Mechano-biology of skeletal muscle hypertrophy and regeneration: Possible mechanism of stretch-induced activation of resident myogenic stem cells

In undamaged postnatal muscle fibers with normal contraction and relaxation activities, quiescent satellite cells of resident myogenic stem cells are interposed between the overlying external lamina and the sarcolemma of a subjacent mature muscle fiber. When muscle is injured, exercised, overused or mechanically stretched, these cells are activated to enter the cell proliferation cycle, divide, differentiate, and fuse with the adjacent muscle fiber, and are responsible for regeneration and work-induced hypertrophy of muscle fibers. Therefore, a mechanism must exist to translate mechanical changes in muscle tissue into chemical signals that can activate satellite cells. Recent studies of satellite cells or single muscle fibers in culture and in vivo demonstrated the essential role of hepatocyte growth factor (HGF) and nitric oxide (NO) radical in the activation pathway. These experiments have also reported that mechanically stretching satellite cells or living skeletal muscles triggers the activation by rapid release of HGF from its extracellular tethering and the subsequent presentation to the receptor c-met. HGF release has been shown to rely on calcium-calmodulin formation and NO radical production in satellite cells and/or muscle fibers in response to the mechanical perturbation, and depend on the subsequent up-regulation of matrix metalloproteinase (MMP) activity. These results indicate that the activation mechanism is a cascade of events including calcium ion influx, calcium-calmodulin formation, NO synthase activation, NO radical production, MMP activation, HGF release and binding to c-met. Better understanding of 'mechano-biology' on the satellite cell activation is essential for designing procedures that could enhance muscle growth and repair activities in meat-animal agriculture and also in neuromuscular disease and aging in humans.     

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 .     

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.     

Comparative analysis of mechanical stretch-induced activation activity of back and leg muscle satellite cells in vitro

It has previously been shown that mechanical stretch induces activation of cultured quiescent satellite cells by rapid release of hepatocyte growth factor (HGF) from its extracellular association with satellite cells and its subsequent presentation to the c‐met receptor. The present study provides evidence that the stretch activation activity varies according to the origin of satellite cells from back and leg skeletal muscles in vitro. Satellite cells were isolated from three muscle groups, back (BK), upper hind limb (UL) and lower hind limb (LL) muscles, of adult male rats and stretch activation activities were compared. In response to stretch, lower hind limb satellite cells showed significantly greater response than upper hind limb and back muscles (LL > UL > BK). Immunoblots of stretched culture media revealed a higher HGF‐releasing capacity of lower hind limb satellite cells than back muscle satellite cells. In addition, lower hind limb satellite cells exhibited a greater activation activity in response to exogenous HGF added to culture media than compared to satellite cells from back and upper hind limb (LL > UL > BK). The increased ability to release HGF and the increased cellular responsiveness might account for higher stretch activation activities of lower hind limb satellite cells. Electrophoretic analysis of myosin heavy chain isoforms verified a higher content of slow muscle fibers in lower limb muscles (LL > UL > BK), suggesting a difference in stretch‐induced activation activity between satellite cells associated with fast and slow muscle fibers.     

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.     

Pseudorabies virus induces a rapid up-regulation of nitric oxide synthases in the nervous system of swine

Nitric oxide (NO) is a free radical gas with important roles in the host's immune response against viral infections. In this study, we examined the kinetics and distribution of nitric oxide synthase (NOS) expression during the early steps of infection of the porcine nervous system by the alphaherpesvirus pseudorabies virus (PRV). To this end, we examined changes in the expression of the three major NOS isoforms, neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS), by immunohistochemistry in the trigeminal ganglia and brain of pigs inoculated intranasally with a virulent PRV strain. The results obtained show that infection of the porcine nervous system by PRV induced a rapid and progressive increment in NOS expression that coincided in timing, location, and magnitude with those of virus propagation in the nervous tissue. A major finding of this study was that PRV caused not only nNOS and iNOS induction in a variety of cell types, but also eNOS up-regulation in endothelial cells and neurons; therefore, all possible sources of NO are activated and probably contribute to the overproduction of NO during infection with the neurotropic alphaherpesvirus PRV in its natural host.     

