Skeletal Muscle Satellite Cells Research Articles

Leu promotes C2C12 cell differentiation by regulating the GSK3β/β-catenin signaling pathway through facilitating the interaction between SESN2 and RPN2.

Leucine (Leu) is an essential amino acid that facilitates skeletal muscle satellite cell differentiation, yet its mechanism remains underexplored. Sestrin2 (SESN2) serves as a Leu sensor, binding directly to Leu, while ribophorin II (RPN2) acts as a signaling factor in multiple pathways. This study aimed to elucidate Leu's impact on mouse C2C12 cell differentiation and skeletal muscle injury repair by modulating RPN2 expression through SESN2, offering a theoretical foundation for clinical skeletal muscle injury prevention and treatment. Leu addition promoted C2C12 cell differentiation compared to the control, enhancing early differentiation via myogenic determinant (MYOD) up-regulation. Sequencing revealed SESN2 binding to and interacting with RPN2. RPN2 overexpression up-regulated MYOD, myogenin and myosin heavy chain 2, concurrently decreased p-GSK3β and increased nuclear β-catenin. Conversely, RPN2 knockdown yielded opposite results. Combining RPN2 knockdown with Leu rescued increased p-GSK3β and decreased nuclear β-catenin compared to Leu absence. Hematoxylin and eosin staining results showed that Leu addition accelerated mouse muscle damage repair, up-regulating Pax7, MYOD and RPN2 in the cytoplasm, and nuclear β-catenin, confirming that the role of Leu in muscle injury repair was consistent with the results for C2C12 cells. Leu, bound with SESN2, up-regulated RPN2 expression, activated the GSK3β/β-catenin pathway, enhanced C2C12 differentiation and expedited skeletal muscle damage repair. © 2024 Society of Chemical Industry.

Eicosapentaenoic acid-mediated activation of PGAM2 regulates skeletal muscle growth and development via the PI3K/AKT pathway

Eicosapentaenoic acid regulates glucose uptake in skeletal muscle and significantly affects whole-body energy metabolism. However, the underlying molecular mechanism remains unclear. Here we report that eicosapentaenoic acid activates phosphoglycerate mutase 2, which mediates the conversion of 2-phosphoglycerate into 3-phosphoglycerate. This enzyme plays a pivotal role in glycerol degradation, thereby facilitating the proliferation and differentiation of satellite cells in skeletal muscle. Interestingly, phosphoglycerate mutase 2 inhibits mitochondrial metabolism, promoting the formation of fast-type muscle fibers. Treatment with eicosapentaenoic acid and phosphoglycerate mutase 2 knockdown induced opposite transcriptomic changes, most of which were enriched in the PI3K-AKT signaling pathway. Phosphoglycerate mutase 2 activated the PI3K-AKT signaling pathway, which inhibited the phosphorylation of FOXO1, and, in turn, inhibited mitochondrial function and promoted the formation of fast-type muscle fibers. Our results suggest that eicosapentaenoic acid promotes skeletal muscle growth and regulates glucose metabolism by targeting phosphoglycerate mutase 2 and activating the PI3K/AKT signaling pathway.

β-hydroxybutyrate exposure restores mitochondrial function in skeletal muscle satellite cells of critically ill patients

Background and AimDysfunction of skeletal muscle satellite cells might impair muscle regeneration and prolong ICU-acquired weakness, a condition associated with disability and delayed death. This study aimed to elucidate the distinct metabolic effects of critical illness and β-OHB on satellite cells isolated from these patients. MethodsSatellite cells were extracted from vastus lateralis muscle biopsies of patients with ICU-acquired weakness (n = 10) and control group of healthy volunteers or patients undergoing elective hip replacement surgery (n = 10). The cells were exposed to standard culture media supplemented with β-OH-butyrate to assess its influence on cell proliferation by ELISA, mitochondrial functions by extracellular flux analysis, electron transport chain complexes by high resolution respirometry, and ROS production by confocal microscopy. ResultsCritical illness led to a decline in maximal respiratory capacity, ATP production and glycolytic capacity and increased ROS production in ICU patients’ cells. Notably, the function of complex II was impaired due to critical illness but restored to normal levels upon exposure to β-OH-butyrate. While β-OH-butyrate significantly reduced ROS production in both control and ICU groups, it had no significant impact on global mitochondrial functions. ConclusionCritical illness induces measurable bioenergetic dysfunction of skeletal muscle satellite cells. β-OH-butyrate displayed a potential in rectifying complex II dysfunction caused by critical illness and this warrants further exploration.

