Myogenic Markers Research Articles

GLP-1RA Liraglutide and Semaglutide Improves Obesity-Induced Muscle Atrophy via SIRT1 Pathway.

Obesity is related to the loss of skeletal muscle mass and function (sarcopenia). The co-existence of obesity and sarcopenia is called sarcopenic obesity (SO). Glucagon like peptide-1 receptor agonists (GLP-1RA) are widely used in the treatment of diabetes and obesity. However, the protective effects of GLP-1RA on skeletal muscle in obesity and SO are not clear. This study investigated the effects of GLP-1RA liraglutide and semaglutide on obesity-induced muscle atrophy and explored the underlying mechanisms. Thirty-six male C57BL/6J mice were randomly divided into two groups and fed a regular diet and a high-fat diet for 18 weeks, respectively. After establishing an obesity model, mice were further divided into six groups: control group, liraglutide (LIRA) group, semaglutide (SEMA) group, high-fat diet (HFD) group, HFD + LIRA group, HFD + SEMA group, and subcutaneous injection for 4 weeks. The body weight, muscle mass, muscle strength, glycolipid metabolism, muscle atrophy markers, myogenic differentiation markers, GLUT4 and SIRT1 were analyzed. C2C12 myotube cells treated with palmitic acid (PA) were divided into four groups: control group, PA group, PA + LIRA group, PA + SEMA group. The changes in glucose uptake, myotube diameter, lipid droplet infiltration, markers of muscle atrophy, myogenic differentiation markers, GLUT4 and SIRT1 were analyzed, and the changes in related indicators were observed after the addition of SIRT1 inhibitor EX527. Liraglutide and semaglutide reduced HFD-induced body weight gain, excessive lipid accumulation and improved muscle atrophy. Liraglutide and semaglutide eliminated the increase of muscle atrophy markers in skeletal muscle and C2C12 myotubes. Liraglutide and semaglutide restored impaired glucose tolerance and insulin resistance. However, these beneficial effects were attenuated by inhibiting SIRT1 expression. Liraglutide and semaglutide protects skeletal muscle against obesity-induced muscle atrophy via the SIRT1 pathway.

Primary Cutaneous Epithelioid Mesenchymal Tumor With a Novel ATP2B4::GLI1 Gene Fusion.

GLI1 gene alterations (rearrangement or amplification) have been found in several bone and soft tissue tumors including pericytic tumors, gastric plexiform fibromyxoma, gastroblastoma, and a various group of epithelioid tumors with regional recurrence or distant metastasis. In this article, we describe a case of primary cutaneous epithelioid mesenchymal tumor harboring hitherto not reported ATP2B4::GLI1 gene fusion. A 42-year-old man presented with a growing firm lesion on the left postauricular scalp. Microscopically, the shave biopsy specimen revealed a dermal-based nodular proliferation of relatively monotonous epithelioid cells with round to ovoid nuclei and pale eosinophilic cytoplasm, accompanied by prominent stromal vasculature. Significant cytologic atypia, necrosis, and mitotic activity were absent. The tumor cells were partially positive for CD34 and S-100 protein, but were negative for other markers, including SOX-10, keratins, and myogenic markers. An ATP2B4::GLI1 gene fusion was identified by next-generation sequencing. Array CGH was also performed, but it did not show relevant chromosomal copy number changes. Awareness of this rare cutaneous tumor, and thus, reporting of additional cases is necessary for further delineating its full clinicopathologic spectrum.

Kinin receptors regulate skeletal muscle regeneration: differential effects for B1 and B2 receptors.

After traumatic skeletal muscle injury, muscle healing is often incomplete and produces extensive fibrosis. Bradykinin (BK) reduces fibrosis in renal and cardiac damage models through the B2 receptor. The B1 receptor expression is induced by damage, and blocking of the kallikrein-kinin system seems to affect the progression of muscular dystrophy. We hypothesized that both kinin B1 and B2 receptors could play a differential role after traumatic muscle injury, and the lack of the B1 receptor could produce more cellular and molecular substrates for myogenesis and fewer substrates for fibrosis, leading to better muscle healing. To test this hypothesis, tibialis anterior muscles of kinin receptor knockout animals were subjected to traumatic injury. Myogenesis, angiogenesis, fibrosis, and muscle functioning were evaluated. Injured B1KO mice showed a faster healing progression of the injured area with a larger amount of central nucleated fiber post-injury when compared to control mice. In addition, they exhibited higher neovasculogenic capacity, maintaining optimal tissue perfusion for the post-injury phase; had higher amounts of myogenic markers with less inflammatory infiltrate and tissue destruction. This was followed by higher amounts of SMAD7 and lower amounts of p-SMAD2/3, which resulted in less fibrosis. In contrast, B2KO and B1B2KO mice showed more severe tissue destruction and excessive fibrosis. B1KO animals had better results in post-injury functional tests compared to control animals. We demonstrate that injured skeletal muscle tissues have a better repair capacity with less fibrosis in the presence of B2 receptor and absence of B1 receptor, including better performances in functional tests.

