     

Time course of changes in angiogenesis-related factors in denervated muscle

Aim: Denervation leads to capillary regression in skeletal muscle. To gain insight into the regulation of this process, we investigated the time course of changes in capillary supply and gene expression of angiogenesis‐related factors during muscle denervation. Method: Female mice underwent surgery to transect the sciatic nerve, and then the gastrocnemius muscles were isolated at 12 h, 1, 3, 5, 10, 20, or 30 days after surgery. The capillary supply was assessed by immunohistochemistry using anti‐PECAM‐1/CD31 antibody. The mRNA levels for angiogenesis‐related factors were analysed using a real‐time polymerase chain reaction. Results: We found that the capillary‐to‐fibre ratio began to decrease 10 days after muscle denervation and decreased by 52% after 30 days. The levels of mRNA for vascular endothelial growth factor (VEGF), its receptors [fms‐like tyrosine kinase (Flt‐1) and a kinase insert domain‐containing receptor/fetal liver kinase‐1 (KDR/Flk‐1)], angiopoietin‐1 and angiopoietin‐2 of denervated muscle were immediately down‐regulated after 12 h and remained lower than control muscle until 30 days after muscle denervation. The levels of mRNA for the VEGF receptor, neuropilin‐1, angiopoietin receptor and Tie‐2 decreased within 12–24 h, but returned to near those of control muscle after 10–20 days, and again decreased after 30 days. Conclusions: These findings suggest that denervation‐induced capillary regression may be associated with down‐regulation of VEGF and angiopoietin signalling.     

The decrease in mature myostatin protein in male skeletal muscle is developmentally regulated by growth hormone

Myostatin inhibits myogenesis and there is reduced abundance of the mature protein in skeletal muscles of adult male compared with female mice. This reduction probably occurs after translation, which suggests that it is a regulated mechanism to reduce the availability of myostatin in males. Reduced myostatin may, thereby, contribute to the development of sexually dimorphic growth of skeletal muscle. Our first objective was to determine if the decrease in mature myostatin protein occurs before the linear growth phase to aid growth, or afterwards to maintain the mass of adult muscle. Mice were killed from 2 to 32 weeks and the gastrocnemius muscle was excised. Myostatin mRNA increased from 2 to 32 weeks and was higher in males than females ( P < 0.001). In contrast, mature protein decreased in males after 6 weeks ( P < 0.001). Our second objective was to determine if growth hormone (GH) induces the decrease in mature myostatin protein. GH increased myostatin mRNA and decreased the abundance of mature protein in hypophysectomised mice ( P < 0.05). Our final objective was to determine if the decrease in mature protein occurs in skeletal muscles of male Stat5b ^−/− mice (Stat5b mediates the actions of GH). As expected, mature myostatin protein was not reduced in Stat5b ^−/− males compared with females. However, myostatin mRNA remained higher in males than females irrespective of genotype. These data suggest that: (1) the decrease in mature myostatin protein is developmentally regulated, (2) GH acting via Stat5b regulates the abundance of mature myostatin and (3) GH acts via a non-Stat5b pathway to regulate myostatin mRNA.     

Remodeling of the vascular bed and progressive loss of capillaries in denervated skeletal muscle

Very little is known regarding structural and functional responses of the vascular bed of skeletal muscle to denervation and about the role of microcirculatory changes in the pathogenesis of post-denervation muscle atrophy. The purpose of the present study was to investigate the changes of the anatomical pattern of vascularization of the extensor digitorum longus muscle in WI/HicksCar rats 1, 2, 4, 7, 12, and 18 months following denervation of the limb. We found that the number of capillaries related to the number of muscle fibers, i.e. the capillary-to-fiber ratio (CFR), decreased by 88%, from 1.55 +/- 0.35 to 0.19 +/- 0.04, during the first 7 months after denervation and then slightly declined at a much lower rate during the next 11 months of observation to 10% of the CFR in normal muscle. Between months 2 and 4 after denervation, the CRF decreased by 2.4 times, from 58% to 24% of the control value. The loss of capillaries during the first 4 months following nerve transection was nearly linear and progressed with an average decrement of 4.16% per week. Electron microscopy demonstrated progressive degeneration of capillaries following nerve transection. In muscle cells close to degenerating capillaries, the loss of subsarcolemmal and intermyofibrillar mitochondria, local disassembly of myofibrils and other manifestations of progressive atrophy were frequently observed. The levels of devascularization and the degree of degenerative changes varied greatly within different topographical areas, resulting in significant heterogeneity of intercapillary distances and local capillary densities within each sample of denervated muscle. Perivascular and interstitial fibrosis that rapidly developed after denervation resulted in the spatial separation of blood vessels from muscle cells and their embedment in a dense lattice of collagen. As a result of this process, diffusion distances between capillaries and the surfaces of muscle fibers increased 10-400 times. Eighteen months after denervation most of the capillaries were heavily cushioned with collagen, and on the average 40% of the muscle cells were completely avascular. Devascularization of the tissue was accompanied by degeneration and death of muscle cells that had become embedded in a dense lattice of collagen. Immunofluorescent staining for the vascular isoform of alpha-actin revealed preservation of major blood vessels and a greater variability in thickness of their medial layer. Hyperplastic growth of the medial layer in some blood vessels resulted in narrowing of their lumens. By the end of month 7 after denervation, large deposits of collagen around arterioles often exceeded their diameters. Identification of oxidative muscle fibers after immunostaining for slow-twitch myosin, as well as using ultrastructural criteria, has shown that after 2 months of denervation oxidative muscle fibers were less susceptible to atrophy than glycolytic fibers. The lower rate of atrophy of type I muscle fibers at early stages of denervation may be explained by their initially better vascularization in normal muscle and their higher capacity to retain capillaries shortly after denervation. Thus, degeneration and loss of capillaries after denervation occurs more rapidly than the loss of muscle fibers, which results in progressive decrease of the CFR in denervated muscle. The change of capillary number in denervated muscle is biphasic: the phase of a rapid decrease of the CFR during the first 7 months after nerve transection is followed by the phase of stabilization. The presence of areas completely devoid of capillaries in denervated muscle and the virtual absence of such areas in normal muscle indicate the development of foci of regional hypoxia during long-term denervation. The anatomical pattern of muscle microvascularization changes dramatically after nerve transection. Each muscle fiber in normal muscle directly contacts on average 3-5 capillaries. (ABSTRACT TRUNCATED)     

