Myosin: Formation and maintenance of thick filaments

Skeletal muscle consists of bundles of myofibers containing millions of myofibrils, each of which is formed of longitudinally aligned sarcomere structures. Sarcomeres are the minimum contractile unit, which mainly consists of four components: Z‐bands, thin filaments, thick filaments, and connectin/titin. The size and shape of the sarcomere component is strictly controlled. Surprisingly, skeletal muscle cells not only synthesize a series of myofibrillar proteins but also regulate the assembly of those proteins into the sarcomere structures. However, authentic sarcomere structures cannot be reconstituted by combining purified myofibrillar proteins in vitro, therefore there must be an elaborate mechanism ensuring the correct formation of myofibril structure in skeletal muscle cells. This review discusses the role of myosin, a main component of the thick filament, in thick filament formation and the dynamics of myosin in skeletal muscle cells. Changes in the number of myofibrils in myofibers can cause muscle hypertrophy or atrophy. Therefore, it is important to understand the fundamental mechanisms by which myofibers control myofibril formation at the molecular level to develop approaches that effectively enhance muscle growth in animals.     

Myosin substitution rate is affected by the amount of cytosolic myosin in cultured muscle cells

In striated muscles, approximately 300 myosin molecules form a single thick filament in myofibrils. Each myosin is continuously displaced by another myosin to maintain the thick filament structure. Our previous study using a fluorescence recovery after photobleaching (FRAP) technique showed that the myosin replacement rate is decreased by inhibition of protein synthesis, but myosin is still exchangeable. This result prompted us to examine whether myosin in the cytoplasm is involved in myosin replacement in myofibrils. To address this, FRAP was measured in green fluorescent protein (GFP)‐tagged myosin heavy chain 3 (Myh3) expressing myotubes that were treated with streptolysin‐O (SLO), which forms pores specifically in the plasma membrane to induce leakage of cytoplasmic proteins. Our biochemical data demonstrated that the cytoplasmic myosin content was reduced in SLO‐permeabilized semi‐intact myotubes. Furthermore, FRAP experiments showed a sluggish substitution rate of GFP‐Myh3 in SLO‐permeabilized myotubes. Taken together, these results demonstrate that the myosin substitution rate is significantly reduced by a decreased amount of myosin in the cytoplasm and that cytoplasmic myosin contributes to myosin replacement in myofibrils.     

Cold exposure increases slow‐type myosin heavy chain 1 (MyHC1) composition of soleus muscle in rats

The aim of this study was to examine the effects of cold exposure on rat skeletal muscle fiber type, according to myosin heavy chain (MyHC) isoform and metabolism‐related factors. Male Wistar rats (7 weeks old) were housed individually at 4 ± 2°C as a cold‐exposed group or at room temperature (22 ± 2°C) as a control group for 4 weeks. We found that cold exposure significantly increased the slow‐type MyHC1 content in the soleus muscle (a typical slow‐type fiber), while the intermediate‐type MyHC2A content was significantly decreased. In contrast to soleus, MyHC composition of extensor digitorum longus (EDL, a typical fast‐type fiber) and gastrocnemius (a mix of slow‐type and fast‐type fibers) muscle did not change from cold exposure. Cold exposure increased mRNA expression of mitochondrial uncoupling protein 3 (UCP3) in both the soleus and EDL. Cold exposure also increased mRNA expression of myoglobin, peroxisome proliferator‐activated receptor gamma coactivator 1α (PGC1α) and forkhead box O1 (FOXO1) in the soleus. Upregulation of UCP3 and PGC1α proteins were observed with Western blotting in the gastrocnemius. Thus, cold exposure increased metabolism‐related factors in all muscle types that were tested, but MyHC isoforms changed only in the soleus.     

Changes in muscle fiber type and expression of mRNA of myosin heavy chain isoforms in porcine muscle during pre‐ and postnatal development

The purpose of this study is to elucidate developmental changes in muscle fiber type in the pig during pre‐ and postnatal development. For this purpose, we performed a histochemical analysis for myosin adenosine triphosphatase activity to assess muscle fiber type and determined abundances of messenger RNA (mRNA) of myosin heavy chain (MHC) isoforms. Samples of Longissimus dorsi (LD) muscle were taken from fetuses on day 90 of the fetal stage. Further, samples of LD, Rhomboideus and Biceps femoris (B. femoris) muscles were taken from pigs when they were 1, 12, 26, 45 or 75 days old. Expression of MHC 2b mRNA in the LD and the B. femoris muscles rapidly and considerably increased from the late fetal stage to the early postnatal stage and this increase was associated with the development of type 2b fibers at least in the LD muscle. As shown by the rapid and considerable changes in expression of MHC 2b mRNA, it seems that a certain plasticity of muscle fiber type still remains in this developmental stage.     