Ascorbic acid reduces Ropivacaine-induced myotoxicity in cultured human osteoporotic skeletal muscle cells

BackgroundOsteoporosis is a worldwide health issue. Loss of bone mass is a potential risk factor for fragility fractures, and osteoporotic fractures place a considerable burden on society. Bone and muscle represent a functional unit in which the two tissues are intimately interconnected. Ropivacaine is a potent local anesthetic used in clinical practice for intraoperative anesthesia and postoperative pain management, in particular for hip surgery. When injected, Ropivacaine can diffuse locally through, in particular in surrounding skeletal muscle tissue, causing dose-dependent cytotoxicity, oxidative stress and myogenesis impairment. Based on those evidences, we focused our attention on Ropivacaine-induced cytotoxicity on cultured human myoblasts.MethodsPrimary human myoblasts and myotubes from healthy subjects, osteoarthritic and osteoporotic patients (OP) were cultured in the presence of Ropivacaine. In some experiments, ascorbic acid (AsA) was added as a potent antioxidant agent. Cell viability and ROS levels were evaluated to investigate the myotoxic activity and Real-Time PCR and Western blot analysis carried out to investigate the expression of proliferation and myogenic markers.ResultsA dose-dependent decrease of cell viability was observed after Ropivacaine exposure in both OP myoblasts and myotubes cultures, whereas those effects were not observed in the presence of Propofol, a general anesthetic. The adding of AsA reduced Ropivacaine negative effects in OP myoblast cultures. In addition, Ropivacaine exposure also increased ROS levels and upregulated Nox4 expression, an enzyme primarily implicated in skeletal muscle ROS generation. AsA treatment counteracted the oxidant activity of Ropivacaine and partially restored the basal condition in cultures. Positive myogenic markers, such as MyoD and Myf5, were downregulated by Ropivacaine exposure, whereas myostatin, a negative regulator of muscle growth and differentiation, was upregulated. The phenotypic deregulation of myogenic controllers in the presence of Ropivacaine was counteracted by AsA treatment.ConclusionsOur findings highlight the oxidative stress-mediated myotoxic effect of Ropivacaine on human skeletal muscle tissue cell cultures, and suggest treatment with AsA as valid strategy to mitigate its negative effects and allowing an ameliorated functional skeletal muscle recovery in patients undergoing hip replacement surgery for osteoporotic bone fracture.

Senescent adipocytes as potential effectors of muscle cells dysfunction: An in vitro model

Recently, there has been a growing body of evidence showing a negative effect of the white adipose tissue (WAT) dysfunction on the skeletal muscle function and quality. However, little is known about the effects of senescent adipocytes on muscle cells.Therefore, to explore potential mechanisms involved in age-related loss of muscle mass and function, we performed an in vitro experiment using conditioned medium obtained from cultures of mature and aged 3 T3-L1 adipocytes, as well as from cultures of dysfunctional adipocytes exposed to oxidative stress or high insulin doses, to treat C2C12 myocytes.The results from morphological measures indicated a significant decrease in diameter and fusion index of myotubes after treatment with medium of aged or stressed adipocytes. Aged and stressed adipocytes presented different morphological characteristics as well as a different gene expression profile of proinflammatory cytokines and ROS production. In myocytes treated with different adipocytes' conditioned media, we demonstrated a significant reduction of gene expression of myogenic differentiation markers as well as a significant increase of genes involved in atrophy. Finally, a significant reduction in protein synthesis as well as a significant increase of myostatin was found in muscle cells treated with medium of aged or stressed adipocytes compared to controls.In conclusion, these preliminary results suggest that aged adipocytes could influence negatively trophism, function and regenerative capacity of myocytes by a paracrine network of signaling.