Meat Science and Muscle Biology Symposium: stem cell niche and postnatal muscle growth

Stem cell niche plays a critical role in regulating the behavior and function of adult stem cells that underlie tissue growth, maintenance, and regeneration. In the skeletal muscle, stem cells, called satellite cells, contribute to postnatal muscle growth and hypertrophy, and thus, meat production in agricultural animals. Satellite cells are located adjacent to mature muscle fibers underneath a sheath of basal lamina. Microenvironmental signals from extracellular matrix mediated by the basal lamina and from the host myofiber both impinge on satellite cells to regulate their activity. Furthermore, several types of muscle interstitial cells, including intramuscular preadipocytes and connective tissue fibroblasts, have recently been shown to interact with satellite cells and actively regulate the growth and regeneration of postnatal skeletal muscles. From this regard, interstitial adipogenic cells are not only important for marbling and meat quality, but also represent an additional cellular component of the satellite cell niche. At the molecular level, these interstitial cells may interact with satellite cells through cell surface ligands, such as delta-like 1 homolog (Dlk1) protein whose overexpression is thought to be responsible for muscle hypertrophy in callipyge sheep. In fact, extracellular Dlk1 protein has been shown to promote the myogenic differentiation of satellite cells. Understanding the cellular and molecular mechanisms within the stem cell niche that regulate satellite cell differentiation and maintain muscle homeostasis may lead to promising approaches to optimizing muscle growth and composition, thus improving meat production and quality.     

猪圆环病毒2型对体外培养的仔猪淋巴细胞抗氧化能力的影响

为进一步了解猪圆环病毒2型(PCV-2)引起淋巴细胞凋亡的机理,测定了PCV-2对体外培养的仔猪淋巴细胞抗氧化能力的影响。取培养后0,2,6,24,48 h的上清液和细胞,用试剂盒测定上清液中总抗氧化能力、一氧化氮(NO)含量、超氧阴离子自由基活性和超氧化物岐化酶活力的变化,同时测定淋巴细胞内一氧化氮合酶(NOS)活力。结果表明,试验组与对照组总抗氧化能力无显著性差异(P>0.05);试验组抗超氧阴离子自由基活性与对照组相比,除了0 h,其他时间点试验组均显著高于对照组(P<0.05);总超氧化物岐化酶活力试验组和对照组均维持在19 U/mL左右,无显著差异(P>0.05);NO含量除24 h时试验组高于对照组外(P<0.05),其余时间两组间差异不显著;细胞内NOS活力除0 h外,其余时间均是对照组高于试验组,且2、24 h显著增高(P<0.05);对照组诱导型一氧化氮合酶(iNOS)活力6 h时显著高于试验组(P<0.05),其余时间(0 h除外)也较试验组高。     

一氧化氮对肉鸡肺动脉高压综合征的影响

肺动脉压升高是肉鸡肺动脉高压综合征(PHS)的重要发病机制。近年来研究表明一氧化氮(NO)在PHS发生发展中发挥着重要作用。本文论述了NO对肉鸡PHS发病过程的影响。一氧化氮合酶(NOS)和NO活性在PHS早期升高而后期下降。NO具有强大的扩张血管的作用,但在PHS过程中,NO合成相对不足,导致肺血管舒缩失衡,引起肺动脉压升高。肺血管重构是肉鸡肺动脉高压综合征的重要病理学变化特征,而NO可促进肺小动脉平滑肌细胞凋亡,在一定程度上抑制肺血管重构的形成。NO作为自由基对机体造成的损伤也是引起PHS的原因之一。在肉鸡日粮中补充NO前体物L-精氨酸可以增加内源性NO的生成,有助于降低PHS的发病率。     

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...     

