Effects of Soybean Isoflavones on In vitro Antioxidative Capacity of Satellite Cells of Porcine Skeletal Muscles

A synthetic isoflavone （ISO-S） or genistein was added in culture medium at different concentrations （0, 10, 20, 30, 40, and 80 p.mol L^-1） to investigate the effects of soybean isoflavones on antioxidative capacity of porcine skeletal muscle satellite cells. After 48 h incubation, the suspension was cryopreserved for the determination of superoxide dismutase （SOD）, catalase （CAT）, glutathione peroxidase （GSH-Px） activities, and malondialdehyde （MDA） content. The mRNA levels of SOD, CAT, and GSH-Px gene in cells were detected with Taqman fluorescent probe method. The results showed that the content of MDA and the activities and the mRNA levels of SOD of porcine skeletal muscle satellite cells were influenced by supplemented soybean isoflavone （P〈0.05） when adding 10-80 μmol L^-1 ISO-S or genistein in the medium. The MDA contents, SOD and CAT activities and their mRNA expression levels of porcine skeletal muscle cells responded quadratically （P〈 0.05） as the level of ISO-S or genistein increased. Pre-incubation of porcine skeletal muscle satellite cells with ISO-S or genistein at 10-40 pmol L-1 elevated the activities and the mRNA expression levels of SOD and CAT in cells concurrently and decreased the cellular content of MDA （P〈 0.05）. The results indicated that pre-incubation of ISO-S or genistein at 10- 40μmol L^-1 could improve the antioxidative capacity of porcine skeletal muscle satellite cells.

MicroRNA-22 inhibits proliferation and promotes differentiation of satellite cells in porcine skeletal muscle

Pig is an important economic animal in China. Improving meat quality and meat productivity is a long time issue in animal genetic breeding. Micro RNAs(mi RNAs) are short non-coding RNAs that participate in various biological processes, such as muscle development and embryogenesis. mi R-22 differentially expresses in embryonic and adult skeletal muscle. However, the underlying mechanism is unclear. In this study, we investigated mi R-22 function in proliferation and differentiation of porcine satellite cells(PSCs) in skeletal muscle. Our data show that mi R-22 expressed in both proliferation and differentiated PSCs and is significantly upregulated(P<0.05) during differentiation. After treated with the mi R-22 inhibitor, PSCs proliferation was significantly increased(P<0.05), as indicated by the up-regulation(P<0.01) of cyclin D1(CCND1), cyclin B1(CCNB1) and down-regulation(P<0.05) of P21. Conversely, over-expression of mi R-22 resulted in opposite results. Differentiation of PSCs was significantly suppressed(P<0.05), evidenced by two major myogenic markers: myogenin(Myo G) and myosin heavy chain(My HC), after transfecting the PSCs with mi R-22 inhibitor. Opposite results were demonstrated in the other way around by transfection with mi R-22 mimics. In conclusion, the data from this study indicated that mi R-22 inhibited the PSCs proliferation but promoted their differentiation.

Protective Effect of ATP on Skeletal Muscle Satellite Cells Damaged by H_2O_2

This study investigated the protective effect of ATP on skeletal muscle satellite cells damaged by H2O2 in neonatal rats and the possible mechanism. The skeletal muscle satellite cells were randomly divided into four groups： normal group, model group（cells treated with 0.1 mmol/L H2O2 for 50 s）, protection group（cells treated with 16, 8, 4, 2, 1, 0.5, or 0.25 mmol/L ATP for 24 h, and then with 0.1 mmol/L H2O2 for 50 s）, proliferation group（cells treated with 16, 8, 4, 2, 1, 0.5, or 0.25 mmol/L ATP for 24 h）. MTT assay, FITC＋PI＋DAPI fluorescent staining, Giemsa staining and immunofluorescence were performed to examine cell viability and apoptosis, and apoptosis-related proteins. The results showed that the survival rate of skeletal muscle satellite cells was decreased and the apoptosis rate was increased after H2O2 treatment（P〈0.01）. Different doses of ATP had different effects on skeletal muscle satellite cells damaged by H2O2： the survival rate of muscle satellite cells treated with ATP at 4, 2, or 1 mmol/L was increased. The protective effect was most profound on cells treated with 2 mmol/L ATP. Immunofluorescence showed that ATP could increase the number of Bcl-2-positive cells（P〈0.01） and decrease the number of the Bax-positive cells（P〈0.01）. It was concluded that ATP could protect skeletal muscle satellite cells against H2O2 damage in neonatal rats, which may be attributed to the up-regulation of the expression of Bcl-2 and down-regulation of Bax, resulting in the suppression of apoptosis.