Clinical, morphological, and immunohistochemical analysis of head and neck rhabdomyosarcomas in pediatric patients

Objective To analyze the clinicopathologic and immunohistochemical features of head and neck rhabdomyosarcomas (HNRMS) in pediatric patients from Brazil, Guatemala, Mexico, and South Africa. Study Design Thirty-eight pediatric HNRMS were obtained from six Oral Pathology services in 23 years. Clinicopathological and immunohistochemical data of all cases were reviewed. Results Most patients were from Brazil and South Africa (12/38; 31.6%, each), ten (26.3%) were from Guatemala, and four (10.5%) were from Mexico. The mean age of patients was 7.6 ± 4.7 years (range 1-19 years) with a slight female predilection. Non-parameningeal tissues (47.4%) were most commonly affected, followed by parameningeal (36.8%) and orbit (13.2%). Microscopically, the most common subtype was embryonal 86.8% (33/38), followed by alveolar 13.2% (5/38). Almost all cases (92.1%; 35/38) were positive for at least two myogenic markers: Desmin, Myogenin, and Myo-D1. Moderate to high Ki-67 proliferation index was identified in most cases. Conclusion Pediatric HNRMS from Brazil, Guatemala, Mexico, and South Africa are more frequently diagnosed in children <10 years old and present classic clinicopathologic and immunohistochemical features for each subtype. To analyze the clinicopathologic and immunohistochemical features of head and neck rhabdomyosarcomas (HNRMS) in pediatric patients from Brazil, Guatemala, Mexico, and South Africa. Thirty-eight pediatric HNRMS were obtained from six Oral Pathology services in 23 years. Clinicopathological and immunohistochemical data of all cases were reviewed. Most patients were from Brazil and South Africa (12/38; 31.6%, each), ten (26.3%) were from Guatemala, and four (10.5%) were from Mexico. The mean age of patients was 7.6 ± 4.7 years (range 1-19 years) with a slight female predilection. Non-parameningeal tissues (47.4%) were most commonly affected, followed by parameningeal (36.8%) and orbit (13.2%). Microscopically, the most common subtype was embryonal 86.8% (33/38), followed by alveolar 13.2% (5/38). Almost all cases (92.1%; 35/38) were positive for at least two myogenic markers: Desmin, Myogenin, and Myo-D1. Moderate to high Ki-67 proliferation index was identified in most cases. Pediatric HNRMS from Brazil, Guatemala, Mexico, and South Africa are more frequently diagnosed in children <10 years old and present classic clinicopathologic and immunohistochemical features for each subtype.

Ginsenoside Rg5 promotes muscle regeneration via p38MAPK and Akt/mTOR signaling

BackgroundSkeletal muscles play a key role in physical activity and energy metabolism. The loss of skeletal muscle mass can cause problems related to metabolism and physical activity. Studies are being conducted to prevent such diseases by increasing the mass and regeneration capacity of muscles. Ginsenoside Rg5 has been reported to exhibit a broad range of pharmacological activities. However, studies on the effects of Rg5 on muscle differentiation and growth are scarce. MethodsTo investigate the effects of Rg5 on myogenesis, C2C12 myoblasts were induced to differentiate with Rg5, followed by immunoblotting, immunostaining, and qRT-PCR for myogenic markers and promyogenic signaling (p38MAPK). Immunoprecipitation confirmed that Rg5 increased the interaction between MyoD and E2A via p38MAPK. To investigate the effects of Rg5 on prevention of muscle mass loss, C2C12 myotubes were treated with dexamethasone to induce muscle atrophy. Immunoblotting, immunostaining, and qRT-PCR were performed for myogenic markers, Akt/mTOR signaling for protein synthesis, and atrophy-related genes (Atrogin-1 and MuRF1). ResultsRg5 promoted C2C12 myoblast differentiation through phosphorylation of p38MAPK and MyoD/E2A heterodimerization. Furthermore, Rg5 stimulated C2C12 myotube hypertrophy via phosphorylation of Akt/mTOR. Phosphorylation of Akt induces FoxO3a phosphorylation, which reduces the expression of Atrogin-1 and MuRF1. ConclusionThis study provides an understanding of how Rg5 promotes myogenesis and hypertrophy and prevents dexamethasone-induced muscle atrophy. The study is the first, to the best of our knowledge, to show that Rg5 promotes muscle regeneration and to suggest that Rg5 can be used for therapeutic intervention of muscle weakness and atrophy, including cancer cachexia.