Relationships between tropomyosin and myosin heavy chain isoforms in bovine skeletal muscle

The composition of tropomyosin (TPM) and myosin heavy chain (MyHC) isoforms was analyzed in 10 physiologically different bovine muscles ( masseter , diaphragm, tongue, semispinalis, pectoralis profundus , biceps femoris, psoas major , semimembranosus, longissimus thoracis and semitendinosus ) to clarify the relationships between TPM and MyHC isoforms in different muscle fiber types. The content of TPM1 and TPM3 was different in muscles according to their function in muscle contraction, although the content of TPM2 was constantly about 50% of the total TPM in all muscles. The content of TPM1 was higher in semimembranosus , longissimus thoracis and semitendinosus, while that of TPM3 was higher in masseter and diaphragm. The high positive correlation between MyHC-slow content and TPM3 content ( r  = 0.92) suggested a coexpression of TPM3 and MyHC-slow isoforms in a muscle fiber. MyHC-slow and TPM3 were expressed at the same level in masseter and diaphragm, whereas there was more TPM3 than MyHC-slow in tongue and semispinalis , so it appears that the excess TPM3 in tongue and semispinalis is expressed with other MyHC isoforms. MyHC-2a was the only fast type isoform expressed in tongue and semispinalis . Therefore, the excess TPM3 was composed of myofibrils with MyHC-2a. The results suggested that a fiber expressing MyHC-2a would be regulated delicately by changing the TPM isoform types.     

Effect of different blood‐guided conditioning programmes on skeletal muscle ultrastructure and histochemistry of sport horses

The effects of three different blood‐guided conditioning programmes on ultrastructural and histochemical features of the gluteus medius muscle of 2‐year‐old sport horses were examined. Six non‐trained Haflinger horses performed three consecutive conditioning programmes of varying lactate‐guided intensities [velocities eliciting blood lactate concentrations of 1.5 (v_1.5), 2.5 (v_2.5) and 4 (v₄) mm respectively] and durations (25 and 45 min). Each conditioning programme lasted 6 weeks and was followed by a 5‐week resting period. Pre‐, post‐ and deconditioning muscle biopsies were analysed. Although training and detraining adaptations were similar in nature, they varied significantly in magnitude among the three different conditioning programmes. Overall, the adaptations consisted in significant increases in size of mitochondria and myofibrils, as well as a hypertrophy of myofibrillar ATPase type IIA muscle fibres and a reduction in number of type IIx low‐oxidative fibres. Together, these changes are compatible with a significant improvement in both muscle aerobic capacity and muscle strength. The use of v_1.5 and v_2.5 as the exercise intensities for 45 min elicited more significant adaptations in muscle, whereas conditioning horses at v₄ for 25 min evoked minimal changes. Most of these muscular adaptations returned towards the pre‐conditioning status after 5 weeks of inactivity. It is concluded that exercises of low or moderate intensities (in the range between v_1.5 and v_2.5) and long duration (45 min) are more effective for improving muscle features associated with stamina and power in sport horses than exercises of higher intensity (equivalent to v₄) and shorter duration (25 min).     

Leucine regulates slow‐twitch muscle fibers expression and mitochondrial function by Sirt1/AMPK signaling in porcine skeletal muscle satellite cells

A previous study demonstrated that leucine upregulates the slow myosin heavy chain mRNA expression in C2C12 cells. However, the role of leucine in slow‐twitch muscle fibers expression and mitochondrial function of porcine skeletal muscle satellite cells as well as its mechanism remain unclear. In this study, porcine skeletal muscle satellite cells cultured in differentiation medium were treated with 2 mM leucine for 3 days. Sirt1 inhibitor EX527, AMPK inhibitor compound C, and AMPKα1 siRNA were used to examine its underlying mechanism. Here we showed that leucine increased slow‐twitch muscle fibers and mitochondrial function‐related gene expression, as well as increased succinic dehydrogenase (SDH) and malate dehydrogenase (MDH) activities. Moreover, leucine increased the protein levels of Sirt1 and phospho‐AMPK. We also found that AMPKα1 siRNA, AMPK inhibitor compound C, or Sirt1 inhibitor EX527 attenuated the positive effect of leucine on slow‐twitch muscle fibers and mitochondrial function‐related gene expression. Finally, we showed that Sirt1 was required for leucine‐induced AMPK activation. Our results provide, for the first time, evidence that leucine induces slow‐twitch muscle fibers expression and improves mitochondrial function through Sirt1/AMPK signaling pathway in porcine skeletal muscle satellite cells.     

