Myogenic Precursor Research Articles

Differential effects of metformin on reductive activity and energy production in pituitary tumor cells compared to myogenic precursors.

Given the multiple targets of metformin within cells, the mechanism by which it may exert a growth-inhibitory action on pituitary tumor cells in vitro remains to be explored. Previous research stressed metformin-induced changes in the activity of signaling pathways regulating cell growth and cell death. In this work, we investigated the effects of metformin on cell viability markers related to cell metabolic activity in rat pituitary tumor cells versus rat myogenic precursors as a model of normal proliferating somatic cells. We designed our experiments in order to use the MTT reduction as a marker of cellular reductive activity and the total cellular ATP levels as a marker of energy supply during short incubations with different metabolic substrates (sodium pyruvate, D-glucose, L-glutamine, sodium citrate). Then, we extended the analysis to extracellular glucose consumption, extracellular medium acidification and pyruvate dehydrogenase (PDH) complex activity. Metformin was found to be effective in both cell types at the same concentrations, although the outcome of the treatment was quite the opposite. Unexpectedly, metformin increased the viability of subconfluent rat myoblasts. Rat pituitary tumor cells and myoblasts differed in the utilization of distinct metabolic substrates and the PDH complex activity. Metformin actions on reductive activity and ATP production were substrate-dependent. Overall, this work points out that metformin actions at the cellular level depend on metabolic features and metabolic requirements of cells. The pyruvate metabolic branch point is most likely to play a main role in the variability of cell response to metformin.

Generation of craniofacial myogenic progenitor cells from human induced pluripotent stem cells for skeletal muscle tissue regeneration

Craniofacial skeletal muscle is composed of approximately 60 muscles, which have critical functions including food uptake, eye movements and facial expressions. Although craniofacial muscles have significantly different embryonic origin, most current skeletal muscle differentiation protocols using human induced pluripotent stem cells (iPSCs) are based on somite-derived limb and trunk muscle developmental pathways. Since the lack of a protocol for craniofacial muscles is a significant gap in the iPSC-derived muscle field, we have developed an optimized protocol to generate craniofacial myogenic precursor cells (cMPCs) from human iPSCs by mimicking key signaling pathways during craniofacial embryonic myogenesis. At each different stage, human iPSC-derived cMPCs mirror the transcription factor expression profiles seen in their counterparts during embryo development. After the bi-potential cranial pharyngeal mesoderm is established, cells are committed to cranial skeletal muscle lineages with inhibition of cardiac lineages and are purified by flow cytometry. Furthermore, identities of iPSC-derived cMPCs are verified with human primary myoblasts from craniofacial muscles using RNA sequencing. These data suggest that our new method could provide not only in vitro research tools to study muscle specificity of muscular dystrophy but also abundant and reliable cellular resources for tissue engineering to support craniofacial reconstruction surgery.