Comprehensive metabolomic analysis of Mangifera indica leaves using UPLC-ESI-Q-TOF-MSE for cell differentiation: An in vitro and in vivo study

The comprehensive metabolic profiling was performed in the leaf extracts of Mangifera indica and assessed for their significant therapeutic application in tissue engineering and regenerative medicine in both in vitro and in vivo studies. About 147 compounds were identified in the ethyl acetate and methanol extracts of M. indica using MS/MS fragmentation analysis and the selected compounds were quantified using LC-QqQ-MS analysis. The in vitro cytotoxic activity showed that the M. indica extracts enhance the proliferation of mouse myoblast cells in concentration-dependent manner. As well, the extracts of M. indica induce the myotube formation by generating oxidative stress in the C2C12 cells was confirmed. The western blot analysis clearly showed that the M. indica induce myogenic differentiation by upregulating the myogenic marker proteins such as PI3K, Akt, mTOR, MyoG, and MyoD. The in vivo studies showed that the extracts expedites the acute wound repair by formation of crust, wound closure and improves the blood perfusion towards the wound area. Together, the leaves of M. indica can be used as excellent therapeutic agent for tissue repair and wound healing applications.

Effects of energy drinks on myogenic differentiation of murine C2C12 myoblasts

Energy drinks, often advertised as dietary supplements that enhance physical and mental performance, have gained increasing popularity among adolescents and athletes. Several studies on individual ingredients such as caffeine or taurine have reported either adverse or favorable influences on myogenic differentiation, a key process in muscle regeneration to repair microtears after an intense workout session. However, the impact of different energy drinks with various formulas on muscle differentiation has never been reported. This study aims to examine the in vitro effects of various energy drink brands on myogenic differentiation. Murine C2C12 myoblast cells were induced to differentiate into myotubes in the presence of one of eight energy drinks at varying dilutions. A dose-dependent inhibition of myotube formation was observed for all energy drinks, supported by reduced percentage of MHC-positive nuclei and fusion index. Moreover, expression of myogenic regulatory factor MyoG and differentiation marker MCK were also decreased. Furthermore, given the variation in formulas of different energy drinks, there were remarkable differences in the differentiation and fusion of myotubes between energy drinks. This is the first study to investigate the impact of various energy drinks on myogenic differentiation and our results suggest an inhibitory effect of energy drinks in muscle regeneration.

MiR-196b-5p promotes myoblast proliferation and differentiation.

MicroRNAs (miRNAs) are a class of non-coding single-stranded RNA molecules about 22 nucleotides in length and are encoded by endogenous genes, and are involved in the regulation of post-transcriptional gene expression in animals and plants. Many studies have shown that microRNAs regulate the development of skeletal muscle, mainly manifested in the activation of muscle satellite cells and biological processes such as proliferation, differentiation, and formation of muscle tubes. In this study, miRNA sequencing screening of longissimus dorsi (LD, mainly fast-twitch fibers) and soleus muscle (Sol, dominated by slow-twitch fibers) identified the miR-196b-5p as a differentially expressed and highly conserved sequence in different skeletal muscles. Studies of miR-196b-5p in skeletal muscle have not been reported. In this study, miR-196b-5p mimics and inhibitor were used in miR-196b-5p overexpression and interference experiments in C2C12 cells. The effect of miR-196b-5p on myoblast proliferation and differentiation was analyzed by western blotting, real-time quantitative RT-PCR, flow cytometry, immunofluorescence staining, and the target gene of miR-196b-5p was identified by bioinformatics prediction and analyzed by dual luciferase reporter assays. The results showed that overexpression of miR-196b-5p could significantly increase the mRNA and protein expression of Cyclin B, Cyclin D and Cyclin E (P<0.05); Cell cycle analysis showed that overexpression of miR-196b-5p significantly increased the proportion of cells in the S phase (P<0.05), indicating that miR-196b-5p could accelerate cell cycle progress. Results of EdU staining showed that overexpression of miR-196b-5p significantly promoted cell proliferation. Conversely, inhibition of miR-196b-5p expression could significantly reduce the proliferation capacity of myoblasts. Further, overexpression of miR-196b-5p could significantly increase the expression levels of myogenic marker genes MyoD, MoyG and MyHC (P<0.05), thereby promoting myoblast fusion and accelerating C2C12 cell differentiation. Bioinformatics predictions and dual luciferase experiments demonstrated that miR-196b-5p could target and inhibit the expression of the Sirt1 gene. Altering the Sirt1 expression could not rescue the effects of miR-196b-5p on the cell cycle, but could weaken the promoting effects of miR-196b-5p on myoblast differentiation, suggesting that miR-196b-5p promoted myoblast differentiation by targeting Sirt1.