小鼠胚胎干细胞向骨骼肌细胞分化的研究进展

近年来,胚胎干细胞的应用越来越广泛,在体外将小鼠胚胎干细胞诱导分化为肌肉细胞,并且利用这些分化得来的肌肉细胞治疗肌肉退行性疾病,一直是胚胎干细胞研究领域的热点,而胚胎干细胞的分化机制更是其中的难点。目前,用于诱导小鼠胚胎干细胞分化为骨骼肌细胞的方法很多,但分化的效率并不是很高,所以研究胚胎干细胞向骨骼肌细胞方向分化的机制显得尤为重要。文章仅就最近几年对小鼠胚胎干细胞向骨骼肌细胞方向分化的一些方法及其机制作一综述。     

Leucine‐induced promotion of post‐absorptive EAA utilization and hepatic gluconeogenesis contributes to protein synthesis in skeletal muscle of dairy calves

The high rate of protein synthesis in skeletal muscle of dairy calves can benefit their first lactation even lifetime milk yield. Since the rate of protein synthesis is relatively low in the post‐absorptive state, the aim of this research was to determine whether leucine supplementation could increase the post‐absorptive essential amino acid (EAA) utilization and protein synthesis in the skeletal muscle. Ten male neonatal dairy calves (38 ± 3 kg) were randomly assigned to either the control (CON, no leucine supplementation, n = 5) or supplementation with 1.435 g leucine/L milk (LEU, n = 5). Results showed that leucine significantly increased the length and protein concentration in longissimus dorsi (LD) muscle, whereas it decreased creatinine concentration and glutamic‐oxalacetic transaminase (GOT) activity. Compared to the control group, leucine supplementation also reduced the glutamic‐pyruvic transaminase (GPT) activity. Supplementation of leucine improved the phosphorylation of mammalian target of rapamycin (mTOR), eukaryotic initiation factor 4E‐binding protein 1 (4EBP1) and substrates ribosomal protein S6 kinase 1 (p70S6K). Supplementation of leucine resulted in increased concentrations of glucose, methionine, threonine, histidine and EAAs and decreased concentration of arginine in serum. Liver glucose concentration was higher and pyranic acid was lower in LEU compared to CON. In conclusion, leucine supplementation can promote post‐absorptive EAA utilization and hepatic gluconeogenesis, which contributes to protein synthesis in skeletal muscle of dairy calves.     

皖南花猪骨骼肌AdpR和MyHCmRNA表达的发育性变化

摘要：为探讨皖南花猪骨骼肌组织中脂联素受体（AdpRl、AdpR2）和不同类型肌球蛋白重链（MyHC）mRNA的发育性变化及性别差异,选择0（出生当天）、30、45、90、180日龄的皖南花猪公母各5头,以B～Actin为内标,采用△△Ct相对定量实时荧光PCR方法对背最长肌和半腱肌中AdpRl、AdpR2、MyHCI、MyHC2a、MyHC2b和My-HC2xmRNA进行定量分析。结果显示,背最长肌和半腱肌AdpRl、AdpR2、MyHCl、MyHC2a、MyHC2b和My—HC2XmRNA的表达都有显著或极显著的发育变化规律（P〈0．05或P〈0．01）。总体上AdpRl、AdpR2、My—HC2a、MyHC2b和MyHC2XmRNA在背最长肌显著或极显著高于半腱肌（P〈0．05或P〈0．01）;MyHClmRNA在背最长肌极显著低于半腱肌（P〈0．01）。半腱肌MyHClmRNA在母猪显著大于公猪（P〈0．05）,而MyHC2amRNA在母猪显著小于公猪（P〈0．05）。背最长肌和半腱肌AdpR2mRNA的表达分别与MyHCl正相关（P〈0．05）,半腱肌AdpRlmRNA的表达与MyHC2x正相关（P〈0．05）。结果表明,皖南花猪骨骼肌组织中AdpR和MyHC的基因表达有特定的发育模式和组织特异性,且有一定性别差异。     