Role of Mitochondrial Calcium in the Maintenance of Skeletal Muscle Homeostasis

Calcium accumulates into the mitochondrial matrix through the mitochondrial calcium uniporter (MCU). Few years ago a MCU homolog has been discovered, which has been called MCUb. MCU and MCUb share a 50% sequence and structure similarity although some conserved differences in the primary sequence prevent MCUb from forming a Ca2+‐permeable channel. Interestingly, MCUb/MCU expression ratio varies greatly among tissues, suggesting that it might contribute to the spatiotemporal control of mitochondrial calcium uptake. We found that MCUb is highly expressed during skeletal muscle regeneration, a process that is mainly regulated by phenotypic skewing of tissue resident and freshly recruited macrophages. We have demonstrated that MCUb expression is specifically induced in macrophages, the most abundant cell population in a regenerating muscle that drive both the proliferation and differentiation of the muscle stem cells, satellite cells, thus affecting the progression of healing after muscle damage. In vitro and in vivo experiments demonstrated that the absence of MCU affects macrophages skewing from a pro (M1) to an anti‐inflammatory profile (M2). Indeed, macrophages of mice lacking MCUb are more prone to acquire a pro‐inflammatory M1 profile. Since M2 macrophages have a pro‐regenerative role and promote myogenic precursor cell differentiation, we hypothesized that MCUb absence, by affecting M2 polarization, might influence the correct remodeling and reorganization of skeletal muscle structure after damage. Our results clearly show that regenerating muscles lacking MCUb show a decrease in the expression level of myogenic regulatory transcription factors involved in satellite cells activation and differentiation. We also observed a decrease in the percentage of regenerating myofibers and a decrease in collagen deposition. In addition, co‐culture experiments demonstrated that macrophage‐conditioned medium from M2 MCUb KO macrophages drastically decreases satellite cell differentiation. Altogether these results demonstrate the role of mitochondrial Ca2+ uptake in promoting the polarization of macrophages towards an M2 pro‐regenerative profile during skeletal muscle regeneration.Moreover, we found that skeletal muscle mitochondria express a unique MCU complex containing an alternative splice isoform of MICU1, MICU1.1, characterized by the addition of a micro‐exon that is sufficient to greatly modify the properties of the MCU. Indeed, MICU1.1 binds Ca2+ one order magnitude more efficiently than MICU1. In skeletal muscle in vivo, MICU1.1 is required for sustained mitochondrial Ca2+ uptake and ATP production. Furthermore, we observed that the MICU1 forced expression in skeletal muscle not only affects energy production but also influences muscle trophism. These results highlight a novel mechanism of the molecular plasticity of the MCU Ca2+ uptake machinery and suggest a previously unexplored pathogenic mechanism in skeletal muscle diseases.Support or Funding InformationThis work is supported by the Italian Telethon ONLUS Foundation (Rome, Italy): GGP16026 to AR and the Italian Ministry of Health (Ricerca Finalizzata) (GR‐2016‐02362779 to AR).

Serial Xenotransplantation in NSG Mice Promotes a Hybrid Epithelial/Mesenchymal Gene Expression Signature and Stemness in Rhabdomyosarcoma Cells.

Serial xenotransplantation of sorted cancer cells in immunodeficient mice remains the most complex test of cancer stem cell (CSC) phenotype. However, we have demonstrated in various sarcomas that putative CSC surface markers fail to identify CSCs, thereby impeding the isolation of CSCs for subsequent analyses. Here, we utilized serial xenotransplantation of unsorted rhabdomyosarcoma cells in NOD/SCID gamma (NSG) mice as a proof-of-principle platform to investigate the molecular signature of CSCs. Indeed, serial xenotransplantation steadily enriched for rhabdomyosarcoma stem-like cells characterized by enhanced aldehyde dehydrogenase activity and increased colony and sphere formation capacity in vitro. Although the expression of core pluripotency factors (SOX2, OCT4, NANOG) and common CSC markers (CD133, ABCG2, nestin) was maintained over the passages in mice, gene expression profiling revealed gradual changes in several stemness regulators and genes linked with undifferentiated myogenic precursors, e.g., SOX4, PAX3, MIR145, and CDH15. Moreover, we identified the induction of a hybrid epithelial/mesenchymal gene expression signature that was associated with the increase in CSC number. In total, 60 genes related to epithelial or mesenchymal traits were significantly altered upon serial xenotransplantation. In silico survival analysis based on the identified potential stemness-associated genes demonstrated that serial xenotransplantation of unsorted rhabdomyosarcoma cells in NSG mice might be a useful tool for the unbiased enrichment of CSCs and the identification of novel CSC-specific targets. Using this approach, we provide evidence for a recently proposed link between the hybrid epithelial/mesenchymal phenotype and cancer stemness.

Differentiation of the human PAX7-positive myogenic precursors/satellite cell lineage in vitro.