Lin28a maintains a subset of adult muscle stem cells in an embryonic-like state.

During homeostasis and after injury, adult muscle stem cells (MuSCs) activate to mediate muscle regeneration. However, much remains unclear regarding the heterogeneous capacity of MuSCs for self-renewal and regeneration. Here, we show that Lin28a is expressed in embryonic limb bud muscle progenitors, and that a rare reserve subset of Lin28a+Pax7- skeletal MuSCs can respond to injury at adult stage by replenishing the Pax7+ MuSC pool to drive muscle regeneration. Compared with adult Pax7+ MuSCs, Lin28a+ MuSCs displayed enhanced myogenic potency in vitro and in vivo upon transplantation. The epigenome of adult Lin28a+ MuSCs showed resemblance to embryonic muscle progenitors. In addition, RNA-sequencing revealed that Lin28a+ MuSCs co-expressed higher levels of certain embryonic limb bud transcription factors, telomerase components and the p53 inhibitor Mdm4, and lower levels of myogenic differentiation markers compared to adult Pax7+ MuSCs, resulting in enhanced self-renewal and stress-response signatures. Functionally, conditional ablation and induction of Lin28a+ MuSCs in adult mice revealed that these cells are necessary and sufficient for efficient muscle regeneration. Together, our findings connect the embryonic factor Lin28a to adult stem cell self-renewal and juvenile regeneration.

Complete mimicry: Rhabdomyosarcoma with FUS::TFCP2 fusion masquerading as carcinoma-diagnostic challenge and report of two cases.

Rhabdomyosarcomas (RMS) are malignant mesenchymal tumors with skeletal muscle differentiation which are classified into alveolar, embryonal, pleomorphic, and spindle cell/sclerosing RMS. Within the spindle cell/sclerosing RMS tumor type there is a recently recognized sub-type categorized as intraosseous spindle cell RMS with TFCP2/NCOA2 gene fusion. This rare tumor is highly aggressive with predominant involvement of the craniofacial and pelvic bones with approximately 30 cases reported to date. Histopathologic features include spindle cell and epithelioid morphology with a characteristic co-expression of epithelial markers, myogenic markers, and ALK1 expression. We report two cases of gnathic spindle cell/sclerosing RMS with FUS::TFCP2 gene fusion that were initially interpreted as carcinomas by referring institutions and later reclassified when encountered in our practice after additional work-up and molecular characterization.

Scanning electrochemical microscopy for the stimulation and investigation of human skeletal muscle-derived mesenchymal stem/stromal cells

IntroductionThe human skeletal muscle-derived mesenchymal stem/stromal cells (SM-MSCs) possess myogenic differentiation potential and participate in the muscle regeneration process. However, the question of how to improve human skeletal muscle regeneration remains actual. In this study, the total SM-MSCs population was separated into subpopulations according to the neural cell adhesion molecule (NCAM, CD56), stimulated with an alternating electric field (AC) using scanning electrochemical microscopy (AC-SECM) and the intracellular redox changes, and myogenic differentiation markers were evaluated. MethodsThe total SM-MSC population was isolated from the postoperative human skeletal muscle biopsies by an enzymatic digestion method. The myogenic differentiation of the total SM-MSCs population before and after AC stimulation was evaluated immunohistochemically by the levels of desmin and myogenin. The effect of AC stimulation on the redox capacity of CD56(+) and CD56(-) cell subpopulations as well as on the level of myogenin on indium tin oxide (ITO) surface were also investigated. ResultsThe total SM-MSCs population grown on a glass coverslip weakly responded to AC stimulus, i.e. the level of desmin was slightly increased by the 3rd day of differentiation, while in the not stimulated cells, it increased only by the 7th day. The level of myogenin did not change after AC stimulation. However, the CD56(+) and CD56(-) subpopulations had different redox activities and myogenic differentiation potential: the CD56(+) cells had stronger natural diffusion and were more redox active compared to the CD56(-) cells; the redox activity of CD56(+) cells was more actively stimulated by an alternating electric field than in CD56(-) cells; at control level, the CD56(+) cells had more myogenic differentiation-regulating transcription factor myogenin, which was more intensively stimulated by AC than in CD56(-) cells. Data show that the total population of human SM-MSCs is heterogeneous with different regenerating potential cells that do not equally respond to extracellular stimuli. The SECM can be used in both ways: (i) for extracellular stimulation and (ii) for the investigation of intracellular redox changes of the human SM-MSCs or their subpopulations allowing a deeper understanding of the mechanisms mediating skeletal tissue regeneration both in vitro and in vivo.

