骨骼肌钝挫伤恢复过程中HIF-1α、AMPKα2表达的改变

目的：观测大鼠骨骼肌钝挫伤（SMBI）恢复进程中腺苷酸活化蛋白激酶α2（AMPKα2）、缺氧诱导因子-1α（HIF-1α）的表达变化,探讨SMBI恢复可能生物学机制。方法：雄性Wistar大鼠48只,随机选取6只作为正常对照组;另42只大鼠重物砸伤后肢小腿建立钝挫伤模型后分7组（n=6）,造模后各组分别在伤后12 h、2 d、5 d、7 d、10 d、15 d、30 d取材,检测小腿三头肌HIF-1α、AMPKα2表达的变化。结果：损伤后12 h HIF-1α、AMPKα2表达均明显升高,在伤后15 d开始回落接近正常;HIF-1α、AMPKα2表达的峰值出现在伤后2 d内,伤后5 d后表达量开始下降;除HIF-1α mRNA在伤后2 d组表达出现峰值外,其余时间点二者mRNA与蛋白表达时程变化基本一致。结论：SMBI后,HIF-1α、AMPKα2可能通过参与调解缺氧适应、肌细胞再生、能量代偿起到促进伤后恢复的作用。     

自噬在急性心肌梗死发病中作用的研究进展

自噬是生物细胞内普遍存在且高度保守的一种生理过程,其通过溶酶体融合降解细胞内的大分子组分、受损的细胞器以及侵入胞内的病原菌,以达到维持细胞稳态的目的。自噬在多种疾病的发生发展中也发挥十分重要的作用,尤其是心血管疾病。自噬对其病程的发展可以发挥两种截然不同的作用。适当的自噬作用可以降低炎症反应和氧化应激促进细胞的存活,以及通过减少泡沫细胞的形成而对维持心血管的正常功能起一个保护作用;但过度的自噬作用会对细胞造成不可逆的损伤,诱导细胞发生不依赖于caspase的自噬性细胞死亡,增加局部的炎症反应,从而促进动脉粥样硬化病变的发展。本文就自噬在急性心肌梗死发生发展中作用的研究进展进行了综述,探讨自噬成为预防及治疗心血管疾病新靶标的可能性。     

鳞翅目昆虫中自噬相关分子Atg8的研究进展

自噬是细胞通过溶酶体自主降解以实现细胞内物质循环利用的过程,在昆虫细胞分化和个体发育中起着重要作用。鳞翅目昆虫属于完全变态昆虫,会通过自噬和凋亡完成蜕变重建过程,是研究自噬机制的模式生物。自噬相关蛋白Atg8是哺乳动物微管相关蛋白1轻链3的同系物,是自噬相关蛋白的核心蛋白家族,对自噬小体形成、膜的延伸、特定物质识别等具有重要意义。文中就鳞翅目昆虫Atg8在自噬信号通路中的作用、Atg8结构特点、Atg8表达分布及Atg8-PE/Atg8水平与自噬活性关系进行了综述。Atg8-PE是自噬信号通路中两个类泛素结合系统之一,在自噬中起着关键作用。序列分析表明,鳞翅目昆虫Atg8与其他真核生物同源蛋白的整体结构相似,尤其与其他昆虫同源蛋白的氨基酸序列高度一致,体现了Atg8的高度保守性。鳞翅目昆虫发育不同阶段,Atg8在中肠、唾液腺、卵巢、脂肪体、丝腺等器官中的表达分布各不相同。并且,Atg8在核质中分布也存在差异,Atg8在细胞核与细胞质之间的穿梭可能存在蛹化前阶段的某些细胞中。通过检测Atg8-PE在细胞内的表达水平或Atg8含量的变化,可以评价细胞自噬的发生程度。     