Satellite cells (SC) are muscle stem cells that can regenerate adult muscles upon injury. Most SC originate from PAX7+ myogenic precursors set aside during development. Although myogenesis has been studied in mouse and chicken embryos, little is known about human muscle development. Here, we report the generation of human induced pluripotent stem cell (iPSC) reporter lines in which fluorescent proteins have been introduced into the PAX7 and MYOG loci. We use single cell RNA sequencing to analyze the developmental trajectory of the iPSC-derived PAX7+ myogenic precursors. We show that the PAX7+ cells generated in culture can produce myofibers and self-renew in vitro and in vivo Together, we demonstrate that cells exhibiting characteristics of human fetal satellite cells can be produced in vitro from iPSC, opening interesting avenues for muscular dystrophy cell therapy. This work provides significant insights into the development of the human myogenic lineage.

Non-fibro-adipogenic pericytes from human embryonic stem cells attenuate degeneration of the chronically injured mouse muscle.

Massive tears of the rotator cuff (RC) are associated with chronic muscle degeneration due to fibrosis, fatty infiltration, and muscle atrophy. The microenvironment of diseased muscle often impairs efficient engraftment and regenerative activity of transplanted myogenic precursors. Accumulating myofibroblasts and fat cells disrupt the muscle stem cell niche and myogenic cell signaling and deposit excess disorganized connective tissue. Therefore, restoration of the damaged stromal niche with non-fibro-adipogenic cells is a prerequisite to successful repair of an injured RC. We generated from human embryonic stem cells (hES) a potentially novel subset of PDGFR-β+CD146+CD34-CD56- pericytes that lack expression of the fibro-adipogenic cell marker PDGFR-α. Accordingly, the PDGFR-β+PDGFR-α- phenotype typified non-fibro-adipogenic, non-myogenic, pericyte-like derivatives that maintained non-fibro-adipogenic properties when transplanted into chronically injured murine RCs. Although administered hES pericytes inhibited developing fibrosis at early and late stages of progressive muscle degeneration, transplanted PDGFR-β+PDGFR-α+ human muscle-derived fibro-adipogenic progenitors contributed to adipogenesis and greater fibrosis. Additionally, transplanted hES pericytes substantially attenuated muscle atrophy at all tested injection time points after injury. Coinciding with this observation, conditioned medium from cultured hES pericytes rescued atrophic myotubes in vitro. These findings imply that non-fibro-adipogenic hES pericytes recapitulate the myogenic stromal niche and may be used to improve cell-based treatments for chronic muscle disorders.

Differentiation of Bioengineered Skeletal Muscle within a 3D Printed Perfusion Bioreactor Reduces Atrophic and Inflammatory Gene Expression.

Bioengineered skeletal muscle tissues benefit from dynamic culture environments which facilitate the appropriate provision of nutrients and removal of cellular waste products. Biologically compatible perfusion systems hold the potential to enhance the physiological biomimicry of in vitro tissues via dynamic culture, in addition to providing technological advances in analytical testing and live cellular imaging for analysis of cellular development. To meet such diverse requirements, perfusion systems require the capacity and adaptability to incorporate multiple cell laden constructs of both monolayer and bioengineered tissues. This work reports perfusion systems produced using additive manufacturing technology for the in situ phenotypic development of myogenic precursor cells in monolayer and bioengineered tissue. Biocompatibility of systems 3D printed using stereolithography (SL), laser sintering (LS), and PolyJet outlined preferential morphological development within both SL and LS devices. When exposed to intermittent perfusion in the monolayer, delayed yet physiologically representative cellular proliferation, MyoD and myogenin transcription of C2C12 cells was evident. Long-term (8 days) intermittent perfusion of monolayer cultures outlined viable morphological and genetic in situ differentiation for the live cellular imaging of myogenic development. Continuous perfusion cultures (13 days) of bioengineered skeletal muscle tissues outlined in situ myogenic differentiation, forming mature multinucleated myotubes. Here, reductions in IL-1β and TNF-α inflammatory cytokines, myostatin, and MuRF-1 atrophic mRNA expression were observed. Comparable myosin heavy chain (MyHC) isoform transcription profiles were evident between conditions; however, total mRNA expression was reduced in perfusion conditions. Decreased transcription of MuRF1 and subsequent reduced ubiquitination of the MyHC protein allude to a decreased requirement for transcription of MyHC isoform transcripts. Together, these data appear to indicate that 3D printed perfusion systems elicit enhanced stability of the culture environment, resulting in a reduced basal requirement for MyHC gene expression within bioengineered skeletal muscle tissue.

