The purpose of this study is to construct a muscle-specific synthetic promoter library, screen out muscle-specific promoters with high activity, analyze the relationship between element composition and activity of highly active promoters, and provide a theoretical basis for artificial synthesis of promoters. In this study, 19 promoter fragments derived from muscle-specific elements, conserved elements, and viral regulatory sequences were selected and randomLy connected to construct a muscle-specific synthetic promoter library. The luciferase plasmids pCMV-Luc and pSPs-Luc were constructed and transfected into the myoblast cell line C2C12. The activities of the synthesized promoters were evaluated by the luciferase activity assay. Two non-muscle-derived cell lines HeLa and 3T3 were used to verify the muscle specificity of the highly active promoters. The sequences of promoters with high activity, good muscle specificity, and correct sequences were analyzed to explore the relationship between the element composition and activity of promoters. We successfully constructed a muscle-specific promoter library and screened out 321 effective synthetic promoter plasmids. Among them, the activity of SP-301 promoter was 5.63 times that of CMV. The 15 promoters with high activity were muscle-specific. In the promoters with high activity and correct sequences, there was a relationship between their element composition and activity. Muscle-specific elements accounted for a high proportion in the promoters, while they had weak correlations with the promoter activity, being tissue-specific determinants. Viral elements accounted for no less than 20% in highly active promoters, which may be the key elements for the promoter activity. The content of conserved elements was proportional to the promoter activity. This study lays a theoretical foundation for the synthesis of tissue-specific efficient promoters and provides a new idea for the construction and application of in-situ gene delivery systems.
Promoter Regions, Genetic ; Humans ; Animals ; Mice ; Gene Library ; Cell Line ; Transfection ; HeLa Cells ; Luciferases/metabolism* ; Muscle, Skeletal/metabolism* ; Plasmids/genetics* ; Myoblasts/cytology*

Sarcopenia is an age-related degenerative skeletal muscle disorder characterized by the loss of skeletal muscle mass and function during aging.Sarcopenia can impair the elderly's ability to perform daily activities and is associated with high risks of falls,fractures,and hospitalization.It seriously affects the quality of life of the elderly and becomes one of the major health problems in the aging society.Skeletal muscle stem cells,also known as muscle satellite cells,play a key role in supporting muscle regeneration and homeostasis maintenance.Studies have suggested that muscle satellite cell functions are tightly regulated by microenvironment signals in the skeletal muscle.Of note,skeletal muscle fibers,serving as an immediate niche of muscle satellite cells,regulate their activation,proliferation,and self-renewal.This article reviews the research progress in the regulatory roles of skeletal muscle stem cells and their microenvironment in sarcopenia during aging,providing theoretical support for potential treatment of sarcopenia via modifying skeletal muscle microenvironment and regulating muscle satellite cell functions.
Sarcopenia/physiopathology* ; Humans ; Satellite Cells, Skeletal Muscle/physiology* ; Muscle, Skeletal/physiopathology* ; Aging/physiology* ; Animals ; Stem Cells

Severe muscle injury is hard to heal and always results in a poor prognosis. Recent studies found that extracellular vesicle-based therapy has promising prospects for regeneration medicine, however, whether extracellular vesicles have therapeutic effects on severe muscle injury is still unknown. Herein, we extracted apoptotic extracellular vesicles derived from mesenchymal stem cells (MSCs-ApoEVs) to treat cardiotoxin induced tibialis anterior (TA) injury and found that MSCs-ApoEVs promoted muscles regeneration and increased the proportion of multinucleated cells. Besides that, we also found that apoptosis was synchronized during myoblasts fusion and MSCs-ApoEVs promoted the apoptosis ratio as well as the fusion index of myoblasts. Furthermore, we revealed that MSCs-ApoEVs increased the relative level of creatine during myoblasts fusion, which was released via activated Pannexin 1 channel. Moreover, we also found that activated Pannexin 1 channel was highly expressed on the membrane of myoblasts-derived ApoEVs (Myo-ApoEVs) instead of apoptotic myoblasts, and creatine was the pivotal metabolite involved in myoblasts fusion. Collectively, our findings firstly revealed that MSCs-ApoEVs can promote muscle regeneration and elucidated that the new function of ApoEVs as passing inter-cell messages through releasing metabolites from activated Pannexin 1 channel, which will provide new evidence for extracellular vesicles-based therapy as well as improving the understanding of new functions of extracellular vesicles.
Creatine/metabolism* ; Extracellular Vesicles ; Muscle, Skeletal/metabolism* ; Myoblasts/metabolism* ; Regeneration ; Connexins/metabolism*