间歇性禁食与运动对骨骼肌自噬的激活及减脂效果的比较

目的:以运动作为对比,观察不同时长(14 d、28 d)间歇性禁食的体重控制效果,探究其对骨骼肌质量及自噬的影响。方法:选取60只SD大鼠(雄)随机分为3组(n=20):安静对照组(Sed组)、间歇性禁食组(InF组)、有氧运动组(Exe组),设实验周期分别为14 d和28 d。InF组采用间歇性禁食(隔日禁食),Exe组施加跑台运动干预,每周记录体重。DEXA检测体脂并计算体脂指数,天平称量比目鱼肌湿重(双侧)并计算湿重指数,免疫荧光检测细胞外基质蛋白laminin反映肌纤维横截面积、检测LC3标记自噬体,透射电镜观察自噬体数量及形态,Western blot检测自噬相关蛋白ULK1、LC3、p62及调控蛋白AMPKα、p-AMPKα(Thr172)的表达情况。结果:①干预7 d开始,InF、Exe组大鼠体重显著低于Sed组,且InF组体重显著低于Exe组(P<0.01),28 d干预后InF、Exe组体脂指数显著低于Sed组,且InF组体脂指数显著低于Exe组(P<0.05)。②干预28 d时Exe组单根肌纤维面积较Sed、InF组明显增大(P<0.01)。③在各...     

运动时长和强度对大鼠骨骼肌线粒体自噬的影响及其机制

本文旨在探讨不同时长、不同强度运动对大鼠骨骼肌线粒体功能和自噬的影响及FUNDC1的作用。选取60只雄性SD大鼠随机分为2周、4周的安静对照组(Con组)、中等强度运动组(M-ex组,跑台运动16 m/min,1 h/d,6 d/week)和大强度运动组(Hi-ex组,跑台运动35 m/min,20 min/d,6 d/week),每组10只。干预结束后分离双侧比目鱼肌,石蜡切片制备透射电镜样本,用ELISA检测柠檬酸合成酶(citrate synthase, CS)的含量,用冰冻切片免疫荧光染色法观察微管相关蛋白1轻链3 (microtubule-associated protein 1 light chain 3, LC3)/细胞色素c氧化酶IV亚型(cytochrome c oxidase IV, COX-IV)、FUNDC1/COX-IV及LC3/FUNDC1的共定位情况。提取骨骼肌线粒体,用Western blot检测过氧化物酶体增殖物激活受体γ共激活因子-1α(peroxisome proliferator-activated receptorγco-activator-1α, PGC-1α)、COX-I、腺苷酸活化蛋白激酶α(AMP-activated protein kinaseα,AMPKα)、p-AMPKα、Unc-51样激酶1 (Unc-51 like kinase 1, ULK1)、FUNDC1、LC3、自噬选择性底物(p62)等自噬相关蛋白表达。结果显示,运动可上调线粒体功能,即PGC-1α、COX-I蛋白表达和CS含量,2周Hi-ex组和2周M-ex组之间无差异,而4周Hi-ex组显著低于4周M-ex组。在运动强度相同的情况下,4周运动组大鼠的骨骼肌线粒体自噬激活程度高于2周运动组;在运动时长相同的情况下,Hi-ex组的线粒体自噬激活程度高于M-ex组。2和4周运动干预均可提高LC3/COX-IV、COX-IV/FUNDC1、FUNDC1/LC3共定位。运动可提高LC3-II/LC3-I比值,下调p62蛋白表达水平,上调FUNDC1、ULK1蛋白表达水平和AMPKα磷酸化水平,4周Hi-ex组的这些蛋白表达变化幅度显著大于4周M-ex组。以上结果提示,运动可诱导骨骼肌线粒体自噬,自噬的激活程度与运动时间和强度有关,运动的诱导机制可能是通过AMPK-ULK1通路影响FUNDC1的表达。     