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。     

Hepatocyte growth factor transduces different intracellular signals in aortic and umbilical venous endothelial cells

Endothelial cells are important for maintenance of vascular integrity by producing a variety of bioactive molecules such as nitric oxide (NO). Recent evidence has suggested that there are some differences in characteristics between endothelial cells from different origins. Here we examined responses of two typical endothelial cells to hepatocyte growth factor (HGF), which induces endothelium-dependent relaxation of microvessels. Stimulation of human umbilical vein endothelial cells (HUVEC) and bovine aortic endothelial cells (BAEC) with HGF increased endothelial NO synthase activity, accompanied with an increase of activity-related site-specific phosphorylation of protein kinase B/Akt. However, HGF stimulated phosphorylation of p38 mitogen-activated protein kinase (MAPK) only in HUVEC, but not in BAEC, while it induced phosphorylation of p44/p42 MAPK in both cells. These results suggest that HGF transduces different intracellular signals between aortic and umbilical venous endothelial cells, and that the differences might represent divergent endothelial responses to growth factors, especially those that activate receptor-tyrosine kinases.     

骨骼肌卫星细胞的研究进展

目前已证实骨骼肌是具有多向分化潜能的成体干细胞的一个储存库。研究者认为骨骼肌中至少有两种干细胞:肌肉卫星细胞(muscle satellite cells)和肌源干细胞(muscle-derived stem cells),由于骨骼肌卫星细胞占了绝大部分,因此肌肉干细胞主要指骨骼肌卫星细胞。作者主要对肌肉卫星细胞的分离、培养、纯化、鉴定及影响其增殖与分化的机制做了简要概述。     

Adipogenic potential of satellite cells from distinct skeletal muscle origins in the rat

The possible relationship between myofiber type composition and adipose tissue development in skeletal muscle in vivo has been suggested. Recent evidence indicated that satellite cells are multipotent cells that can undergo not only myogenic, but also adipogenic differentiation. In the present study, rat satellite cells were isolated from soleus, back, extensor digitorum longus, tibialis anterior and quadriceps muscles, and their adipogenic potentials were compared by culturing them under adipogenic conditions in vitro. Cells from soleus muscle exhibited the highest adipogenic potential as judged from Oil Red-staining and immunocytochemical C/EBPalpha-staining. The adipogenic potential of satellite cells was positively correlated with type I myofiber distribution in the corresponding muscle of origin. These results demonstrated that the adipogenic potential of satellite cells differs according to the muscle of origin and suggested that its possible correlation to type I myofiber distribution may account for preferential adipose tissue development in slow oxidative muscles.     

Extrinsic regulation of domestic animal-derived myogenic satellite cells II

The existence of myogenic satellite cells was reported some 47 years ago, and, since that time, satellite cell research has flourished. So much new information is generated (daily) on these cells that it can be difficult for individuals to keep abreast of important issues related to their activation and proliferation, the modulation of the activity of other cell types, the differentiation of the cells to facilitate normal skeletal muscle growth and development, or to the repair of damaged myofibers. The intent of this review is to summarize new information about the extrinsic regulation of myogenic satellite cells and to provide specific mechanisms involved in altering satellite cell physiology. Where possible, examples from agriculturally important animals are used for illustrative purposes.     

Sensitivity of skeletal muscle to pro-apoptotic factors

In mononuclear cells, apoptosis leads to DNA fragmentation and cell destruction, regardless of the activated pathway. As regards multinuclear cells, e.g. skeletal muscle fibers, apoptosis rarely induces the death of the entire cell, and it generally affects single nuclei. This process, referred to as nuclear apoptosis, has a negative effect on the expression of genes in the myonuclear domain. Apoptosis may be initiated in muscle cells by external stimuli which activate cell membrane death receptors as well as by internal stimuli which stimulate the mitochondrial release of pro-apoptotic proteins. Reactive oxygen species also play an important role in the initiation of apoptosis. In muscle cells, ROS are produced in response to extracellular reactions or by cell mitochondria. It is, therefore, believed that mitochondria play a central role in apoptosis within skeletal muscle. Skeletal muscles have a well-developed system that protects them against oxidative damage. Myogenic stem cells are an integral part of multinucleated myofibers, and they are critically important for the maintenance of normal muscle mass, muscle growth, regeneration and hypertrophy. The latest research results indicate that myogenic cells are more sensitive to oxidative stress and pro-apoptotic factors than well-differentiated cells, such as myotubes. The complex structure and activity of skeletal muscle prompted research into the role of apoptosis and its intensity under various physiological and pathological conditions. This review summarizes the results of research investigating control mechanisms and the apoptosis process in skeletal muscle fibers, and indicates unresearched areas where further work is required.     