OBJECTIVE: To observe the effects of electroacupuncture on threshold of pain, gait, proliferation and differentiation of muscle satellite cell in rats with acute blunt trauma of gastrocnemius muscle, and to explore the possible mechanism of electroacupuncture in promoting the repair of acute injury of skeletal muscle. METHODS: A total of 48 SD rats were randomly divided into a blank group (6 rats), a model group (24 rats) and an electroacupuncture group (18 rats). In the model group and the electroacupuncture group, the model of acute blunt trauma of gastrocnemius muscle was established by self-made impactor. In the electroacupuncture group, electroacupuncture was applied at "Chengshan" (BL 57) and "Yanglingquan" (GB 34) on the right side, with disperse-dense wave, in frequency of 2 Hz/100 Hz, once a day, 30 min each time. Electroacupuncture intervention was performed for 3, 7 and 14 days according to the sampling time. On the 1st, 3rd, 7th and 14th days after modeling, the mechanical withdrawal pain threshold of hindfoot was detected by Von Frey method; the standing time and the maximum contact area of the right hindfoot were recorded by Cat Walk XT^TM animal gait analysis instrument; the morphology of the right gastrocnemius muscle and the number of inflammatory cells were observed by HE staining; the positive expression of paired box gene 7 (Pax7) and myogenic differentiation (MyoD) of the right gastrocnemius muscle was detected by immunofluorescence. RESULTS: After modeling, the muscle fiber rupture and massive infiltration of red blood cells and inflammatory cells were observed in the right gastrocnemius muscle; after electroacupuncture intervention, the morphology of muscle fiber was intact and the infiltration of inflammatory cells was improved. Compared with the blank group, in the model group, the differences of mechanical withdrawal pain threshold between the left and right foot were increased (P<0.05), the standing time was shortened and the maximum contact area of the right hindfoot was decreased (P<0.05), the number of inflammatory cells and the positive expression of Pax7 and MyoD of the right gastrocnemius muscle were increased (P<0.05) on the 1st, 3rd, 7th and 14th days after modeling. Compared with the model group, in the electroacupuncture group, the differences of mechanical withdrawal pain threshold were decreased (P<0.05), the standing time was prolonged (P<0.05), the number of inflammatory cells of right gastrocnemius muscle was decreased (P<0.05) on the 7th and 14th days after modeling; the maximum contact area of the right hindfoot was increased (P<0.05), the positive expression of MyoD of the right gastrocnemius muscle was increased (P<0.05) on the 3rd, 7th and 14th days after modeling; the positive expression of Pax7 of the right gastrocnemius muscle was increased (P<0.05) on the 3rd day after modeling. CONCLUSION Electroacupuncture can effectively improve the pain threshold and gait in rats with acute blunt trauma of gastrocnemius muscle, and promote the repair of skeletal muscle injury, the mechanism may be related to the up-regulation of Pax7 and MyoD, so as to promoting the proliferation and differentiation of muscle satellite cell.
Animals ; Rats ; Rats, Sprague-Dawley ; Satellite Cells, Skeletal Muscle ; Electroacupuncture ; Muscle, Skeletal ; Gait ; Wounds, Nonpenetrating ; Pain ; Cell Differentiation ; Cell Proliferation