Biological Characteristics of Skeletal Muscle Satellite Cells and Its Application Prospects in Muscle Trauma Repair

As the only source of stem cells, satellite cells play and extraordinary role in the remediation process of skeletal muscle after injury. This paper overviewed the biological characteristic of skeletal muscle satellite cells and its role in repairing muscle injury, and put forward the prospects of its application in muscle trauma repair.

Muscle satellite cells:one of the important factors in the occurrence and development of sarcopenia

Sarcopenia,or muscle loss,has been one of the hot topics in the medical field in recent years.Due to limited attention and effective treatments for sarcopenia in the past,many patients,especially the elderly,suffered irreversible damage to their motor function caused by sarcopenia.However,recent scientific studies have found that the occurrence and development of sarcopenia are closely related to the function and quantity of muscle satellite cells.This article briefly discusses the relationship between muscle satellite cells and sarcopenia.

Crosstalk among canonical Wnt and Hippo pathway members in skeletal muscle and at the neuromuscular junction

Skeletal muscles are essential for locomotion,posture,and metabolic regulation.To understand physiological processes,exercise adaptation,and muscle-related disorders,it is critical to understand the molecular pathways that underlie skeletal muscle function.The process of muscle contra ction,orchestrated by a complex interplay of molecular events,is at the core of skeletal muscle function.Muscle contraction is initiated by an action potential and neuromuscular transmission requiring a neuromuscular junction.Within muscle fibers,calcium ions play a critical role in mediating the interaction between actin and myosin filaments that generate force.Regulation of calcium release from the sarcoplasmic reticulum plays a key role in excitation-contraction coupling.The development and growth of skeletal muscle are regulated by a network of molecular pathways collectively known as myogenesis.Myogenic regulators coordinate the diffe rentiation of myoblasts into mature muscle fibers.Signaling pathways regulate muscle protein synthesis and hypertrophy in response to mechanical stimuli and nutrient availability.Seve ral muscle-related diseases,including congenital myasthenic disorders,sarcopenia,muscular dystrophies,and metabolic myopathies,are underpinned by dys regulated molecular pathways in skeletal muscle.Therapeutic interventions aimed at preserving muscle mass and function,enhancing regeneration,and improving metabolic health hold promise by targeting specific molecular pathways.Other molecular signaling pathways in skeletal muscle include the canonical Wnt signaling pathway,a critical regulator of myogenesis,muscle regeneration,and metabolic function,and the Hippo signaling pathway.In recent years,more details have been uncovered about the role of these two pathways during myogenesis and in developing and adult skeletal muscle fibers,and at the neuromuscular junction.In fact,research in the last few years now suggests that these two signaling pathways are interconnected and that they jointly control physiological and pathophysiological processes in muscle fibers.In this review,we will summarize and discuss the data on these two pathways,focusing on their concerted action next to their contribution to skeletal muscle biology.However,an in-depth discussion of the noncanonical Wnt pathway,the fibro/a dipogenic precursors,or the mechanosensory aspects of these pathways is not the focus of this review.

Impact of Bovine Skeletal Muscle Satellite Cell Differentiation by Small Interfering RNA Targeting Myogenin Gene

To examine the effect of myogenin gene on the differentiation of bovine skeletal muscle satellite cell, we constructed small interfering RNA plasmid vector to obtain myogenin knockdown bovine skeletal muscle cells, then used cell transfection, real time RCR and Western Blot to detect the influence of myogenin to cell differentiation. Results showed that the knockdown of myogenin significantly decreased its expression and other muscle-specific genes. Compared to the control, it could differentiate into few myotubes when challenged by low serum in the medium. These findings provided an important theoretical basis for further explore of the genetic mechanism in adult skeletal muscle, the remedy of muscle injuries and the cultivation of high-yield transgenic cattle.