运动训练及饮食限制对小鼠骨骼肌线粒体自噬Bnip3/Nix表达的影响*

目的:探讨运动训练和饮食限制对小鼠骨骼肌线粒体自噬蛋白Bnip3/Nix表达的影响。方法:C57雄性小鼠按体重随机分为对照组(C),不做干预;饮食限制组(D),饮食控制在C组的60%;运动训练组(E),10周递增负荷耐力训练;饮食限制加运动训练组(DE),进行饮食限制和耐力训练。8只/组,干预10周后,麻醉处死提取腓肠肌,Western blot技术检测Bnip3、Nix蛋白表达。结果:与C组比较,干预后D、E及DE组的体重和体重增长值均显著降低(P＜0.01),腓肠肌Bnip3、Nix蛋白表达升高,其中仅DE组Nix蛋白表达升高具有统计学意义(P＜0.05)。结论:运动训练加饮食限制能有效促进小鼠骨骼肌Nix蛋白表达,有利于骨骼肌线粒体自噬发生。     

自噬的相关分子机制

细胞自噬是真核生物中高度保守的一类生物学途径,它通过降解细胞浆内不同组分,维持细胞自身平衡并帮助细胞在应激情况下生存。自噬在生物体生长发育、免疫防御、肿瘤抑制及神经退行性疾病中都有重大的意义。哺乳动物细胞中,自噬过程主要由自噬相关蛋白（Atg）所形成的一系列复合物所调控,这些蛋白质分别在自噬的启动、自噬泡的形成、延伸及成熟和降解过程中发挥重要的作用。在此,本文针对一些重要的自噬相关蛋白质对近年来自噬分子机制的研究进展做一总结。     

线粒体自噬的研究进展

由于线粒体在生物氧化和能量转换过程中会产生活性氧,线粒体DNA又比核DNA更容易发生突变,因此线粒体是一种比较容易受到损伤的细胞器.及时清除细胞内受损的线粒体对细胞维持正常的状态具有重要的作用.细胞主要通过自噬来清除损伤线粒体,维持细胞稳态.越来越多的研究表明,线粒体自噬是一种特异性的过程,线粒体通透性孔道通透性的改变在这个过程中起着重要的作用.线粒体自噬在维持细胞内线粒体的正常功能和基因组稳定性上起着重要作用,但是线粒体发生自噬的信号通路及其调控机制还有待进一步深入研究.     

细胞自噬及真菌中自噬研究概述

细胞自噬是真核生物中广泛存在的、主要依赖于溶酶体或液泡的保守的降解途径,通过降解细胞内过多或异常的蛋白、细胞器等以维持正常的细胞功能。近10年来自噬研究方面的飞速进展显示出自噬与癌症、神经退行性疾病、衰老及心脏病等人类疾病相关。与此同时,自噬在丝状真菌的生长、形态和发育等方面发挥着重要作用,特别是在丝状真菌的细胞分化过程中,自噬起到了关键性作用,如致病性生长、程序性细胞死亡及孢子形成。本文主要论述了什么是自噬,自噬的检测方法及以真菌为对象的自噬研究进展。     

Wnt信号通路在骨骼肌损伤修复过程中的作用及机制研究CSCD

Wnt信号通路分为经典Wnt信号通路和非经典Wnt信号通路,而非经典Wnt信号通路又可分为Wnt/Ca^(2+)信号通路、Wnt/PCP信号通路和Wnt/PI3K信号通路。经典Wnt信号通路的恰当激活可有效抑制Notch信号通路,促进成肌分化和肌管融合。但经典Wnt信号通路过早或持续性激活,可通过调节多种细胞因子的表达,加重损伤骨骼肌纤维化,损害骨骼肌再生。而Wnt7a通过多条非经典Wnt信号通路刺激肌卫星细胞扩增、迁移,促进骨骼肌损伤修复,并能激活Akt/mTOR信号通路而诱导肌纤维肥大。     