UBE2L3对牛骨骼肌卫星细胞增殖分化的影响

将分离的牛骨骼肌卫星细胞(BSMSCs)进行体外培养,首先检测泛素结合酶UBE2L3在BSMSCs增殖分化过程中mRNA以及蛋白表达水平的变化.设计UBE2L3的3个干扰RNA(si-UBE2 L3-1、si-UBE2L3-2、si-UBE2L3-3),对干扰效果进行筛选.构建UBE2L3过表达质粒载体pcDNA3.1...     

Identification and characterization of proteoglycans in bovine neonatal skeletal muscle

In order to provide background for understanding biological roles of proteoglycans (PG) in developing skeletal muscle, we have isolated and characterized PG in bovine neonatal skeletal muscle. Two types of PG were isolated from skeletal muscle by density gradient ultracentrifugation and ion‐exchange chromatography. One was a small PG (PG‐S) with a molecular size of 100–130 kDa, another was a large PG (PG‐L) with a molecular size of 300–500 kDa. The glycosaminoglycan chains of PG‐S and PG‐L were dermatan sulfate and chondroitin sulfate, respectively, judged by cellulose acetate membrane electrophoresis. Immunoblot assays revealed that both PG bound to type I, II, III and IV collagen, laminin and fibronectin. Unlike PG‐S, PG‐L bound to type V collagen and hyaluronic acid. Small proteoglycans had a core protein of 45 kDa, which reacted with the antibody against the decorin core protein. The N‐terminal amino acid sequence of the PG‐S core protein was consistent with that of decorin from bovine bone and tendon. Thus, PG‐S from neonatal skeletal muscle was identified as decorin in bovines. Immunohistochemical analysis with antibodies against PG‐L and PG‐S demonstrated that PG‐L was located both in the perimysium and endomysium, but PG‐S was localized exclusively in the perimysium. These findings suggest that the characterized PG may have distinct roles in the ECM construction of developing skeletal muscle.     

Effects of eccentric exercise on branched‐chain amino acid profiles in rat serum and skeletal muscle

Supplementation of branched‐chain amino acid (BCAA) is often used to attenuate exercise‐induced skeletal muscle damage and promote adaptation, but no definitive conclusion on the benefits of BCAA on muscle recovery after injurious exercise can be drawn. Exploration of the systematic BCAA alteration in muscular injury‐repair stage per se without any BCAA supplement should provide some useful information in favour of BCAA application in muscle regeneration after injury. One bout of 90‐min downhill‐running exercise was performed to cause rat skeletal muscle injury. After exercise, myofibrillar BCAA concentrations showed minor changes compared with exercise before, while serum concentrations of BCAA were lower after exercise. Especially, serum leucine, isoleucine and total BCAA concentrations 2 weeks post‐run were significantly lower than normal values of exercise before (p = 0.008, p = 0.041, p = 0.015). The data demonstrate that a single eccentric exercise can significantly decrease the serum BCAA concentrations, which mean high utilization of BCAA for myogenesis after injurious exercise.     

Preliminary experiments on mechanical stretch-induced activation of skeletal muscle satellite cells in vivo

We have shown in vitro that mechanical stretch triggers activation of quiescent satellite cells of skeletal muscle to enter the cell cycle through an intracellular cascade of events including nitric oxide (NO) synthesis that results in the release of hepatocyte growth factor (HGF) from its extracellular association and its subsequent presentation to signaling receptors. In order to explore the activation mechanism in vivo, stretch experiments were conducted in the living animal using our suspension model developed. This system used the weight of the hind portion of rats to stretch the inside muscles of the left hind limb suspended for a period of 0.5–2.0 h. At the end of the stretch period, the rats received an intraperitoneal injection of bromodeoxyuridine followed by immunocytochemistry for its incorporation as an index of satellite cell activation in vivo. Depending on the period of stretch, bromodeoxyuridine labeling was increased significantly over the contralateral unstretched leg or control muscle from untreated rats. A stretched muscle extract prepared from the 2 h stretched tissue by incubating it in PBS, showed the active form of HGF as revealed by immunoblotting and it could stimulate the activation of unstretched satellite cells. Also, administering NO synthase inhibitor L‐NAME prior to muscle stretch abolished the stretch activation of satellite cells. Therefore, the results from these experiments demonstrate that stretching muscle triggers NO synthesis and HGF release, which could activate satellite cells in vivo.     