Modified cell-electrospinning for 3D myogenesis of C2C12s in aligned fibrin microfiber bundles

Electrospinning methods can generate scaffolds with alignment cues to guide the development of myogenic precursors into 3D skeletal muscle grafts. However, cells seeded onto these scaffolds adhere to the exterior resulting in regions of acellularity within the scaffold interior. To overcome this limitation, we modified an aqueous solution-electrospinning method to encapsulate C2C12s and electrospin them into fibrin/polyethylene oxide (PEO) microfiber bundles. We demonstrated that loading C2C12s as cellular aggregates (80–90 μm in diameter) and modifying several other electrospinning parameters dramatically increased cell viability following exposure to the 4.5 kV electric field. C2C12-seeded fibrin/PEO microfiber bundles were cultured for up to seven days. Uninduced and myogenically induced C2C12s proliferated, elongated and became multinucleated. Myogenic induction increased the number of myotube-associated nuclei (36.4 ± 12% vs. 6.2 ± 1.9%), myotube length (122.4 ± 10.9 μm vs. 59.9 ± 8.3 μm), and myotube diameter (16.76 ± 2.06 μm vs. 12.49 ± 0.93 μm). The data presented in this study demonstrates for the first time that cells can be loaded inside the aligned fibrin hydrogel 3D construct during aqueous solution electrospinning while retaining their potential for de novo tissue formation.

Differentiation of Human Embryonic Stem Cells into Engrafting Myogenic Precursor Cells

Degenerative muscle diseases affect muscle tissue integrity and function. Human embryonic stem cells (hESC) are an attractive source of cells to use in regenerative therapies due to their unlimited capacity to divide and ability to specialize into a wide variety of cell types. A practical way to derive therapeutic myogenic stem cells from hESC is lacking. In this study, we demonstrate the development of two serum-free conditions to direct the differentiation of hESC towards a myogenic precursor state. Using TGFß and PI3Kinase inhibitors in combination with bFGF we showed that one week of differentiation is sufficient for hESC to specialize into PAX3+/PAX7+ myogenic precursor cells. These cells also possess the capacity to further differentiate in vitro into more specialized myogenic cells that express MYOD, Myogenin, Desmin and MYHC, and showed engraftment in vivo upon transplantation in immunodeficient mice. Ex vivo myomechanical studies of dystrophic mouse hindlimb muscle showed functional improvement one month post-transplantation. In summary, this study describes a promising system to derive engrafting muscle precursor cells solely using chemical substances in serum-free conditions and without genetic manipulation.

Emerin anchors Msx1 and its protein partners at the nuclear periphery to inhibit myogenesis

BackgroundPrevious studies have shown that in myogenic precursors, the homeoprotein Msx1 and its protein partners, histone methyltransferases and repressive histone marks, tend to be enriched on target myogenic regulatory genes at the nuclear periphery. The nuclear periphery localization of Msx1 and its protein partners is required for Msx1’s function of preventing myogenic precursors from pre-maturation through repressing target myogenic regulatory genes. However, the mechanisms underlying the maintenance of Msx1 and its protein partners’ nuclear periphery localization are unknown.ResultsWe show that an inner nuclear membrane protein, Emerin, performs as an anchor settled at the inner nuclear membrane to keep Msx1 and its protein partners Ezh2, H3K27me3 enriching at the nuclear periphery, and participates in inhibition of myogenesis mediated by Msx1. Msx1 interacts with Emerin both in C2C12 myoblasts and mouse developing limbs, which is the prerequisite for Emerin mediating the precise location of Msx1, Ezh2, and H3K27me3. The deficiency of Emerin in C2C12 myoblasts disturbs the nuclear periphery localization of Msx1, Ezh2, and H3K27me3, directly indicating Emerin functioning as an anchor. Furthermore, Emerin cooperates with Msx1 to repress target myogenic regulatory genes, and assists Msx1 with inhibition of myogenesis.ConclusionsEmerin cooperates with Msx1 to inhibit myogenesis through maintaining the nuclear periphery localization of Msx1 and Msx1’s protein partners.