Identification of mulberry leaf flavonoids and evaluating their protective effects on H2O2-induced oxidative damage in equine skeletal muscle satellite cells.

Introduction: Horses are susceptible to oxidative stress during strenuous endurance exercise, leading to muscle fatigue and damage. Mulberry leaf flavonoids (MLFs) possess significant antioxidant properties. However, the antioxidant efficacy of MLFs can be influenced by the extraction process, and their impact on H2O2-induced oxidative stress in equine skeletal muscle satellite cells (ESMCs) remains unexplored. Methods: Our study employed three extraction methods to obtain MLFs: ultrasound-assisted extraction (CEP), purification with AB-8 macroporous resin (RP), and n-butanol extraction (NB-EP). We assessed the protective effects of these MLFs on H2O2-induced oxidative stress in ESMCs and analyzed the MLF components using metabolomics. Results: The results revealed that pre-treatment with MLFs dose-dependently protected ESMCs against H2O2-induced oxidative stress. The most effective concentrations were 0.8mg/mL of CEP, 0.6mg/mL of RP, and 0.6mg/mL of NB-EP, significantly enhancing EMSC viability (p < 0.05). These optimized MLF concentrations promoted the GSH-Px, SOD and T-AOC activities (p < 0.05), while reducing MDA production (p < 0.05) in H2O2-induced ESMCs. Furthermore, these MLFs enhanced the gene expression, including Nrf2 and its downstream regulatory genes (TrxR1, GPX1, GPX3, SOD1, and SOD2) (p < 0.05). In terms of mitochondrial function, ESMCs pre-treated with MLFs exhibited higher basal respiration, spare respiratory capacity, maximal respiration, ATP-linked respiration compared to H2O2-induced ESMCs (p < 0.05). Additionally, MLFs enhanced cellular basal glycolysis, glycolytic reserve, and maximal glycolytic capacity (p < 0.05). Metabolomics analysis results revealed significant differences in mulberrin, kaempferol 3-O-glucoside [X-Mal], neohesperidin, dihydrokaempferol, and isobavachalcone among the three extraction processes (p < 0.05). Discussion: Our study revealed that MLFs enhance antioxidant enzyme activity, alleviate oxidative damage in ESMCs through the activation of the Nrf2 pathway, and improve mitochondrial respiration and cell energy metabolism. Additionally, we identified five potential antioxidant flavonoid compounds, suggesting their potential incorporation into the equine diet as a strategy to alleviate exercise-induced oxidative stress.

Glutenin-chitosan 3D porous scaffolds with tunable stiffness and systematized microstructure for cultured meat model

A glutenin (G)-chitosan (CS) complex (G-CS) was cross-linked by water annealing with aim to prepare structured 3D porous cultured meat scaffolds (CMS) here. The CMS has pore diameters ranging from 18 to 67 μm and compressive moduli from 16.09 to 60.35 kPa, along with the mixing ratio of G/CS. SEM showed the porous organized structure of CMS. FTIR and CD showed the increscent content of α-helix and β-sheet of G and strengthened hydrogen-bondings among G-CS molecules, which strengthened the stiffness of G-CS. Raman spectra exhibited an increase of G concentration resulted in higher crosslinking of disulfide-bonds in G-CS, which aggrandized the bridging effect of G-CS and maintained its three-dimensional network. Cell viability assay and immuno-fluorescence staining showed that G-CS effectively facilitated the growth and myogenic differentiation of porcine skeletal muscle satellite cells (PSCs). CLSM displayed that cells first occupied the angular space of hexagon and then ring-growth circle of PSCs were orderly formed on G-CS. The texture and color of CMS which loaded proliferated PSCs were fresh-meat like. These results showed that physical cross-linked G-CS scaffolds are the biocompatible and stable adaptable extracellular matrix with appropriate architectural cues and natural micro-environment for structured CM models.