Chemerin‐induced mitochondrial dysfunction in skeletal muscle

Chemerin is a novel adipocyte‐derived factor that induces insulin resistance in skeletal muscle. However, the effect of chemerin on skeletal muscle mitochondrial function has received little attention. In the present study, we investigated whether mitochondrial dysfunction is involved in the pathogenesis of chemerin‐mediated insulin resistance. In this study, we used recombinant adenovirus to express murine chemerin in C57BL/6 mice. The mitochondrial function and structure were evaluated in isolated soleus muscles from mice. The oxidative mechanism of mitochondrial dysfunction in cultured C2C12 myotubes exposed to recombinant chemerin was analysed by western blotting, immunofluorescence and quantitative real‐time polymerase chain reaction. The overexpression of chemerin in mice reduced the muscle mitochondrial content and increased mitochondrial autophagy, as determined by the increased conversion of LC3‐I to LC3‐II and higher expression levels of Beclin1 and autophagy‐related protein‐5 and 7. The chemerin treatment of C2C12 myotubes increased the generation of mitochondrial reactive oxygen species, concomitant with a reduced mitochondrial membrane potential and increased the occurrence of mitochondrial protein carbonyls and mitochondrial DNA deletions. Knockdown of the expression of chemokine‐like receptor 1 or the use of mitochondria‐targeting antioxidant Mito‐TEMPO restored the mitochondrial dysfunction induced by chemerin. Furthermore, chemerin exposure in C2C12 myotubes not only reduced the insulin‐stimulated phosphorylation of protein kinase B (AKT) but also dephosphorylated forkhead box O3α (FoxO3α). Chemerin‐induced mitochondrial autophagy likely through an AKT‐FoxO3α‐dependent signalling pathway. These findings provide direct evidence that chemerin may play an important role in regulating mitochondrial remodelling and function in skeletal muscle.     

低氧习服大鼠骨骼肌毛细血管密度和血流供应的变化特点

目的：观察大鼠在低氧习服过程中,骨骼肌毛细血管密度和血流供应的变化规律。方法：大鼠在模拟海拔5000m低氧5、15和30d后,用肌球蛋白ATP酶（mATPase)组织化学方法显示骨骼肌Ⅰ、Ⅱ型纤维和毛细血管并进行图像分析;用放射性微球法测定骨骼肌血流量。结果：低氧5d组大鼠骨骼肌纤维即出现明显萎缩,15d和30d组大鼠毛细血管密度显著增高,但单位面积内毛细血管数/肌纤维数（C/F）的比值无明显变化。在所观测的时间内,各组大鼠骨骼肌血流量未见明显变化。结论：大鼠在低氧习服过程中,毛细血管并未发生真正的增生,而由于骨骼肌纤维出现萎缩,使毛细敌国管数目相对增多。     

Cellular responses in exertion-induced skeletal muscle injury

Muscle injury is a common result of muscle exertion caused by overload and over-activity. In this presentation, an attempt was made to discuss models of muscle injury which involve exertion but not excessive strain, although most functional activities of the extremities require some eccentric muscle actions. Muscle injury is characterized by cellular and extracellular matrix responses which appear to be common to all types of muscle trauma - even in the absence of bleeding. Using tenotomy and functional over-load of the rat hindlimb muscles as examples, illustrations of several of these responses are presented and discussed.     

Leptin expression in human primary skeletal muscle cells is reduced during differentiation

We found leptin to be strongly expressed in undifferentiated human myoblasts derived from biopsies of the thigh (Musculus vastus lateralis). Both mRNA expression and secretion of leptin were reduced during in vitro differentiation into primary myotubes. However, the expression of the leptin receptor (OB-Rb) mRNA, was unchanged during differentiation of the muscle cells. Administration of recombinant leptin had no effect on leptin, myogenin, myoD, or GLUT4 mRNA expressions during the period of cellular differentiation. A functional leptin receptor was demonstrated by an acute leptin-induced 1.5-fold increase in ERK activity (P = 0.029). Although mRNA expression of regulation of suppressor of cytokine signaling-3 (SOCS-3) mRNA expression was unaltered, leptin significantly stimulated fatty acid oxidation after 6 h measured as acid soluble metabolites (ASM). Palmitic acid (PA), oleic acid (OA), and eicosapentaenoic acid (EPA), known to modulate leptin expression in other tissues, had no effect on mRNA expression or secretion of leptin from human myotubes. In conclusion, we demonstrate that leptin is highly expressed in undifferentiated human myoblasts and the expression is reduced during differentiation to mature myotubes. The role of leptin in these cells needs to be further characterized.     