The homeoprotein Msx1 cooperates with Pkn1 to prevent terminal differentiation in myogenic precursor cells

The homeoprotein Msx1 plays a critical role in embryonic patterning, particularly to maintain myogenic progenitor cell fate. However, the mechanisms underlying Msx1-mediated inhibition of myogenesis remain largely unknown. Here, we show that Msx1 cooperates with the protein kinase C-related kinase 1 (Pkn1), a member of the protein kinase C-related kinase family, to prevent the terminal differentiation of myogenic precursor cells. In mouse C2C12 cells, Pkn1 knockout partly impaired Msx1-mediated inhibition of myogenic differentiation, indicating a role for Pkn1 in this process. Furthermore, we found that Pkn1 was required for Msx1 enrichment at the promoter of Myf5, a myogenic regulatory gene. In Pkn1 knockout cells, this reduced Msx1 enrichment at the Myf5 promoter coincided with attenuated repression of Myf5 transcription. Together with our observation that Msx1 and Pkn1 were associated in a protein complex, these findings strongly suggest that Msx1 cooperates with Pkn1 to down-regulate Myf5 and, therefore, prevent the differentiation of myogenic precursor cells. Collectively, our data provide key insights into the mechanisms underlying Msx1 function in the prevention of myogenic differentiation.

Zfp423 Regulates Skeletal Muscle Regeneration and Proliferation.

Satellite cells (SCs) are skeletal muscle stem cells that proliferate in response to injury and provide myogenic precursors for growth and repair. Zfp423 is a transcriptional cofactor expressed in multiple immature cell populations, such as neuronal precursors, mesenchymal stem cells, and preadipocytes, where it regulates lineage allocation, proliferation, and differentiation. Here, we show that Zfp423 regulates myogenic progression during muscle regeneration. Zfp423 is undetectable in quiescent SCs but becomes expressed during SC activation. After expansion, Zfp423 is gradually downregulated as committed SCs terminally differentiate. Mice with satellite-cell-specific Zfp423 deletion exhibit severely impaired muscle regeneration following injury, with aberrant SC expansion, defective cell cycle exit, and failure to transition efficiently from the proliferative stage toward commitment. Consistent with a cell-autonomous role of Zfp423, shRNA-mediated knockdown of Zfp423 in myoblasts inhibits differentiation. Surprisingly, forced expression of Zfp423 in myoblasts induces differentiation into adipocytes and arrests myogenesis. Affinity purification of Zfp423 in myoblasts identified Satb2 as a nuclear partner of Zfp423 that cooperatively enhances Zfp423 transcriptional activity, which in turn affects myoblast differentiation. In conclusion, by controlling SC expansion and proliferation, Zfp423 is essential for muscle regeneration. Tight regulation of Zfp423 expression is essential for normal progression of muscle progenitors from proliferation to differentiation.