Molecular Regulation of Porcine Skeletal Muscle Development: Insights from Research on CDC23 Expression and Function.

Cell division cycle 23 (CDC23) is a component of the tetratricopeptide repeat (TPR) subunit in the anaphase-promoting complex or cyclosome (APC/C) complex, which participates in the regulation of mitosis in eukaryotes. However, the regulatory model and mechanism by which the CDC23 gene regulates muscle production in pigs are largely unknown. In this study, we investigated the expression of CDC23 in pigs, and the results indicated that CDC23 is widely expressed in various tissues and organs. In vitro cell experiments have demonstrated that CDC23 promotes the proliferation of myoblasts, as well as significantly positively regulating the differentiation of skeletal muscle satellite cells. In addition, Gene Set Enrichment Analysis (GSEA) revealed a significant downregulation of the cell cycle pathway during the differentiation process of skeletal muscle satellite cells. The protein-protein interaction (PPI) network showed a high degree of interaction between genes related to the cell cycle pathway and CDC23. Subsequently, in differentiated myocytes induced after overexpression of CDC23, the level of CDC23 exhibited a significant negative correlation with the expression of key factors in the cell cycle pathway, suggesting that CDC23 may be involved in the inhibition of the cell cycle signaling pathway in order to promote the differentiation process. In summary, we preliminarily determined the function of CDC23 with the aim of providing new insights into molecular regulation during porcine skeletal muscle development.

The telocytes relationship with satellite cells: Extracellular vesicles mediate the myotendinous junction remodeling.

The peripheral nerve injury (PNI) affects the morphology of the whole locomotor apparatus, which can reach the myotendinous junction (MTJ) interface. In the injury condition, the skeletal muscle satellite cells (SC) are triggered, activated, and proliferated to repair their structure, and in the MTJ, the telocytes (TC) are associated to support the interface with the need for remodeling; in that way, these cells can be associated with SC. The study aimed to describe the SC and TC relationship after PNI at the MTJ. Sixteen adult Wistar rats were divided into Control Group (C, n = 8) and PNI Group (PNI, n = 8), PNI was performed by the constriction of the sciatic nerve. The samples were processed for transmission electron microscopy and immunostaining analysis. In the C group was evidenced the arrangement of sarcoplasmic evaginations and invaginations, the support collagen layer with a TC inside it, and an SCthrough vesicles internally and externally to then. In the PNI group were observed the disarrangement of invaginations and evaginations and sarcomeres degradation at MTJ, as the disposition of telopodes adjacent and in contact to the SCwith extracellular vesicles and exosomes in a characterized paracrine activity. These findings can determine a link between the TCs and the SCs at the MTJ remodeling. RESEARCH HIGHLIGHTS: Peripheral nerve injury promotes the myotendinous junction (MTJ) remodeling. The telocytes (TC) and the satellite cells (SC) are present at the myotendinous interface. TC mediated the SCactivity at MTJ.

An Identification of Functional Genetic Variants in B4GALNT2 Gene and Their Association with Growth Traits in Goats.