Localization of eNOS in the olfactory epithelium of the rat

Nitric oxide (NO) is a free radical and produced from L-arginine by nitric oxide synthase (NOS). Since NO is recently suggested to be involved in olfactory perception, the expression of eNOS, an isoform of NOS, was examined in the rat olfactory epithelium. The activity of NADPH-diaphorase was also examined as a marker of NOS. In the dorsomedial region of the nasal cavity, intensely positive reactions for NADPH-diaphorase were observed in the entire cytoplasm of sensory cells (olfactory cells). By immunohistochemistry, intensely positive reactions for eNOS were also found in the dorsomedial region of the nasal cavity. These reactions were observed on the free border of the olfactory epithelium. By immunoelectron microscopy, positive reactions for eNOS were found in the cilia of olfactory cells. In addition, in situ hybridization analysis of the olfactory epithelium revealed the expression of eNOS mRNA in the olfactory cells. These results indicate the presence of eNOS in the olfactory cells of the rat, and differential expression of eNOS in the olfactory epithelium depending on the regions of the nasal cavity. In addition, NO produced by eNOS may be involved in olfactory perception in the cilia of olfactory cells.     

Neuronal nitric oxide synthase is heterogeneously distributed in equine myofibers and highly expressed in endurance trained horses

Mammalian skeletal muscle expresses splice variants of neuronal nitric oxide synthase (nNOS). Skeletal muscles have a metabolically heterogeneous population of myofibers, and fiber composition in equine skeletal muscle is correlated with athletic ability in endurance events. In this study, we investigated whether nNOS expression in equine skeletal muscle is related to fiber type and endurance training. Biopsy samples obtained from the gluteus medius of sedentary- (SH) and endurance-trained (TH) horses were examined for the electrophoretic mobility of myosin heavy chain (MHC) and NOS activity. Serial tissue cross-sections were stained for myosin ATPase and nicotinamide adenine dinucleotide (NADH) reductase, and also immunostained for nNOS. The gluteus medius of TH had higher levels of nNOS expression and activity when compared to muscle from SH. In SH, nNOS was restricted to the subsarcolemmal area while in TH nNOS was also present at cytoplasmic sites. A splice variant of nNOS was heterogeneously distributed among the different myofibers, its expression being higher in fast-oxidative-glycolytic type IIA fibers than in fast-glycolytic type IIX fibers and absent in slow-twitch type I fibers. Trained horses had a significantly higher relative content of type IIA fibers, a greater oxidative capacity, and a lower percentage of type IIX fibers when compared with SH. The differences in muscle fiber typing between the 2 groups of horses reflected alterations that probably resulted from the endurance-training program. Overall, these results show that nNOS is differentially expressed and localized in the gluteus medius according to the fiber type and the athletic conditioning of the horses.     

动物成肌分化及非编码RNA调控研究进展

肌细胞分化密切关系到肉用动物的肌肉产量,也与人类的一系列疾病(如肌肉萎缩、心脏病等)密切相关。胚胎成肌分化期决定了肌纤维数量,是动物骨骼肌发育的关键时期。动物成肌分化及骨骼肌发育严格受各种细胞信号分子和转录因子调控,其中microRNA(miRNA)和lncRNA发挥着重要作用。本文从动物胚胎成肌分化及调控途径、卫星细胞的分化及调控、非编码RNA对肌肉形成的调控等方面进行综述,并展望了畜禽动物骨骼肌生长发育分子调控机制的研究方向,为提高畜禽肌肉产量与质量提供一定的分子理论参考。