Top 10 Significant Spindled Head and Neck Lesions to Scrutinze.

Spindled lesions are a challenging area in head and neck pathology. This is particularly true in the sinonasal tract, where several uncommon entities with both unique and overlapping morphologic, immunophenotypic, and/or molecular features can occur. Review. The clinicopathologic characteristics of biphenotypic sinonasal sarcoma and nine important differential diagnostic considerations with one or more overlapping feature are summarized to establish a practical framework for approaching spindled lesions of the sinonasal tract. Morphologic evaluation is central to the work up of sinonasal spindle cell lesions-in particular, cellular morphology, tumor architecture and growth pattern, and the presence of admixed epithelial elements - however, focused immunohistochemical analysis of neural, myogenic, rhabdomyoblastic, epithelial, and/or melanocytic marker expression and/or ancillary tests for tumor-specific molecular alterations may be necessary for definitive diagnosis.

MiR-424(322)-5p targets Ezh1 to inhibit the proliferation and differentiation of myoblasts.

The proliferation and differentiation of myoblasts are considered the key biological processes in muscle development and muscle-related diseases, in which the miRNAs involved remain incompletely understood. Previous research reported that miR-424(322)-5p is highly expressed in mouse skeletal muscle. Therefore, C2C12 cells are used as a model to clarify the effect of miR-424(322)-5p on the proliferation and differentiation of myoblasts. The data show that miR-424(322)-5p exhibits a decreasing trend upon myogenic differentiation. Overexpression of miR-424(322)-5p inhibits the proliferation of myoblasts, manifested by downregulation of proliferation marker genes ( CCNB1, CCND2, and CDK4), decreased percentage of EdU + cells, and reduced cell viability. In contrast, these phenotypes are promoted in myoblasts treated with an inhibitor of miR-424(322)-5p. Interestingly, its gain of function inhibits the expression of myogenic regulators, including MyoD, MyoG, MyHC, and Myf5. Additionally, immunofluorescence staining of MyHC and MyoD shows that overexpression of miR-424(322)-5p reduces the number of myotubes and decreases the myotube fusion index. Consistently, inhibition of its function mediated by an inhibitor promotes the expressions of myogenic markers and myotube fusion. Mechanistically, gene prediction and dual-luciferase reporter experiments confirm that enhancer of zeste homolog 1 ( Ezh1) is one of the targets of miR-424(322)-5p. Furthermore, knockdown of Ezh1 inhibits the proliferation and differentiation of myoblasts. Compared with NC and inhibitor treatment, inhibitor+si- EZH1 treatment rescues the phenotypes of proliferation and differentiation mediated by the miR-424(322)-5p inhibitor. Taken together, these data indicate that miR-424(322)-5p targets Ezh1 to negatively regulate the proliferation and differentiation of myoblasts.

Electrospun Poly(L-lactide-co-ε-caprolactone) Scaffold Potentiates C2C12 Myoblast Bioactivity and Acts as a Stimulus for Cell Commitment in Skeletal Muscle Myogenesis.