Knockdown of CSRP3 inhibits differentiation of chicken satellite cells by promoting TGF-β/Smad3 signaling

Muscle LIM protein (MLP/CSRP3/CRP3) is a microtubule-associated protein preferentially expressed in cardiac and skeletal muscle and has a central role during muscle development and for architectural maintenance of muscle cells. LIM-domain proteins act as both modulators and downstream targets of TGF-β signaling, which is well documented to negatively regulate differentiation of myogenic precursor cells or myoblasts. Herein, we determined whether CSRP3 regulates chicken satellite cell proliferation and differentiation in vitro, and examined its mechanism of action by focusing on the TGF-β signaling pathway. Interference of CSRP3 mRNA expression had no effect on the proliferation of satellite cells, but significantly inhibited satellite cell differentiation into myotubes at 24, 48, and 72 h after initiation of differentiation. However, CSRP3 overexpression did not affect the proliferation or differentiation of satellite cells. Moreover, knockdown of CSRP3 caused up-regulation of TGF-β and Smad3 mRNA and protein levels. The phosphorylation of Smad3 in CSRP3-knockdown cells was greater than that in wild-type cells at 24, 48, and 72 h after initiation of differentiation. Collectively, knockdown of CSRP3 suppressed chicken satellite cell differentiation by regulating Smad3 phosphorylation in the TGF-β signaling pathway. Our results indicate that CSRP3 might play an important role in promoting satellite cell differentiation in chicken.

Glucose regulates protein turnover and growth-related mechanisms in rainbow trout myogenic precursor cells

Rainbow trout are considered glucose intolerant because they are poor utilizers of glucose, despite having functional insulin receptors and glucose transporters. Following high carbohydrate meals, rainbow trout are persistently hyperglycemic, which is likely due to low glucose utilization in peripheral tissues including the muscle. Also, rainbow trout myogenic precursor cells (MPCs) treated in vitro with insulin and IGF1 increase glucose uptake and protein synthesis, whereas protein degradation is decreased. Given our understanding of glucose regulation in trout, we sought to understand how glucose concentrations affect protein synthesis, protein degradation; and expression of genes associated with muscle growth and proteolysis in MPCs. We found that following 24 h and 48 h of treatment with low glucose media (5.6 mM), myoblasts had significant decreases in protein synthesis. Also, low glucose treatments affected the expression of both mstn2a and igfbp5. These findings support that glucose is a direct regulator of protein synthesis and growth-related mechanisms in rainbow trout muscle.

The Nuclear Receptor and Clock Repressor Rev-erb\u03b1 Suppresses Myogenesis

Rev-erbα is a ligand-dependent nuclear receptor and a key repressor of the molecular clock transcription network. Accumulating evidence indicate that the circadian clock machinery governs diverse biological processes in skeletal muscle, including muscle growth, repair and mass maintenance. The physiological function of Rev-erbα in myogenic regulation remains largely unknown. Here we show that Rev-erbα exerts cell-autonomous inhibitory effects on proliferation and differentiation of myogenic precursor cells, and these actions concertedly inhibit muscle regeneration in vivo. Mechanistic studies reveal Rev-erbα direct transcriptional control of two major myogenic mechanisms, proliferative pathway and the Wnt signaling cascade. Consistent with this finding, primary myoblasts lacking Rev-erbα display significantly enhanced proliferative growth and myogenic progression. Furthermore, pharmacological activation of Rev-erbα activity attenuates, whereas its inhibition by an antagonist promotes these processes. Notably, upon muscle injury, the loss-of-function of Rev-erbα in vivo augmented satellite cell proliferative expansion and regenerative progression during regeneration. Collectively, our study identifies Rev-erbα as a novel inhibitory regulator of myogenic progenitor cell properties that suppresses postnatal myogenesis. Pharmacological interventions to dampen Rev-erbα activity may have potential utilities to enhance regenerative capacity in muscle diseases.