OBJECTIVE: To investigate the feasibility of a dual-crosslinked injectable hydrogel derived from acellular musclar matrix (AMM) for promoting myoblasts proliferation and myogenic differentiation. METHODS: Firstly, hyaluronic acid was oxidized with NaIO ₄ and methylated to prepare methacrylamidated oxidized hyaluronic acid (MOHA). Then, AMM obtained by washing enzymatically treated muscle tissue was aminolyzed to prepare aminated AMM (AAMM). MOHA hydrogel and AAMM were crosslinked using Schiff based reaction and UV radiation to prepare a dual-crosslinked MOHA/AAMM injectable hydrogel. Fourier transform infrared spectroscopy (FTIR) was used to characterize MOHA, AAMM, and MOHA/AAMM hydrogels. The injectability of MOHA/AAMM hydrogel were evaluated by manual injection, and the gelation performance was assessed by UV crosslinking. The rheological properties and Young's modulus of the hydrogel were examined through mechanical tests. The degradation rate of the hydrogel was assessed by immersing it in PBS. The active components of the hydrogel were verified using immunofluorescence staining and ELISA assay kits. The promotion of cell proliferation by the hydrogel was tested using live/dead staining and cell counting kit 8 (CCK-8) assays after co-culturing with C2C12 myoblasts for 9 days. The effect of the hydrogel on myogenic differentiation was evaluated by immunofluorescence staining and real time quantitative polymerase chain reaction (RT-qPCR). RESULTS: FTIR spectra confirmed the successful preparation of MOHA/AAMM hydrogel. The hydrogel exhibited good injectability and gelation ability. Compared to MOHA hydrogel, MOHA/AAMM hydrogel exhibited higher viscosity and Young's modulus, a reduced degradation rate, and contained a higher amount of collagen (including collagen type Ⅰ and collagen type Ⅲ) as well as bioactive factors (including epidermal growth factor, fibroblast growth factor 2, vascular endothelial growth factor, and insulin-like growth factor 1). The live/dead cell staining and CCK-8 assay indicated that with prolonged incubation time, there was a significant increase in viable cells and a decrease in dead cells in the C2C12 myoblasts within the MOHA/AAMM hydrogel. Compared with MOHA hydrogel, the difference was significant at each time point ( P<0.05). Immunofluorescence staining and RT-qPCR analysis demonstrated that the deposition of IGF-1 and expression levels of myogenic-related genes (including Myogenin, Troponin T, and myosin heavy chain) in the MOHA/AAMM group were significantly higher than those in the MOHA group ( P<0.05). CONCLUSION The MOHA/AAMM hydrogel prepared based on AMM can promote myoblasts proliferation and myogenic differentiation, providing a novel dual-crosslinked injectable hydrogel for muscle tissue engineering.
Hydrogels ; Hyaluronic Acid/pharmacology* ; Vascular Endothelial Growth Factor A/metabolism* ; Tissue Engineering/methods* ; Cell Differentiation ; Myoblasts/metabolism* ; Cell Proliferation

BACKGROUND: The liver is an organ with remarkable regenerative capacity; however, once chronic fibrosis occurs, liver failure follows, with high mortality and morbidity rates. Continuous exposure to proinflammatory stimuli exaggerates the pathological process of liver failure; therefore, immune modulation is a potential strategy to treat liver fibrosis. Mesenchymal stem cells (MSCs) with tissue regenerative and immunomodulatory potential may support the development of therapeutics for liver fibrosis. METHODS: Here, we induced hepatic injury in mice by injecting carbon tetrachloride (CCl₄) and investigated the therapeutic potential of conditionedmedium from tonsil-derivedMSCs (T-MSCCM). In parallel, we used recombinant human IL-1Ra,which, as we have previously shown, is secreted exclusively from T-MSCs and resolves the fibrogenic activation of myoblasts. Hepatic inflammation and fibrosis were determined by histological analyses using H&E and Picro-Sirius Red staining. RESULTS: The results demonstrated that T-MSC CM treatment significantly reduced inflammation as well as fibrosis in the CCl₄-injured mouse liver. IL-1Ra injection showed effects similar to T-MSC CM treatment, suggesting that T-MSC CM may exert anti-inflammatory and anti-fibrotic effects via the endogenous production of IL-1Ra. The expression of genes involved in fibrosis was evaluated, and the results showed significant induction of alpha-1 type I collagen, transforming growth factor beta, and tissue inhibitor of metalloproteases 1 upon CCl₄ injection, whereas treatment with T-MSC CM or IL-1Ra downregulated their expression. CONCLUSION: Taken together, these data support the therapeutic potential of T-MSC CM and/or IL-1Ra for the alleviation of liver fibrosis, as well as in treating diseases involving organ fibrosis.
Animals ; Carbon Tetrachloride ; Collagen Type I ; Culture Media, Conditioned ; Fibrosis ; Humans ; Inflammation ; Interleukin 1 Receptor Antagonist Protein ; Liver Cirrhosis ; Liver Failure ; Liver ; Mesenchymal Stromal Cells ; Metalloproteases ; Mice ; Mortality ; Myoblasts ; Transforming Growth Factor beta