Tissue engineering combines a scaffold, cells and regulatory signals, reproducing a biomimetic extracellular matrix capable of supporting cell attachment and proliferation. We examined the role of an electrospun scaffold made of a biocompatible polymer during the myogenesis of skeletal muscle (SKM) as an alternative approach to tissue regeneration. The engineered nanostructure was obtained by electrospinning poly(L-lactide-co-ε-caprolactone) (PLCL) in the form of a 3D porous nanofibrous scaffold further coated with collagen. C2C12 were cultured on the PLCL scaffold, and cell morphology and differentiation pathways were thoroughly investigated. The functionalized PLCL scaffold recreated the SKM nanostructure and performed its biological functions, guiding myoblast morphogenesis and promoting cell differentiation until tissue formation. The scaffold enabled cell-cell interactions through the development of cellular adhesions that were fundamental during myoblast fusion and myotube formation. Expression of myogenic regulatory markers and muscle-specific proteins at different stages of myogenesis suggested that the PLCL scaffold enhanced myoblast differentiation within a shorter time frame. The functionalized PLCL scaffold impacts myoblast bioactivity and acts as a stimulus for cell commitment, surpassing traditional 2D cell culture techniques. We developed a screening model for tissue development and a device for tissue restoration.

In vitro myogenesis activation of specific muscle-derived stem cells from patients with Duchenne muscular dystrophy

BackgroundMuscle-derived stem cells (MDSCs) contribute to the repair of injured muscles. However, the myogenicity of MDSCs generated from patients with Duchenne muscular dystrophy (DMD) relative to healthy individuals remains unclear. MethodsA human DMD model was established using the stem cells prepared from muscle derived from patients with DMD (DMD-hMDSCs). The expression of myogenic lineage-specific markers in MDSCs was examined with immunofluorescence, real-time polymerase chain reaction, and western blotting. ResultsIt was demonstrated that, compared with cells from healthy subjects, DMD-hMDSCs are primed to self-differentiate in growth-inducing medium (GM) and robustly differentiate into myotubes in differentiation-inducing medium(DM). This feature was termed “myogenesis activation,” and it was speculated that it contributes to the depletion of myogenic progenitors. Furthermore, MDSCs consistently express pax7, but the time-course of this expression does not correlate with the expression of the myogenic lineage-specific markers. ConclusionsThe myogenesis activation in DMD-hMDSCs demonstrated in this study may provide novel mechanistic insights into DMD pathogenesis and potential therapies.

TRNA-derived small RNA, 5'tiRNA-Gly-CCC, promotes skeletal muscle regeneration through the inflammatory response.

Increasing evidence shows that tRNA-derived small RNAs (tsRNAs) are not only by-products of transfer RNAs, but they participate in numerous cellular metabolic processes. However, the role of tsRNAs in skeletal muscle regeneration remains unknown. Small RNA sequencing revealed the relationship between tsRNAs and skeletal muscle injury. The dynamic expression level of 5'tiRNA-Gly after muscle injury was confirmed by real-time quantitative PCR (q-PCR). In addition, q-PCR, flow cytometry, the 5-ethynyl-2'-deoxyuridine (Edu), cell counting kit-8, western blotting and immunofluorescence were used to explore the biological function of 5'tiRNA-Gly. Bioinformatics analysis and dual-luciferase reporter assay were used to further explore the mechanism of action under the biological function of 5'tiRNA-Gly. Transcriptome analysis revealed that tsRNAs were significantly enriched during inflammatory response immediately after muscle injury. Interestingly, we found that 5'tiRNA-Gly was significantly up-regulated after muscle injury (P<0.0001) and had a strong positive correlation with inflammation in vivo. In vitro experiments showed that 5'tiRNA-Gly promoted the mRNA expression of proinflammatory cytokines (IL-1β, P=0.0468; IL-6, P=0.0369) and the macrophages of M1 markers (TNF-α, P=0.0102; CD80, P=0.0056; MCP-1, P=0.0002). On the contrary, 5'tiRNA-Gly inhibited the mRNA expression of anti-inflammatory cytokines (IL-4, P=0.0009; IL-10, P=0.0007; IL-13, P=0.0008) and the mRNA expression of M2 markers (TGF-β1, P=0.0016; ARG1, P=0.0083). Flow cytometry showed that 5'tiRNA-Gly promoted the percentage of CD86+ macrophages (16%, P=0.011) but inhibited that of CD206+ macrophages (10.5%, P=0.012). Immunofluorescence showed that knockdown of 5'tiRNA-Gly increased the infiltration of M2 macrophages to the skeletal muscles (13.9%, P=0.0023) and inhibited the expression of Pax7 (P=0.0089) in vivo. 5'tiRNA-Gly promoted myoblast the expression of myogenic differentiation marker genes (MyoD, P=0.0002; MyoG, P=0.0037) and myotube formation (21.3%, P=0.0016) but inhibited the positive rate of Edu (27.7%, P=0.0001), cell viability (22.6%, P=0.003) and the number of myoblasts in the G2 phase (26.3%, P=0.0016) in vitro. Mechanistically, we found that the Tgfbr1 gene is a direct target of 5'tiRNA-Gly mediated by AGO1 and AGO3. 5'tiRNA-Gly dysregulated the expression of downstream genes related to inflammatory response, activation of satellite cells and differentiation of myoblasts through the TGF-β signalling pathway by targeting Tgfbr1. These results reveal that 5'tiRNA-Gly potentially regulated skeletal muscle regeneration by inducing inflammation via the TGF-β signalling pathway. The findings of this study uncover a new potential target for skeletal muscle regeneration treatment.