Isolation and characterization of myogenic precursor cells from human cremaster muscle

Human myogenic precursor cells have been isolated and expanded from a number of skeletal muscles, but alternative donor biopsy sites must be sought after in diseases where muscle damage is widespread. Biopsy sites must be relatively accessible, and the biopsied muscle dispensable. Here, we aimed to histologically characterize the cremaster muscle with regard number of satellite cells and regenerative fibres, and to isolate and characterize human cremaster muscle-derived stem/precursor cells in adult male donors with the objective of characterizing this muscle as a novel source of myogenic precursor cells. Cremaster muscle biopsies (or adjacent non-muscle tissue for negative controls; N = 19) were taken from male patients undergoing routine surgery for urogenital pathology. Myosphere cultures were derived and tested for their in vitro and in vivo myogenic differentiation and muscle regeneration capacities. Cremaster-derived myogenic precursor cells were maintained by myosphere culture and efficiently differentiated to myotubes in adhesion culture. Upon transplantation to an immunocompromised mouse model of cardiotoxin-induced acute muscle damage, human cremaster-derived myogenic precursor cells survived to the transplants and contributed to muscle regeneration. These precursors are a good candidate for cell therapy approaches of skeletal muscle. Due to their location and developmental origin, we propose that they might be best suited for regeneration of the rhabdosphincter in patients undergoing stress urinary incontinence after radical prostatectomy.

EGFR-Aurka Signaling Rescues Polarity and Regeneration Defects in Dystrophin-Deficient Muscle Stem Cells by Increasing Asymmetric Divisions.

Loss of dystrophin expression in Duchenne muscular dystrophy (DMD) causes progressive degeneration of skeletal muscle, which is exacerbated by reduced self-renewing asymmetric divisions of muscle satellite cells. This, in turn, affects the production of myogenic precursors and impairs regeneration and suggests that increasing such divisions may be beneficial. Here, through a small-molecule screen, we identified epidermal growth factor receptor (EGFR) and Aurora kinase A (Aurka) as regulators of asymmetric satellite cell divisions. Inhibiting EGFR causes a substantial shift from asymmetric to symmetric division modes, whereas EGF treatment increases asymmetric divisions. EGFR activation acts through Aurka to orient mitotic centrosomes, andinhibiting Aurka blocks EGF stimulation-inducedasymmetric division. Invivo EGF treatment markedlyactivates asymmetric divisions of dystrophin-deficientsatellite cells in mdx mice, increasing progenitor numbers, enhancing regeneration, and restoring muscle strength. Therefore, activating anEGFR-dependent polarity pathway promotes functional rescue of dystrophin-deficient satellite cells and enhances muscle force generation.

Dynamic membrane proteome of adipogenic and myogenic precursors in skeletal muscle highlights EPHA2 may promote myogenic differentiation through ERK signaling

The balance of myogenic and adipogenic differentiation is crucial for skeletal muscle homeostasis. Given the vital role of membrane proteins (MBPs) in cell signal perception, membrane proteomics was conducted to delineate mechanisms regulating differentiation of adipogenic and myogenic precursors in skeletal muscle. Adipogenic and myogenic precursors with divergent differentiation potential were isolated from the longissimus dorsi muscle of neonatal pigs by the preplate method. A total of 85 differentially expressed MBPs (P < 0.05 and fold change ≥1.2 or ≤0.83) between 2 precursors were detected via isobaric tags for relative and absolute quantitation (iTRAQ) assay, including 67 up-regulated and 18 down-regulated in myogenic precursors. Functional enrichment analysis uncovered that myogenic and adipogenic precursors showed significant differences in cytoskeleton organization, syncytium formation, environmental information processing, and organismal systems. Furthermore, key MBPs in regulating cell differentiation were also characterized, including ITGB3, ITGAV, ITPR3, and EPHA2. Noteworthily, EPHA2 was required for myogenic differentiation, and it may promote myogenic differentiation through ERK signaling. Collectively, our study provided an insight into the distinct MBP profile between myogenic and adipogenic precursors in skeletal muscle and served as a solid basis for supporting the role of MBPs in regulating differentiation.—Zhang, X., Wang, L., Qiu, K., Xu, D., Yin, J. Dynamic membrane proteome of adipogenic and myogenic precursors in skeletal muscle highlights EPHA2 may promote myogenic differentiation through ERK signaling.