Effect of dexamethasone on the expression of atrogin‐1/MAFbx in chick skeletal muscle

Expression of atrogin‐1/MAFbx, a muscle‐specific E3 ubiquitin ligase, is high under catabolic conditions, that result in muscle atrophy. Messenger RNA (mRNA) expression of atrogin‐1/MAFbx is increased by the glucocorticoid dexamethasone in mammalian skeletal muscle. This study investigated the effects of dexamethasone on expression of atrogin‐1/MAFbx in skeletal muscle of neonatal chicks and in chick myotubes. Chicks were given a single intraperitoneal injection of dexamethasone at a concentration of 10 mg/kg body weight. Twenty‐four hours after dexamethasone administration, the Pectoralis muscle weight of chicks was decreased. mRNA expression of atrogin‐1/MAFbx in skeletal muscle of chicks was significantly increased by dexamethasone administration. Expression of other proteolytic‐related genes (20S proteasome C2 subunit, m‐calpain large subunit, and cathepsin B) in skeletal muscle of chicks was not increased by dexamethasone administration. Chick myotubes were incubated with dexamethasone (1, 10 or 100 µmol/L) for 6 h. Expression of atrogin‐1/MAFbx mRNA in chick myotubes was increased in the presence of all concentrations of dexamethasone. However, expression of other proteolytic‐related genes (20S proteasome C2 subunit, m‐calpain large subunit and cathepsin B) in chick myotubes was not affected by dexamethasone treatment. These results indicate that dexamethasone enhances atrogin‐1/MAFbx expression in chick skeletal muscle, resulting in increased muscle atrophy.     

牛骨骼肌卫星细胞的分离培养及诱导分化方法的建立

为了给牛骨骼肌卫星细胞的分离培养及诱导分化方法及进一步揭示肌肉分化的机理及转基因肉牛的研究提供重要帮助,试验以新生胎牛的骨骼肌为试验材料,分别采用胶原酶Ⅰ和胶原酶Ⅺ与胰蛋白酶结合对其进行消化,并通过差速贴壁法对骨骼肌卫星细胞进行分离纯化,同时采用免疫荧光染色、RT-PCR、Western-blot法对骨骼肌卫星细胞进行鉴定。结果表明:研究成功获得了大量的牛骨骼肌卫星细胞;该细胞的标志性分子的mRNA及其蛋白表达的纯度在98%以上;细胞生长状态良好,可稳定传至90代并保持旺盛的增殖活力;细胞分化效率高,2%的马血清能够诱导细胞分化使其融合形成多核肌管,数量众多的肌管可自发融合为更粗的肌管,并可观察到其具有收缩现象。     

Mechanical stretch-induced activation of skeletal muscle satellite cells is dependent on nitric oxide production in vitro

Mechanical stretch induces activation of cultured quiescent satellite cells and the activation response is owing to rapid release of hepatocyte growth factor (HGF) from its extracellular association with satellite cells and its subsequent presentation to the c-met receptor. We provide new evidence that the stretch activation is dependent on nitric oxide (NO) production. Stretch activation could be abolished by the addition of N ^G-nitro- L -arginine methyl ester (L-NAME), a competitive inhibitor of NO synthesis, but not by N ^G-nitro- D -arginine methyl ester hydrochloride, a less active enantiomer of L-NAME. Adding HGF to the L-NAME culture restored the activation response, indicating that L-NAME does not directly inhibit satellite cell activation, but acts upstream from the HGF release. In addition, immunoblots of satellite cell lysate revealed the presence of nitric oxide synthase. These experiments suggest that NO is involved in linking mechanical perturbation of satellite cells to chemical signaling responsible for HGF release from its sequestration in vitro .     

干扰DHX9对牛骨骼肌卫星细胞增殖与成肌分化的影响

为研究天冬氨酸-谷氨酸-丙氨酸-组氨酸盒解旋酶9 (DEAH (Asp-Glu-Ala-His)-box helicase 9,DHX9)对牛骨骼肌细胞增殖与分化的影响,利用已经建立的牛骨骼肌卫星细胞体外成肌分化模型,设计合成DHX9的si-RNA,采用荧光定量PCR和Western blot技术检测DHX9基因在成肌...