OBJECTIVE: To apply hypoxia of different oxygen concentration on C2C12 cells to study the changes of Nrf2 antioxidant system under HO. METHODS: The perfect simulative effect time and concentration of HO were chosen. Cell vitality was tested after C2C12 cells cultured in 0.1 mmol/L, 0.25 mmol/L, 0.5 mmol/L, 0.75 mmol/L, 1 mmol/L and 2 mmol/L HO for 1 or 2 h respectively. The C2C12 cells were divided into different oxygen concentration group: 21%O, 12%O, 8%O, 5%O respectively. And then cells were treated with HO for 1 h, and collected for determination. Immunofluorescence of Nrf2 and the protein expression of Nrf2 were detected. The expressions of antioxidant enzymes superoxide dismutase 1 (SOD1), superoxide dismutase 2 (SOD2), catalase(CAT), NADPH quinine oxidoreductase-1 (NQO-1), glutathione peroxidase-1 (GPX-1), Heme oxygenase-1 (HO-1) mRNA and cellular ROS levels were tested by high quality fluorescence assay. RESULTS: 0.5 mmol/L HO for 1 h was selected as the conditions of HOstimulation. Compared with 21% O group, the expressions of Nrf2 mRNA and protein, antioxidant enzymes SOD1, SOD2, CAT, HO-1, NQO-1, GPX-1 mRNA were increased significantly (P＜0.05 or P＜0.01), and ROS level was lower (P＜0.01) in 12%O group cells; only the expression of GPX-1 mRNA was increased (P＜0.05) in 8%O group; the expressions of Nrf2 mRNA and protein expression, antioxidant enzymes SOD1, SOD2, NQO-1, GPX-1 mRNA were decreased significantly(P＜0.05 or P＜0.01), and ROS level was higher (P＜0.01) in 5%O group. CONCLUSION Hypoxia can affect the Nrf2 antioxidant system, and the different oxygen concentrations have different impact. In addition, 12% O for 12 h could promote the Nrf2 antioxidant system, and 5% extremely low oxygen may inhibit it.
Animals ; Antioxidants ; metabolism ; Cell Line ; Cell Survival ; Hydrogen Peroxide ; Mice ; Myoblasts ; enzymology ; NF-E2-Related Factor 2 ; metabolism ; Oxidative Stress ; Oxygen ; Reactive Oxygen Species ; metabolism

OBJECTIVE: To investigate the expression of EGR1 gene and the localization of EGR1 protein in bovine skeletal muscle-derived satellite cells (MDSCs), as well as to investigate the mechanism that EGR1 protein enters the nucleus. METHODS: Bovine MDSCs were cultured in differentiation medium for 1 day, 3 days and 5 days, respectively, and each group was triplicate. The expression of EGR1 gene and the localization of EGR1 protein were studied at different differentiation period in MDSCs by qRT-PC and Western blot. Moreover, the changes on the expression of endogenous EGR1 gene and EGR1 proteins were explored by CRISPRi, site-directed mutagenesis and laser confocal method. RESULTS: The results from the qRT-PCR and Western blot showed that the expressions of EGR1 gene on transcription level and translation level were significantly higher in differentiated cells than those in undifferentiated cells. The highest expression was found on the third day after the differentiation, and then began to decline. Immunofluorescence assays showed that EGR1 proteins were preferentially expressed in differentiated MDSCs, and increased along with the increase of number of myotubes. Confocal observation revealed that some EGR1 proteins were transferred into the nucleus in the differentiation of cells, however, the EGR1 proteins would not be detected in the differentiated MDSCs nuclei if a site directed mutagenesis (serine) on EGR1 protein occurred. CONCLUSION During the differentiation of bovine skeletal muscle satellite cells, the transcriptional level of EGR1 gene is increased, and some EGR1 proteins are transferred into the nucleus. The serine phosphorylation at position 533 of the C terminal of EGR1 protein is necessary for the nucleus transfer.
Animals ; Cattle ; Cell Differentiation ; Cell Nucleus ; Cells, Cultured ; Early Growth Response Protein 1 ; genetics ; metabolism ; Muscle Fibers, Skeletal ; Satellite Cells, Skeletal Muscle ; metabolism