Effect of p38 inhibitor on the proliferation of chicken muscle stem cells and differentiation into muscle and fat

Inhibiting the p38 mitogen-activated protein kinase (MAPK) signaling pathway delays differentiation and increases proliferation of muscle stem cells in most species. Here, we aimed to investigate the effect of p38 inhibitor (p38i) treatment on the proliferation and differentiation of chicken muscle stem cells. Chicken muscle stem cells were collected from the muscle tissues of Hy-line Brown chicken embryos at embryonic day 18, then isolated by the preplating method. Cells were cultured for 4 days in growth medium supplemented with dimethyl sulfoxide or 1, 10, 20 μM of p38i, then subcultured for up to 4 passages. Differentiation was induced for 3 days with differentiation medium. Each treatment was replicated 3 times. The proliferation and mRNA expression of paired box 7 gene and myogenic factor 5 gene, as well as the mRNA expression of myogenic differentiation marker gene myogenin were significantly higher in p38i-treated cultures than in control (p<0.05), but immunofluorescence staining and mRNA expression of myosin heavy chain (MHC) were not significantly different between the two groups. Oil red O staining of accumulated lipid droplets in differentiated cell cultures revealed a higher lipid density in p38i-treated cultures than in control; however, the expression of the adipogenic marker gene peroxisome proliferator activated receptor gamma was not significantly different between the two groups. p38 inhibition in chicken muscle stem cells improves cell proliferation, but the effects on myogenic differentiation and lipid accumulation require additional analysis. Further studies are needed on the chicken p38-MAPK pathway to understand the muscle and fat development mechanism.

Nuclear factor 1 X-type-associated regulation of myogenesis in developing mouse tongue

ObjectivesThe tongue contains skeletal myofibers that differ from those in the trunk, limbs, and other orofacial muscles. However, the molecular basis of myogenic differentiation in the tongue muscles remains unclear. In this study, we conducted comprehensive gene expression profiling of the developing murine tongue. MethodsTongue primordia were dissected from mouse embryos at embryonic day (E)10.5–E18.5, while myogenic markers were detected via microarray analysis and quantitative polymerase chain reaction (PCR). In addition to common myogenic regulatory factors such as Myf5, MyoD, myogenin, and Mrf4, we focused on Nfix, which acts as a unique molecular switch triggering the shift from embryonic to fetal myoblast lineage during limb myogenesis. Nfix inhibition was performed using a specific antisense oligonucleotide in the organ culture of tongue primordia. ResultsMicroarray and ingenuity pathway analyses confirmed the significant upregulation of myogenic signaling molecules, including Nfix, associated with the differentiation of myoblasts from myogenic progenitor cells during E10.5–E11.5. Quantitative PCR confirmed that Nfix expression started at E10.5 and peaked at E14.5. Fetal myoblast-specific genes, such as Mck and Myh8, were upregulated after E14.5, whereas embryonic myoblast-specific genes, such as Myh3 and Myh7, were downregulated. When Nfix was inhibited in the organ culture of tongue primordia, subtle morphological differences were noted in the tongue. Such an observation was only noted in the cultures of E10.5-derived tongue primordia. ConclusionsThese results reveal the contribution of Nfix to tongue myogenesis. Nfix expression during early tongue development may play a vital role in tongue muscle development.