β-1,4-N-acetylgalactosamine transferase 2 (B4GALNT2) is a vital candidate gene that affects the growth traits in sheep. However, whether it has the same function in goats remains to be investigated further. This study selected 348 Nanjiang Yellow goats, screened all exons, and conserved non-coding regions of the B4GALNT2 gene for single-nucleotide polymorphisms (SNPs). Our results revealed the presence of a synonymous mutation, rs672215506, within the exon of the B4GALNT2 gene in the Nanjiang Yellow goat population. The mutation resulted in a decrease in the mRNA stability of the B4GALNT2 gene. The results of SNP detection of the conserved non-coding region of the B4GALNT2 gene showed five potential regulatory SNPs in the Nanjiang Yellow goat population. Except for rs66095343, the ~500 bp fragments of the other four SNPs (rs649127714, rs649573228, rs652899012, and rs639183528) significantly increased the luciferase activity both in goat skeletal muscle satellite cells (MuSCs) and 293T cells. The genetic diversity indexes indicated low or intermediate levels for all six SNPs analyzed, and the genotype frequencies were in Hardy-Weinberg equilibrium. Association analysis showed that rs660965343, rs649127714, and rs649573228 significantly correlate with growth traits in the later stage of growth and development of Nanjiang Yellow goats. The haplotype combinations of H2H3 and H2H2 had higher body weight and greater body size. Moreover, H2H2 haplotype combinations significantly correlated with the litter size of the Nanjiang Yellow goats. The results of our study demonstrate the potential role of the B4GALNT2 gene as a functional genetic marker in the breeding programs of Nanjiang Yellow goats.

Adiponectin attenuates H2O2-induced apoptosis in chicken skeletal myoblasts through the lysosomal-mitochondrial axis.

Adiponectin has previously been investigated for exerting its protective effect against myocardial injury through anti-apoptotic and anti-oxidative actions. Therefore, the present study aimed to investigate the nature and mechanism of adiponectin inhibition of H2O2-induced apoptosis in chicken skeletal myoblasts. Skeletal muscle satellite cells were differentiated and assigned into three groups. Group C was on the blank control group, group H was stimulated with the H2O2 (500μmol/L, 4h) alone group, group A + H was pre-treated with adiponectin (10μg/mL, 24h) and stimulated with the H2O2 (500μmol/L, 4h) group. Cytotoxicity inhibited by adiponectin was evaluated by the CCK-8 assay. The degree of apoptosis and oxidative damage was investigated by the TdT-mediated dUTP nick end labeling (TUNEL) and reactive oxygen species (ROS) staining assays. Oxidative stress was assessed by evaluating lipid peroxidation, superoxide dismutase, and reduced glutathione. Acridine orange (AO) staining detected lysosomal membrane permeability. The changes in mitochondrial membrane potential (MMP) were analyzed using 5,5,6,6'-tetrachloro-1,1,3,3-tetraethylimidacarbocyanine iodide (JC-1) dye under a fluorescence microscope. The lysosomal function, mitochondrial function, and apoptosis-related mRNA and protein expression levels were quantified by real-time quantitative PCR and western blot, respectively. The results suggested that adiponectin treatment attenuated H2O2-induced cytotoxicity and oxidative stress in skeletal myoblasts. Compared with H2O2 treatment, TUNEL and ROS staining demonstrated lower apoptosis upon adiponectin treatment. AO staining confirmed the amelioration of lysosomal membrane damage, and JC-1 staining revealed an increase in mitochondrial membrane potential after adiponectin treatment. At the molecular level, adiponectin treatment inhibited the expression of the lysosomal apoptotic factors cathepsin B, chymotrypsin B, and the mitochondrial apoptotic pathway cytochrome-c (cyt-c) and caspase-8; decreased the apoptotic marker gene Bax; and increased the expression of the anti-apoptotic marker gene Bcl-2. Adiponectin treatment attenuated H2O2-induced apoptosis in skeletal myoblasts, possibly by inhibiting oxidative stress and apoptosis through the lysosomal-mitochondrial axis.

Studying the Effect of MBNL1 and MBNL2 Loss in Skeletal Muscle Regeneration.