Generation of Yellow Fever virus vaccine in skeletal muscle cells of chicken embryos

BACKGROUND The Yellow Fever (YF) vaccine is produced by the inoculation of embryonated chicken eggs with YF17DD virus on the ninth day of development. Full embryos are collected on the twelfth day of development for vaccine formulation. Skeletal muscle tissue is the main site where biosynthesis of viral particles occurs.OBJECTIVES This study aimed to analyse the experimental infection of skeletal muscle cells of chicken embryos by the 17DD Yellow Fever virus (YFV) in vivo and in vitro.METHODS Chicken embryos infected with YF17DD virus were analysed by immunofluorescence using confocal and super-resolution microscopes. Primary cultures of skeletal muscle cells of non-infected chicken embryos were evaluated for susceptibility and permissiveness to YF17DD virus using different protocols. This evaluation was performed based on morphological, viral titration, molecular biology, and colorimetric techniques.FINDINGS The present work phenotypically characterises embryonic chicken skeletal muscle cells as myogenic precursors expressing the Pax7 transcription factor in some cases. We demonstrated that these cells are susceptible to in vitro infection at different multiplicities of infection (MOIs), reproducing the same infection pattern observed in vivo. Furthermore, myogenic precursors and myoblasts are preferred infection targets, but establishment of infection does not depend on the presence of these cells. The peak of viral production occurred at 48 hpi, with decay occurring 72 hpi, when the cytopathic effect can be observed.MAIN CONCLUSIONS In conclusion, the primary culture of chicken skeletal muscle cells is a good model for studying muscle cells infected with YF17DD virus. This culture system displays satisfactory emulation of the in vitro phenomenon observed, contributing to our understanding of virus infection dynamics and leading to the development of alternative methods of vaccine production.

Histological, transcriptomic and in vitro analysis reveal an intrinsic activated state of myogenic precursors in hyperplasic muscle of trout

BackgroundThe dramatic increase in myotomal muscle mass in post-hatching fish is related to their ability to lastingly produce new muscle fibres, a process termed hyperplasia. The molecular and cellular mechanisms underlying fish muscle hyperplasia largely remain unknown. In this study, we aimed to characterize intrinsic properties of myogenic cells originating from hyperplasic fish muscle. For this purpose, we compared in situ proliferation, in vitro cell behavior and transcriptomic profile of myogenic precursors originating from hyperplasic muscle of juvenile trout (JT) and from non-hyperplasic muscle of fasted juvenile trout (FJT) and adult trout (AT).ResultsFor the first time, we showed that myogenic precursors proliferate in hyperplasic muscle from JT as shown by in vivo BrdU labeling. This proliferative rate was very low in AT and FJT muscle. Transcriptiomic analysis revealed that myogenic cells from FJT and AT displayed close expression profiles with only 64 differentially expressed genes (BH corrected p-val < 0.001). In contrast, 2623 differentially expressed genes were found between myogenic cells from JT and from both FJT and AT. Functional categories related to translation, mitochondrial activity, cell cycle, and myogenic differentiation were inferred from genes up regulated in JT compared to AT and FJT myogenic cells. Conversely, Notch signaling pathway, that signs cell quiescence, was inferred from genes down regulated in JT compared to FJT and AT. In line with our transcriptomic data, in vitro JT myogenic precursors displayed higher proliferation and differentiation capacities than FJT and AT myogenic precursors.ConclusionsThe transcriptomic analysis and examination of cell behavior converge to support the view that myogenic cells extracted from hyperplastic muscle of juvenile trout are intrinsically more potent to form myofibres than myogenic cells extracted from non-hyperplasic muscle. The generation of gene expression profiles in myogenic cell extracted from muscle of juvenile trout may yield insights into the molecular and cellular mechanisms controlling hyperplasia and provides a useful list of potential molecular markers of hyperplasia.