THE IMPROVEMENT OF INFARCTED MYOCARDIAL CONTRACTILE FORCE AFTER AUTOLOGOUS SKELETAL MUSCLE SATELLITE CELL IMPLANTATION

Objective To study the improvement of infarcted myocardial contractile force after autologous skeletal muscle satellite cell implantation via intracoronary arterial perfusion. Methods Skeletal muscle cells were harvested from gluteus max of adult mongrel dogs and the cells were cultured and expanded before being labeled with DAPI (4’, 6-diamidino-2-phenylindone). The labeled cells were then implanted into the acute myocardial infarct site via the ligated left anterior descending (LAD) coronary artery. Specimens were taken at 2nd, 4th, 8th week after myoblast implantation for histologic and contractile force evaluation, respectively. Results The satellite cells with fluorescence had been observed in the infarct site and also in papi- llary muscle with consistent oriented direction of host myocardium. A portion of the implanted cells had differen- tiated into muscle fibers. Two weeks after implantation, the myocardial contractile force showed no significant difference between the cell implant group and control group. At 4 and 8 week, the contractile force in the cell implant group was better than that in control group. Conclusion The skeletal muscle satellite cells, implanted into infarct myocardium by intracoronary arterial perfusion, could disseminate through the entire infarcted zone with myocardial regeneration and improve the contractile function of the infarcted myocardium.

Denervated muscle extract promotes recovery of muscle atrophy through activation of satellite cells. An experimental study

Purpose: The objective of the present study was to determine whether a denervated muscle extract(DmEx) could stimulate satellite cell response in denervated muscle.Methods: Wistar rats were divided into 4 groups: normal rats, normal rats treated with DmEx, denervated rats, and denervated rats treated with DmEx. The soleus muscles were examined using immunohistochemical techniques for proliferating cell nuclear antigen, desmin, and myogenic differentiation antigen(MyoD), and electron microscopy was used for analysis of the satellite cells.Results: The results indicate that while denervation causes activation of satellite cells, DmEx also induces myogenic differentiation of cells localized in the interstitial space and the formation of new muscle fibers. Although DmEx had a similar effect in nature on innervated and denervated muscles, this response was of greater magnitude in denervated vs. intact muscles.Conclusion: Our study shows that treatment of denervated rats with DmEx potentiates the myogenic response in atrophic denervated muscles.

Temporal and spatial regulation of biomimetic vascularization in 3D-printed skeletal muscles

In the intricate skeletal muscle tissue,the symbiotic relationship between myotubes and their supporting vasculature is pivotal in delivering essential oxygen and nutrients.This study explored the complex interplay between skeletal muscle and endothelial cells in the vascularization ofmuscle tissue.By harnessing the capabilities of three-dimensional(3D)bioprinting and modeling,we developed a novel approach involving the co-construction of endothelial and muscle cells,followed by their subsequent differentiation.Our findings highlight the importance of the interaction dynamics between these two cell types.Notably,introducing endothelial cells during the advanced phases of muscle differentiation enhanced myotube assembly.Moreover,it stimulated the development of the vascular network,paving the way for the early stages of vascularized skeletal muscle development.The methodology proposed in this study indicates the potential for constructing large-scale,physiologically aligned skeletal muscle.Additionally,it highlights the need for exploring the delicate equilibrium and mutual interactions between muscle and endothelial cells.Based on the multicell-type interaction model,we can predict promising pathways for constructing even more intricate tissues or organs.