Loss of function of members of the muscleblind-like (MBNL) family of RNA binding proteins has been shown to play a key role in the spliceopathy of RNA toxicity in myotonic dystrophy type 1 (DM1), the most common muscular dystrophy affecting adults and children. MBNL1 and MBNL2 are the most abundantly expressed members in skeletal muscle. A key aspect of DM1 is poor muscle regeneration and repair, leading to dystrophy. We used a BaCl2-induced damage model of muscle injury to study regeneration and effects on skeletal muscle satellite cells (MuSCs) in Mbnl1∆E3/∆E3 and Mbnl2∆E2/∆E2 knockout mice. Similar experiments have previously shown deleterious effects on these parameters in mouse models of RNA toxicity. Muscle regeneration in Mbnl1 and Mbnl2 knockout mice progressed normally with no obvious deleterious effects on MuSC numbers or increased expression of markers of fibrosis. Skeletal muscles in Mbnl1∆E3/∆E3/ Mbnl2∆E2/+ mice showed increased histopathology but no deleterious reductions in MuSC numbers and only a slight increase in collagen deposition. These results suggest that factors beyond the loss of MBNL1/MBNL2 and the associated spliceopathy are likely to play a key role in the defects in skeletal muscle regeneration and deleterious effects on MuSCs that are seen in mouse models of RNA toxicity due to expanded CUG repeats.

Satellite Cell-Derived Exosomes: A Novel Approach to Alleviate Skeletal Muscle Atrophy and Fibrosis.

Skeletal muscle atrophy coincides with extensive fibrous tissue hyperplasia in muscle-atrophied patients, and fibrous tissue plays a vital role in skeletal muscle function and hinders muscle fiber regeneration. However, effective drugs to manage skeletal muscle atrophy and fibrosis remain elusive. This study isolated and characterized exosomes derived from skeletal muscle satellite cells (MuSC-Exo). The study investigated their effects on denervated skeletal muscle atrophy and fibrosis in Sprague Dawley (SD) rats via intramuscular injection. MuSC-Exo demonstrated the potential to alleviate skeletal muscle atrophy and fibrosis. The underlying mechanism using single-cell RNA sequencing data and functional analysis are analyzed. Mechanistic studies reveal close associations between fibroblasts and myoblasts, with the transforming growth factor β1 (TGF-β1)-Smad3-Pax7 axis governing fibroblast activation in atrophic skeletal muscle. MuSC-Exo intervention inhibited the TGF-β1/Smad3 pathway and improved muscle atrophy and fibrosis. In conclusion, MuSC-Exo-based therapy may represent a novel strategy to alleviate skeletal muscle atrophy and reduce excessive fibrotic tissue by targeting Pax7 through the TGF-β1/Smad3 pathway.

RASGRP1 targeted by H3K27me3 regulates myoblast proliferation and differentiation in mice and pigs.

Skeletal muscle is not only the largest organ in the body that is responsible for locomotion and exercise but also crucial for maintaining the body's energy metabolism and endocrine secretion. The trimethylation of histone H3 lysine 27 (H3K27me3) is one of the most important histone modifications that participates in muscle development regulation by repressing the transcription of genes. Previous studies indicate that the RASGRP1 gene is regulated by H3K27me3 in embryonic muscle development in pigs, but its function and regulatory role in myogenesis are still unclear. In this study, we verify the crucial role of H3K27me3 in RASGRP1 regulation. The gain/loss function of RASGRP1 in myogenesis regulation is performed using mouse myoblast C2C12 cells and primarily isolated porcine skeletal muscle satellite cells (PSCs). The results of qPCR, western blot analysis, EdU staining, CCK-8 assay and immunofluorescence staining show that overexpression of RASGRP1 promotes cell proliferation and differentiation in both skeletal muscle cell models, while knockdown of RASGRP1 leads to the opposite results. These findings indicate that RASGRP1 plays an important regulatory role in myogenesis in both mice and pigs.

CREG1 deficiency impaired myoblast differentiation and skeletal muscle regeneration.