Although atopic dermatitis (AD) is known to be a representative skin disorder, it also affects the systemic immune response. In a recent study, myoblasts were shown to be involved in the immune regulation, but the roles of muscle cells in AD are poorly understood. We aimed to identify the relationship between mitochondria and atopy by genome-wide analysis of skeletal muscles in mice. We induced AD-like symptoms using house dust mite (HDM) extract in NC/Nga mice. The transcriptional profiles of the untreated group and HDM-induced AD-like group were analyzed and compared using microarray, differentially expressed gene and functional pathway analyses, and protein interaction network construction. Our microarray analysis demonstrated that immune response-, calcium handling-, and mitochondrial metabolism-related genes were differentially expressed. In the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology pathway analyses, immune response pathways involved in cytokine interaction, nuclear factor-kappa B, and T-cell receptor signaling, calcium handling pathways, and mitochondria metabolism pathways involved in the citrate cycle were significantly upregulated. In protein interaction network analysis, chemokine family-, muscle contraction process-, and immune response-related genes were identified as hub genes with many interactions. In addition, mitochondrial pathways involved in calcium signaling, cardiac muscle contraction, tricarboxylic acid cycle, oxidation-reduction process, and calcium-mediated signaling were significantly stimulated in KEGG and Gene Ontology analyses. Our results provide a comprehensive understanding of the genome-wide transcriptional changes of HDM-induced AD-like symptoms and the indicated genes that could be used as AD clinical biomarkers.
Animals ; Biomarkers ; Calcium ; Calcium Signaling ; Citric Acid ; Citric Acid Cycle ; Cytokines ; Dermatitis, Atopic ; Gene Ontology ; Genome ; Metabolism ; Mice ; Microarray Analysis ; Mitochondria ; Muscle Cells ; Muscle Contraction ; Muscle, Skeletal ; Myoblasts ; Myocardium ; Oxidation-Reduction ; Protein Interaction Maps ; Pyroglyphidae ; Receptors, Antigen, T-Cell ; Skin

Anoctamin 5 (ANO5)/TMEM16E belongs to a member of the ANO/TMEM16 family member of anion channels. However, it is a matter of debate whether ANO5 functions as a genuine plasma membrane chloride channel. It has been recognized that mutations in the ANO5 gene cause many skeletal muscle diseases such as limb girdle muscular dystrophy type 2L (LGMD2L) and Miyoshi muscular dystrophy type 3 (MMD3) in human. However, the molecular mechanisms of the skeletal myopathies caused by ANO5 defects are poorly understood. To understand the role of ANO5 in skeletal muscle development and function, we silenced the ANO5 gene in C2C12 myoblasts and evaluated whether it impairs myogenesis and myotube function. ANO5 knockdown (ANO5-KD) by shRNA resulted in clustered or aggregated nuclei at the body of myotubes without affecting differentiation or myotube formation. Nuclear positioning defect of ANO5-KD myotubes was accompanied with reduced expression of Kif5b protein, a kinesin-related motor protein that controls nuclear transport during myogenesis. ANO5-KD impaired depolarization-induced [Ca²⁺]i transient and reduced sarcoplasmic reticulum (SR) Ca²⁺ storage. ANO5-KD resulted in reduced protein expression of the dihydropyridine receptor (DHPR) and SR Ca²⁺-ATPase subtype 1. In addition, ANO5-KD compromised co-localization between DHPR and ryanodine receptor subtype 1. It is concluded that ANO5-KD causes nuclear positioning defect by reduction of Kif5b expression, and compromises Ca²⁺ signaling by downregulating the expression of DHPR and SERCA proteins.
Active Transport, Cell Nucleus ; Calcium Channels, L-Type ; Cell Membrane ; Chloride Channels ; Humans ; Muscle Development ; Muscle Fibers, Skeletal ; Muscle, Skeletal ; Muscular Diseases ; Muscular Dystrophies ; Muscular Dystrophies, Limb-Girdle ; Myoblasts ; RNA, Small Interfering ; Ryanodine Receptor Calcium Release Channel ; Sarcoplasmic Reticulum