Catalpa bignonioides extract improves exercise performance through regulation of growth and metabolism in skeletal muscles

Objective:To evaluate the effects of Catalpa bignonioides fruit extract on the promotion of muscle growth and muscular capacity in vitro and in vivo.Methods:Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.Cell proliferation was assessed using a 5-bromo-2’-deoxyuridine(BrdU)assay kit.Western blot analysis was performed to determine the protein expressions of related factors.The effects of Catalpa bignonioides extract were investigated in mice using the treadmill exhaustion test and whole-limb grip strength assay.Chemical composition analysis was performed using high-performance liquid chromatography(HPLC).Results:Catalpa bignonioides extract increased the proliferation of C2C12 mouse myoblasts by activating the Akt/mTOR signaling pathway.It also induced metabolic changes,increasing the number of mitochondria and glucose metabolism by phosphorylating adenosine monophosphate-activated protein kinase.In an in vivo study,the extract-treated mice showed improved motor abilities,such as muscular endurance and grip strength.Additionally,HPLC analysis showed that vanillic acid may be the main component of the Catalpa bignonioides extract that enhanced muscle strength.Conclusions:Catalpa bignonioides improves exercise performance through regulation of growth and metabolism in skeletal muscles,suggesting its potential as an effective natural agent for improving muscular strength.

The Influence of SelW on GSH and GPx after Post-Transcriptional Gene Silencing in Mouse Skeletal Muscle Cells

The expression of Selenoprotein W(SelW)in C2C12 skeletal muscle cells was specifically decreased to examine its influence on the amount of glutathione(GSH)and the activity of glutathione peroxidase(GPx).SelW knock-down was performed by RNA interference(RNAi)in cultured muscle cells and verified by Real-time PCR and Western blotting.In addition,cell viability,GSH content and GPx activity were assayed.The results showed that the mRNA level and protein expression of SelW were decreased successfully by 71.9%and 68.8%relative to control values,cell viability decreased by 21.5%,GSH increased by 29.76%,and GPx increased by 47.58%.WST assay showed that compared with blank control,the value of positive group dropped 21.5%;In GSH and GPx assay,compared with blank control the positive group increased29.76%and 47.58%separately.In conclusion,SelW knock-down by RNAi caused significant cytotoxity in skeletal muscle cells and led to compensatory increases in GSH content and GPx activity.These findings are consistent with the suggestions from bioinformatics indicating an antioxidative role for SelW in skeletal muscle cells.

Vessel-associated stem cells from skeletal muscle:From biology to future uses in cell therapy

Over the last years,the existence of different stem cells with myogenic potential has been widely investigated.Be-sides the classical skeletal muscle progenitors represented by satellite cells,numerous multipotent and embryologi-cally unrelated progenitors with a potential role in muscle differentiation and repair have been identified.In order to conceive a therapeutic approach for degenerative muscle disorders,it is of primary importance to identify an ideal stem cell endowed with all the features for a possible use in vivo.Among all emerging populations,vessel-associated stem cells are a novel and promising class of multipotent progenitors of mesodermal origin and with high myogenic potential which seem to best fit all the requirements for a possible cell therapy.In vitro and in vivo studies have already tested the effectiveness and safety of vessel-associated stem cells in animal models.This leads to the concrete possibility in the future to start pilot human clinical trials,hopefully opening the way to a turn-ing point in the treatment of genetic and acquired muscle disorders.

Critical Limb Ischaemia in Adult Human Skeletal Muscle Increases Satellite Cell Proliferation but Not Differentiation

Critical Limb Ischaemia (CLI) is defined as the presence of rest pain, ulcers and/or gangrene in a limb for over 2 weeks. Associated exercise intolerance is caused by muscle fibre atrophy, fibro- fatty infiltration, nerve dysfunction, mitochondrial damage and myofibril disorder. We aimed to determine the behaviour of satellite cells, responsible for the repair and regeneration of damaged muscle, in repairing the damage caused to critically ischaemic adult human skeletal muscle. CD34, pax7 and MyoD are all markers of satellite cells at different stages of the cell cycle and allow for an assessment of their number and activity in ischaemia. Local ethical committee approval and informed consent was obtained. Samples of harvested gastrocnemius muscle of patients undergoing major perigenicular amputation for CLI (n = 10) were analysed and compared to a control group undergoing coronary artery bypass grafting (n = 10). Using immunohistochemistry, the expression of pax7, CD34 and MyoD was assessed in five sequential blinded randomly generated fields. Statistical testing of the data collected was made via the Mann Whitney U test. Protein electrophoresis was used to confirm overall protein expression of the satellite cell markers. There was a significant increase in the number of satellite cells observed in CLI muscle sections as demonstrated by the expression of pax7 (2.4×?fold p ?Haematopoietic Stem Cells?(HSCs) and satellite cells were also more abundant, with a 2×?fold increase observed (p < 0.0001) whilst those cells expressing both CD34 and pax7 and identified as quiescent satellite cells, were significantly greater in number in the CLI samples (2.9×?fold p < 0.0001), confirmed via immunohistochemistry and protein electrophoresis. There was a significant decrease in the expression of MyoD positive or activated satellite cells (p < 0.0001). This indicates an increase in the proliferation of the satellite cell population as a response to CLI but less active cells are observed.