CREG1 (cellular repressor of E1A-stimulated genes 1) is a protein involved in cellular differentiation and homeostasis regulation. However, its role in skeletal muscle satellite cells differentiation and muscle regeneration is poorly understood. This study aimed to investigate the role of CREG1 in myogenesis and muscle regeneration. RNA sequencing data (GSE8479) was analysed from the Gene Expression Omnibus database (GEO, https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi). We generated Creg1 knockdown and skeletal muscle satellite cells specific Creg1 overexpression mice mediated by adeno-associated virus serotype 9 (AAV9), skeletal muscle mature myofibre Creg1 knockout mice (myoblast/Creg1MKO), and control mice Creg1flox/flox (Creg1fl/fl) as in vivo models. The mice were injected into tibialis anterior (TA) muscle with 100μL of 10μM cardiotoxin to establish a muscle regeneration model. Creg1fl/fl and Creg1MKO mice were treated with AAV-sh-C-Cbl (2×1010 genomic copies/mouse) to silence C-Cbl in the TA muscle. 293T and C2C12 cells were transfected with plasmids using lipofectamine RNAi MAX in vitro. Mass spectrometry analyses and RNA sequencing transcriptomic assay were performed. We analysed the transcriptional profiles of the skeletal muscle biopsies from healthy older (N=25) and younger (N=26) adult men and women in GSE8479 database, and the results showed that Creg1 was associated with human sarcopenia. We found that Creg1 knockdown mice regenerated less newly formed fibres in response to cardiotoxin injection (~30% reduction, P<0.01); however, muscle satellite cells specific Creg1 overexpression mice regenerated more newly formed fibres (~20% increase, P<0.05). AMPKa1 is known as a key mediator in the muscle regeneration process. Our results revealed that CREG1 deficiency inhibited AMPKa1 signalling through C-CBL E3-ubiquitin ligase-mediated AMPKa1 degradation (P<0.01). C-CBL-mediated AMPKa1 ubiquitination was attributed to the K48-linked polyubiquitination of AMPKa1 at K396 and that the modification played an important role in the regulation of AMPKa1 protein stability. We also found that Creg1MKO mice regenerated less newly formed fibres compared with Creg1fl/fl mice (~30% reduction, P<0.01). RNA-seq analysis showed that CREG1 deletion in impaired muscles led to the upregulation of inflammation and DKK3 expression. The TA muscles of Creg1MKO mice were injected with AAV-vector or AAV-shC-Cbl, silencing C-CBL (P<0.01) in the skeletal muscles of Creg1MKO mice significantly improved muscle regeneration induced by CTX injury (P<0.01). Our findings suggest that CREG1 may be a potential therapeutic target for skeletal muscle regeneration.

Establishment and analysis of immortalized chicken skeletal muscle satellite cell lines1

Skeletal muscle satellite cells are stem cells that are known for their multipotency and ability to proliferate in vitro. However, primary skeletal muscle satellite cells have limited proliferative capacity in vitro, which hinders their study in poultry skeletal muscle. The emergence of immortalization techniques for cells has provided a useful tool to overcome this limitation and explore the functions of skeletal muscle satellite cells. In this study, we achieved the immortalization of chicken skeletal muscle satellite cells by transducing primary cells with TERT (Telomerase reverse transcriptase) amplified from chicken (chTERT) using a lentiviral vector through reconstitution of telomerase activity. The cells successfully bypassed replicative senescence but did not achieve true immortalization. Preliminary functional characterization of the established cell line revealed that the proliferative characteristics and cell cycle profile of the immortalized chicken skeletal muscle satellite cell lines (ICMS) were similar to those of chicken primary muscle satellite cells (CPMSCs). Serum dependency analysis and soft agar assays indicated that ICMS did not undergo malignant transformation. Induced differentiation results demonstrated that ICMS retained their capacity for differentiation. The cell lines established in this study provide an important basis for the establishment of immortalized poultry cell lines and a cell model for the study of poultry skeletal muscle-related functional genes.

Organ Regeneration Without Relying on Regeneration-Dedicated Stem Cells.