去神经支配骨骼肌萎缩的多种治疗

背景：骨骼肌去神经支配后出现肌萎缩和收缩功能的丧失,肌纤维发生与萎缩相关的一系列的形态学和组织学的变化。近年来随着技术水平及理论研究的进展,治疗效果有了较大的提高,但仍未达到满意的效果。 目的：总结并讨论近年来有关去神经支配骨骼肌萎缩的治疗新进展,为肌肉功能的恢复提供理论基础。 方法：由第一作者用计算机检索中国期刊全文数据库(CNKI：2000/2010)和Medline数据库(2000/2010),检索词分别为“去神经支配,骨骼肌,肌萎缩,治疗”和“denervated,skeletal muscles,atrophy,treatment”。共检索到135篇文章,按纳入和排除标准对文献进行筛选,共纳入30篇文章,从物理治疗、神经修复和植入、细胞移植、药物、基因和中药治疗等方面进行总结和分析。 结果与结论：近年来去神经支配骨骼肌的治疗取得了很大进展,但这些方法只能在一定程度上缓解骨骼肌萎缩,在一段时间内维持肌肉的生理功能。目前研究随着去神经支配骨骼肌萎缩机制的阐明,针对性的治疗措施将会取得更好的效果。     

Myostatin and follistatin expression in skeletal muscles of rats with chronic heart failure

Skeletal muscle abnormalities can contribute to decreased exercise capacity in heart failure. Although muscle atrophy is a common alteration in heart failure, the mechanisms responsible for muscle mass reduction are not clear. Myostatin, a member of TGF-β family (transforming growth factor), regulates muscle growth and mass. Several studies have shown a negative correlation between myostatin expression and muscle mass. The aim of this study was to evaluate myostatin expression in skeletal muscles of rats with heart failure. As myostatin gene expression can be modulated by follistatin, we also evaluated its expression. Heart failure was induced by myocardial infarction (MI, n  = 10); results were compared to Sham-operated group ( n  = 10). Ventricular function was assessed by echocardiogram. Gene expression was analyzed by real-time PCR and protein levels by Western blotting in the soleus and gastrocnemius muscles; fibre trophism was evaluated by morphometric analysis. MI group presented heart failure evidence such as pleural effusion and right ventricular hypertrophy. Left ventricular dilation and dysfunction were observed in MI group. In the soleus muscle, cross-sectional area ( P  = 0.006) and follistatin protein levels (Sham 1.00 ± 0.36; MI 0.18 ± 0.06 arbitrary units; P  = 0.03) were lower in MI and there was a trend for follistatin gene expression to be lower in MI group ( P  = 0.085). There was no change in myostatin expression between groups. In gastrocnemius, all MI group parameters were statistically similar to the Sham. In conclusion, our data show that during chronic heart failure, decreased skeletal muscle trophism is combined with unchanged myostatin and reduced follistatin expression.     

Role of the motor nerve in activity-induced enzymatic adaptation in skeletal muscle

The sciatic nerve was cut on one side in 11 male cats, and a piece of the nerve was removed. The cats were then divided at random into two groups, a stimulation group (S) of five cats and a control group (C) of six cats. Bilateral electrical stimulation (2 Hz) of the gastrocnemius muscle (directly or via the motor nerve) was carried out in the S cats 4 h/day, 3 days/wk for 4 wk. The voltage delivered was adjusted in each cat so that both gastrocnemius muscles lifted identical loads the same distance. The activity of the tricarboxylic acid cycle marker enzyme succinate dehydrogenase (SDH) per unit of muscle weight more than doubled in response to stimulation both in the intact and the denervated gastrocnemius muscle. Stimulation did not affect the activity of the glycolytic marker enzyme 6-phosphofructokinase (PFK) or muscle capillarization. Denervation resulted in pronounced (approx 50%) fiber atrophy, which was not prevented by the stimulation. It is concluded that the presence of the motor nerve per se is not necessary for an activity-induced adaptation of the oxidative capacity of skeletal muscle.