Time-dependent gene expression analysis after mouse skeletal muscle contusion

Background:Though the mechanisms of skeletal muscle regeneration are deeply understood,those involved in muscle contusion,one of the most common muscle injuries in sports medicine clinics,are not.The objective of this study is to explore the mechanisms involved in muscle regeneration after contusion injury.Methods:In this study,a total of 72 mice were used.Eight of them were randomly chosen for the control group,while the rest were subjected to muscle contusion.Subsequently,their gastrocnemius muscles were harvested at different time points.The changes in muscle morphology were assessed by hematoxylin and eosin(HE) stain.In addition,the gene expression was analyzed by real-time polymerase chain reaction.Results:The data showed that the expression of many genes,i.e.,specific markers of immune cells and satellite cells,regulatory factors for muscle regeneration,cytokines,and chemokines,increased in the early stages of recovery,especially in the first 3 days.Furthermore,there were strict rules in the expression of these genes.However,almost all the genes returned to normal at 14 days post-injury.Conclusion:The sequence of immune cells invaded after muscle contusion was neutrophils,M1 macrophages and M2 macrophages.Some CC(CCL2,CCL3,and CCL4) and CXC(CXCL10) chemokines may be involved in the chemotaxis of these immune cells.HGF may be the primary factor to activate the satellite cells after muscle contusion.Moreover,2 weeks are needed to recover when acute contusion happens as used in this study.

Simple, Reliable Isolation, Purification and Cultivation of Murine Skeletal Muscle Microvascular Endothelial Cells

Objectives: Microvascular dysfunction in skeletal muscle is involved in metabolic and vascular diseases. Microvascular endothelial cells (MEC) are poorly characterized in the progression of associated diseases in part due to lack of availability of MEC from various animal models. The objective was to provide a fast, simple, and efficient method to isolate murine MEC derived from skeletal muscle. Methods: Dissected abdominal skeletal muscles from C57BL/6J mice at 8 - 12 weeks of age were enzymatically dissociated. MEC were isolated using a modified two-step Dynabeads™-based purification method. With a combination of Dynabeads™ - Griffonia simplicifolia lectin-I and Dynabeads™ - monoclonal antibody against CD31/PECAM-1, MEC were isolated and purified twice followed by cultivation. Results: Isolated and purified cells were viable and cultured. MEC were characterized by using immunofluorescence to identify CD31/PECAM-1, an EC marker, and two specific functional assays, which include a capillary-like tube formation and the uptake of Dil-Ac-LDL. The purity of isolated cell populations from skeletal muscle microvessels, which was assessed by flow cytometry, was 88.02% ± 2.99% (n = 6). Conclusions: This method is simple, fast, and highly reproducible for isolating MEC from murine skeletal muscle. The method will enable us to obtain primary cultured MEC from various genetic or diseased murine models, contributing to insightful knowledge of diseases associated with the dysfunction of microvessels.

IGF-1, bFGF EXPRESSION AND VASCULAR REGENERATION IN ACUTE INFARCTED CANINE MYOCARDIUM AFTER AUTOLOGUS SKELETAL MUSCLE SATELLITE CELL IMPLANTATION

Objective To study the cell growth factor secretion and vascular regeneration in acute in-farcted myocardium after autologous skeletal muscle satellite cell implantation. Methods Autologous skeletal muscle satellite cells from adult mongrel canine were implanted into the acute myocardial infarct site via the ligated left anterior descending (LAD) artery. Specimens were harvested at 2, 4 , 8 weeks after implantation for the expression of insulin-like growth factor-1 (IGF-1), basic fibroblast growth factor ( bFGF) and the vascular density. Results The expression of IGF-1, bFGF and the vascular density in skeletal muscle satellite cell implant group were higher than that in the control group. Conclusion The skeletal muscle satellite cells, after being implanted into the acute myocardial infarction, not only showed myocardial regeneration, but also showed the ability to secrete the cell factors, hence representing a positive effect on the regeneration of the infarcted myocardium.