The classic conception of tissue regeneration assumed the existence of tissue-proper regeneration stem cells that are set aside during normal tissue development and reserved as stem cells for regeneration. However, modern studies using cell tracing and other approaches have ruled out the presence of regeneration-proper stem cells in most cases in vertebrate tissue regeneration. The only experimentally validated regeneration-dedicated reserve cells are the satellite cells in skeletal muscle (e.g., Michele 2022) (see Sect. 5.2.3 ). Here, we will first discuss examples of large-scale tissue regeneration, liver regeneration in mammals, and lens and limb regeneration in newts. Then, attempts to widen the tissue regeneration capacity in mammals with exogenous transcription factor genes will be reviewed.

Manual Therapy Exerts Local Anti-Inflammatory Effects Through Neutrophil Clearance.

Background: Manual therapy (MT) has been widely used in China to treat local tissue inflammation for a long time. However, there is a lack of scientific evidence for using MT in anti-inflammatory therapy, and its anti-inflammatory mechanism needs further clarification. Methods: We utilized MT to treat cardiotoxin (CTX) injury-induced skeletal muscle inflammation in C57BL6/J mice. We analyzed the underlying mechanism by integrating single-cell RNA sequencing (scRNA-seq) with molecular techniques. Hematoxylin and eosin (H&E) and immunohistochemical (IHC) staining were used to assess skeletal muscle inflammation and muscle fiber cross-sectional area (CSA). scRNA-seq, immunofluorescence, and western blot were performed to determine cellular and molecular outcome changes. Results: Compared with CTX injury-induced skeletal muscle inflammatory mice, MT intervention significantly reduced proinflammatory cytokines interleukin (IL)-1β, IL-6, and tumor necrosis factor alpha (TNF-α) expression levels; scRNA-seq detected that neutrophil numbers and activity were maximum proportions increased in injured skeletal muscle among macrophage, T cells, B cells, endothelial cells, fast muscle cells, fibroblasts, and skeletal muscle satellite cells; and S100A9 gene expression was supreme in neutrophils. However, after treatment with MT, S100A9 protein expression and the numbers and activity of Ly6g+/Mpo+ neutrophils were significantly inhibited, thus reducing the inflammatory cytokine levels and exerting an anti-inflammatory effect by early clearing neutrophils. Conclusion: MT can mitigate localized inflammation induced by injured skeletal muscle, achieved by decreasing S100A9 protein expression and clearing neutrophils in mice, which may help advance therapeutic strategies for skeletal muscle localized inflammation.

LncRNA GTL2 regulates myoblast proliferation and differentiation via the PKA-CREB pathway in Duolang sheep.

Long non-coding RNAs (lncRNAs), which are RNA molecules longer than 200 nucleotides that do not encode proteins, are implicated in a variety of biological processes, including growth and development. Despite research into the role of lncRNAs in skeletal muscle development, the regulatory mechanisms governing ovine skeletal muscle development remain unclear. In this study, we analyzed the expression profiles of lncRNAs in skeletal muscle from 90-day-old embryos (F90), 1-month-old lambs (L30), and 3-year-old adult sheep (A3Y) using RNA sequencing. In total, 4 738 lncRNAs were identified, including 997 that were differentially expressed. Short-time series expression miner analysis identified eight significant expression profiles and a subset of lncRNAs potentially involved in muscle development. Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed that the predicted target genes of these lncRNAs were primarily enriched in pathways associated with muscle development, such as the cAMP and Wnt signaling pathways. Notably, the expression of lncRNA GTL2 was found to decrease during muscle development. Moreover, GTL2 was highly expressed during the differentiation of skeletal muscle satellite cells (SCs) and was shown to modulate ovine myogenesis by affecting the phosphorylation levels of PKA and CREB. Additionally, GTL2 was found to regulate both the proliferation and differentiation of SCs via the PKA-CREB signaling pathway. Overall, this study provides a valuable resource and offers novel insights into the functional roles and regulatory mechanisms of lncRNAs in ovine skeletal muscle growth and development.