Protein synthesis in skeletal muscle measured at different times during a 24 hour period

Six groups of 5 male rats (starting body weight 109 g) were allowed free access to a conventional rat diet. At 4 hourly intervals, starting at 10.00 h muscle protein synthesis was measured. By relating the weights of the gastrocnemius and soleus muscles to the initial body weights of the animals (i.e., at 09.30, day 1), a linear increase in muscle weight throughout the day was demonstrated. The fractional rate of muscle protein synthesis varied from 16.8% per day to 20.3% per day in gastrocnemius muscle and from 17.9% per day and 22.1% per day in the soleus. It was calculated that the maximum error incurred in estimating daily muscle protein synthesis by extrapolation of the value at any one time was 6% in gastrocnemius and 9% in soleus. It is concluded that calculations of the average rate of muscle protein degradation based on the difference between the rates of synthesis and deposition are generally valid in rats allowed free access to an adequate diet.     

Alcohol-induced autophagy contributes to loss in skeletal muscle mass

Patients with alcoholic cirrhosis and hepatitis have severe muscle loss. Since ethanol impairs skeletal muscle protein synthesis but does not increase ubiquitin proteasome-mediated proteolysis, we investigated whether alcohol-induced autophagy contributes to muscle loss. Autophagy induction was studied in: A) Human skeletal muscle biopsies from alcoholic cirrhotics and controls, B) Gastrocnemius muscle from ethanol and pair-fed mice, and C) Ethanol-exposed murine C2C12 myotubes, by examining the expression of autophagy markers assessed by immunoblotting and real-time PCR. Expression of autophagy genes and markers were increased in skeletal muscle from humans and ethanol-fed mice, and in myotubes following ethanol exposure. Importantly, pulse-chase experiments showed suppression of myotube proteolysis upon ethanol-treatment with the autophagy inhibitor, 3-methyladenine (3MA) and not by MG132, a proteasome inhibitor. Correspondingly, ethanol-treated C2C12 myotubes stably expressing GFP-LC3B showed increased autophagy flux as measured by accumulation of GFP-LC3B vesicles with confocal microscopy. The ethanol-induced increase in LC3B lipidation was reversed upon knockdown of Atg7, a critical autophagy gene and was associated with reversal of the ethanol-induced decrease in myotube diameter. Consistently, CT image analysis of muscle area in alcoholic cirrhotics was significantly reduced compared with control subjects. In order to determine whether ethanol per se or its metabolic product, acetaldehyde, stimulates autophagy, C2C12 myotubes were treated with ethanol in the presence of the alcohol dehydrogenase inhibitor (4-methylpyrazole) or the acetaldehyde dehydrogenase inhibitor (cyanamide). LC3B lipidation increased with acetaldehyde treatment and increased further with the addition of cyanamide. We conclude that muscle autophagy is increased by ethanol exposure and contributes to sarcopenia.     

Regenerative potential of human skeletal muscle during aging

In this study, we have investigated the consequences of aging on the regenerative capacity of human skeletal muscle by evaluating two parameters: (i) variation in telomere length which was used to evaluate the in vivo turn-over and (ii) the proportion of satellite cells calculated as compared to the total number of nuclei in a muscle fibre. Two skeletal muscles which have different types of innervation were analysed: the biceps brachii, a limb muscle, and the masseter, a masticatory muscle. The biopsies were obtained from two groups: young adults (23 +/- 1.15 years old) and aged adults (74 +/- 4.25 years old). Our results showed that during adult life, minimum telomere lengths and mean telomere lengths remained stable in the two muscles. The mean number of myonuclei per fibre was lower in the biceps brachii than in the masseter but no significant change was observed in either muscle with increasing age. However, the number of satellite cells, expressed as a proportion of myonuclei, decreased with age in both muscles. Therefore, normal aging of skeletal muscle in vivo is reflected by the number of satellite cells available for regeneration, but not by the mean number of myonuclei per fibre or by telomere lengths. We conclude that a decrease in regenerative capacity with age may be partially explained by a reduced availability of satellite cells.     

