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 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

Myoblast fusion depends on mitochondrial integrity and intracellular Ca²⁺ signaling regulated by various ion channels. In this study, we investigated the ionic currents associated with [Ca²⁺]i regulation in normal and mitochondrial DNA-depleted (ρ0) L6 myoblasts. The ρ0 myoblasts showed impaired myotube formation. The inwardly rectifying K⁺ current (I(Kir)) was largely decreased with reduced expression of KIR2.1, whereas the voltage-operated Ca²⁺ channel and Ca²⁺-activated K⁺ channel currents were intact. Sustained inhibition of mitochondrial electron transport by antimycin A treatment (24 h) also decreased the I(Kir). The ρ0 myoblasts showed depolarized resting membrane potential and higher basal [Ca²⁺]ᵢ. Our results demonstrated the specific downregulation of I(Kir) by dysfunctional mitochondria. The resultant depolarization and altered Ca²⁺ signaling might be associated with impaired myoblast fusion in ρ0 myoblasts.
Antimycin A ; Down-Regulation ; Electron Transport ; Ion Channels ; Membrane Potentials ; Mitochondria ; Muscle Development ; Muscle Fibers, Skeletal ; Myoblasts* ; Oxidative Phosphorylation*

Sumoylation, the conjugation of a small ubiquitin-like modifier (SUMO) protein to a target, has diverse cellular effects. However, the functional roles of the SUMO modification during myogenesis have not been fully elucidated. Here, we report that basal sumoylation of histone deacetylase 1 (HDAC1) enhances the deacetylation of MyoD in undifferentiated myoblasts, whereas further sumoylation of HDAC1 contributes to switching its binding partners from MyoD to Rb to induce myocyte differentiation. Differentiation in C2C12 skeletal myoblasts induced new immunoblot bands above HDAC1 that were gradually enhanced during differentiation. Using SUMO inhibitors and sumoylation assays, we showed that the upper band was caused by sumoylation of HDAC1 during differentiation. Basal deacetylase activity was not altered in the SUMO modification-resistant mutant HDAC1 K444/476R (HDAC1 2R). Either differentiation or transfection of SUMO1 increased HDAC1 activity that was attenuated in HDAC1 2R. Furthermore, HDAC1 2R failed to deacetylate MyoD. Binding of HDAC1 to MyoD was attenuated by K444/476R. Binding of HDAC1 to MyoD was gradually reduced after 2 days of differentiation. Transfection of SUMO1 induced dissociation of HDAC1 from MyoD but potentiated its binding to Rb. SUMO1 transfection further attenuated HDAC1-induced inhibition of muscle creatine kinase luciferase activity that was reversed in HDAC1 2R. HDAC1 2R failed to inhibit myogenesis and muscle gene expression. In conclusion, HDAC1 sumoylation plays a dual role in MyoD signaling: enhancement of HDAC1 deacetylation of MyoD in the basally sumoylated state of undifferentiated myoblasts and dissociation of HDAC1 from MyoD during myogenesis.
Creatine Kinase, MM Form ; Gene Expression ; Histone Deacetylase 1 ; Histone Deacetylases ; Histones ; Luciferases ; Muscle Cells ; Muscle Development ; Myoblasts ; Myoblasts, Skeletal ; Sumoylation ; Transfection

We examined the effect of a combination of astaxanthin (AX) supplementation, repeated heat stress, and intermittent reloading (IR) on satellite cells in unloaded rat soleus muscles. Forty-nine male Wistar rats (8-week-old) were divided into control, hind-limb unweighting (HU), IR during HU, IR with AX supplementation, IR with repeated heat stress (41.0-41.5 °C for 30 min), and IR with AX supplementation and repeated heat stress groups. After the experimental period, the antigravitational soleus muscle was analyzed using an immunohistochemical technique. Our results revealed that the combination of dietary AX supplementation and heat stress resulted in protection against disuse muscle atrophy in the soleus muscle. This protective effect may be partially due to a higher satellite cell number in the atrophied soleus muscle in the IR/AX/heat stress group compared with the numbers found in the other groups. We concluded that the combination treatment with dietary AX supplementation and repeated heat stress attenuates soleus muscle atrophy, in part by increasing the number of satellite cells.
Animals ; Body Weight ; Dietary Supplements ; Fibrinolytic Agents/pharmacology* ; Heat-Shock Response ; Hindlimb ; Hot Temperature ; Immunohistochemistry ; Male ; Muscle, Skeletal ; Muscular Atrophy/drug therapy* ; Oxidative Stress ; Rats ; Rats, Wistar ; Satellite Cells, Skeletal Muscle/cytology* ; Xanthophylls/pharmacology*