Cyclin D3 Colocalizes with Myogenin and p21 in Skeletal Muscle Satellite Cells during Early-Stage Functional Overload.

Myogenic cell differentiation is modulated by multiple regulatory factors, such as myogenin, p21, and cyclin D3 during myogenesis in vitro. It is also recognized that myogenin and p21 play important roles in regulating muscle satellite cell (SC) differentiation during overload-induced muscle hypertrophy in vivo. However, the expression patterns and functional role of cyclin D3 in the progress of muscle hypertrophy remain unclear. Thus, the present study investigated cyclin D3 expression in skeletal muscles during early-stage functional overload. Plantaris muscles were exposed to functional overload due to ablation of the gastrocnemius and soleus muscles. As a result, cyclin D3 expression was detected in the nuclei of SCs but not in myonuclei on day 1 after surgery. Cyclin D3 expression, after functional overload, gradually increased, reaching a maximum on day 7 along with myogenin expression. Moreover, in response to the functional overload, cyclin D3 was expressed simultaneously with myogenin and p21 in SC nuclei. Therefore, the present study suggests that cyclin D3 with myogenin and p21 may interactively regulate SC differentiation during early-stage functional overload.

Bovine Skeletal Muscle Satellite Cells: Isolation, Growth, and Differentiation.

Skeletal muscle in cattle occupies a large part of the animal's body mass and develops into an important source of nutrients for human nutrition. Recently, the attention on bovine myogenic cells is increased to develop strategies of cultured in vitro meat as an alternative food source, more sustainable, ethical, and healthy than traditional meat production. At present, investigating the proliferation and differentiation of bovine skeletal muscle myogenic cells in vitro maintains its importance in the study of the mechanisms underlying the physiological and pathological events affecting the skeletal muscle, but it is of particular interest in animal husbandry and the food industry fields.In cell-based biological research, cell lines are one of the favored experimental tools because a population of cells could proliferate indefinitely in vitro under different stimuli, but they are limited to addressing the relevant biological properties of a cell population. On the other hand, primary cells from normal animal tissues undergo a limited number of divisions in vitro before they enter senescence but preserve their original characteristics and functions, and researchers can acquire the opportunity to study the individual donors and not just cells.In this chapter, we provide a basic protocol to isolate satellite cells from the skeletal muscle of cattle to obtain a good number of myogenic cells that can grow in in vitro conditions and undergo multiple rounds of cell division (myoblasts) before entering differentiation (myotubes). Furthermore, the robust expansion of these cells leads to the possibility to investigate physiological events or disorders related to the skeletal muscle tissue.

Isolation, identification, and induced differentiation of satellite cells from skeletal muscle of adult tree shrews.

A method for the in vitro isolation, purification, identification, and induced differentiation of satellite cells from adult tree shrew skeletal muscle was established. The mixed enzyme digestion method and differential adhesion method were used to obtain skeletal muscle satellite cells, which were identified and induced to differentiate to verify their pluripotency. The use of a mixture of collagenase II, hyaluronidase IV, and DNase I is an efficient method for isolating adult tree shrew skeletal muscle satellite cells. The P3 generation of cells had good morphology, rapid proliferation, high viability, and an "S"-shaped growth curve. Reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence staining indicated that marker genes or proteins were expressed in skeletal muscle satellite cells. After myogenic differentiation was induced, multiple-nucleated myotubes were observed, and the MyHC protein was expressed. The expression of myogenic marker genes changed with the differentiation process. After the induction of adipogenic differentiation, orange-red lipid droplets were observed, and the expression of adipogenic marker genes increased gradually with the differentiation process. In summary, satellite cells from adult tree shrew skeletal muscle were successfully isolated using a mixed enzyme digestion method, and their potential for differentiation into myogenic and adipogenic cells was confirmed, laying a foundation for further in vitro study of tree shrew muscle damage.