Key signalling factors and pathways in the molecular determination of skeletal muscle phenotype

The molecular basis and control of the biochemical and biophysical properties of skeletal muscle, regarded as muscle phenotype, are examined in terms of fibre number, fibre size and fibre types. A host of external factors or stimuli, such as ligand binding and contractile activity, are transduced in muscle into signalling pathways that lead to protein modifications and changes in gene expression which ultimately result in the establishment of the specified phenotype. In skeletal muscle, the key signalling cascades include the Ras-extracellular signal regulated kinase-mitogen activated protein kinase (Erk-MAPK), the phosphatidylinositol 3'-kinase (PI3K)-Akt1, p38 MAPK, and calcineurin pathways. The molecular effects of external factors on these pathways revealed complex interactions and functional overlap. A major challenge in the manipulation of muscle of farm animals lies in the identification of regulatory and target genes that could effect defined and desirable changes in muscle quality and quantity. To this end, recent advances in functional genomics that involve the use of micro-array technology and proteomics are increasingly breaking new ground in furthering our understanding of the molecular determinants of muscle phenotype.     

Tetanic contraction induces enhancement of fatigability and sarcomeric damage in atrophic skeletal muscle and its underlying molecular mechanisms

Muscle unloading due to long-term exposure of weightlessness or simulated weightlessness causes atrophy, loss of functional capacity, impaired locomotor coordination, and decreased resistance to fatigue in the antigravity muscles of the lower limbs. Besides reducing astronauts＇ mobility in space and on returning to a gravity environment, the molecular mechanisms for the adaptation of skeletal muscle to unloading also play an important medical role in conditions such as disuse and paralysis. The tail-suspended rat model was used to simulate the effects of weightlessness on skeletal muscles and to induce muscle unloading in the rat hindlimb. Our series studies have shown that the maximum of twitch tension and the twitch duration decreased significantly in the atrophic soleus muscles, the maximal tension of high-frequency tetanic contraction was significantly reduced in 2-week unloaded soleus muscles, however, the fatigability of high- frequency tetanic contraction increased after one week of unloading. The maximal isometric tension of intermittent tetanic contraction at optimal stimulating frequency did not alter in 1- and 2-week unloaded soleus, but significantly decreased in 4-week unloaded soleus. The 1-week unloaded soleus, but not extensor digitorum Iongus （EDL）, was more susceptible to fatigue during intermittent tetanic contraction than the synchronous controls. The changes in K＋ channel characteristics may increase the fatigability during high-frequency tetanic contraction in atrophic soleus muscles. High fatigability of intermittent tetanic contraction may be involved in enhanced activity of sarcoplasmic reticulum Ca2＋-ATPase （SERCA） and switching from slow to fast isoform of myosin heavy chain, tropomyosin, troponin I and T subunit in atrophic soleus muscles. Unloaded soleus muscle also showed a decreased protein level of neuronal nitric oxide synthase （nNOS）, and the reduction in nNOS-derived NO increased frequency of calcium sparks and elevated intracellular resting Ca2＋ concentration （[Ca2＋]i） in unloaded soleus muscles. High [Ca2＋]i activated calpain-1 which induced a higher degradation of desmin. Desmin degradation may loose connections between adjacent myofibrils and further misaligned Z-disc during repeated tetanic contractions. Passive stretch in unloaded muscle could preserve the stability of sarcoplasmic reticulum Ca2＋ release channels by means of keeping nNOS activity, and decrease the enhanced protein level and activity of calpain to control levels in unloaded soleus muscles. Therefore, passive stretch restored normal appearance of Z-disc and resisted in part atrophy of unloaded soleus muscles. The above results indicate that enhanced fatigability of high-frequency tetanic contraction is associated to the alteration in K＋ channel characteristics, and elevated SERCA activity and slow to fast transition of myosin heavy chain （MHC） isoforms increases fatigability of intermittent tetanic contraction in atrophic soleus muscle. The sarcomeric damage induced by tetanic contraction can be retarded by stretch in atrophic soleus muscles.