The vascular endothelial growth factor expression and vascular regeneration in infarcted myocardium by skeletal muscle satellite cells

Background Myocardial infarction results in tissue necrosis, leading to cell loss and ultimately to cardiac failure. Implantation of skeletal muscle satellite cells into the scar area may compensate for the cell loss and provides a new strategy for infarct therapy. Vascular endothelial growth factor （VEGF） is a promising reagent for inducing myocardial angiogenesis. Skeletal myoblast transplantation has been shown to improve cardiac function in chronic heart failure models by regenerating muscle. We hypothesized that VEGF expression and vascular regeneration increased in infarcted myocardium by skeletal muscle satellite cells, which can promote vascular producing and improve survival environment in infarcted myocardium. Methods The skeletal muscle satellite cells were implanted into the infarcted myocardium in a model through ligated left anterior artery in Louis Inbrad Strain rat. Specimens were got for identifying the expression of VEGF and the density of vascular by immunochemical method at two weeks after implantation. Results The proliferation and differentiation of the skeletal muscle satellite cell was very well. The expression of VEGF was higher in the implanted group （146.83±2.49） than that in the control group （134.26±6.84） （P〈0.05）. The vascular density in the implanted group （13,00± 1.51） was also higher than that in the control （10.68 ± 1.79） （P〈0.05）. Conclusion The implanted satellite cell could excrete growth factor that would induce angiogenesis and improve cell survival environment in infarcted myocardium.

Influence of skeletal muscle satellite cells implanted into infarcted myocardium on remnant myocyte volumes

Objective To study the effects of skeletal muscle satellite cells implanted into infarcted myocardium on the volume of remnant myocytes Methods Thirty six adult mongrel canines were divided randomly into implantation group and control group In the implantation group, skeletal muscle satellite cells taken from the gluteus maximus muscles of the dogs were cultured, proliferated and labeled with 4', 6 diamidino 2 phenylindone (DAPI) in vitro In both groups, a model of acute myocardial infarction was established in every dog In the implantation group, each dog was injected with M199 solution containing autologous skeletal muscle satellite cells The dogs in the control group received M199 solution without skeletal muscle satellite cells The dogs of both groups were killed 2, 4 and 8 weeks after implantation (six dogs in a separate group each time) Both infarcted myocardium and normal myocytes distal from the infracted regions isolated were observed under optical and fluorescent microscope Their volumes were determined using a confocal microscopy image analysis system and analyzed using SAS A P <0 05 was considered significant Results A portion of the implanted cells differentiated into muscle fiber with striations and were connected with intercalated discs Cross sectional area and cell volume were increased in normal myocardium Hypertrophy of remnant myocytes in the infarcted site after skeletal muscle cell implantation was much more evident than in the control group. Cross sectional area, cell area and cell volume differed significantly from those of the control group ( P < 0.05) Hypertrophy of the cells occurred predominantly in terms of width and thickness, whereas cell length remained unchanged Conclusion Skeletal muscle satellite cells implanted into infarct myocardium, could induce the hypertrophy of remnant myocyte cells in the infarcted site and could also aid in the recovery of the contractile force of the infarcted myocardium

Effects of Massage on Satellite Cells of Acute Contusive Skeletal Muscles

Objective： To study the mechanism of Tuina in the treatment of skeletal muscle injury. Methods： Rabbits were heavily beaten at gastrocnemius muscle to make acute contusion model and then treated respectively by early Tuina and routine Tuina. The number of satellite cells of skeletal muscles was observed. Results： The number of the satellite cells continued to grow in both groups, and it began to increase significantly 3-5 days after Tuina treatment. Early Tuina treatment produces larger number of satellite cells than routine Tuina treatment. Conclusion： Early Tuina treatment is helpful to the marked recovery of skeletal muscles by increasing the number of satellite cell.