RESUMO
Sestrin2 (Sesn2) has been previously confirmed to be a stress-response molecule. However, the influence of Sesn2 on myogenic differentiation remains elusive. This study was conducted to analyze the role of Sesn2 in the myogenic differentiation of C2C12 myoblasts and related aspects in mdx mice, an animal model of Duchenne muscular dystrophy (DMD). Our results showed that knockdown of Sesn2 reduced the myogenic differentiation capacity of C2C12 myoblasts. Predictive analysis from two databases suggested that miR-182-5p is a potential regulator of Sesn2. Further experimental validation revealed that overexpression of miR-182-5p decreased both the protein and mRNA levels of Sesn2 and inhibited myogenesis of C2C12 myoblasts. These findings suggest that miR-182-5p negatively regulates myogenesis by repressing Sesn2 expression. Extending to an in vivo model of DMD, knockdown of Sesn2 led to decreased Myogenin (Myog) expression and increased Pax7 expression, while its overexpression upregulated Myog levels and enhanced the proportion of slow-switch myofibers. These findings indicate the crucial role of Sesn2 in promoting myogenic differentiation and skeletal muscle regeneration, providing potential therapeutic targets for muscular dystrophy.
Assuntos
Diferenciação Celular , Camundongos Endogâmicos mdx , MicroRNAs , Desenvolvimento Muscular , Mioblastos , Miogenina , Animais , Mioblastos/metabolismo , Camundongos , Desenvolvimento Muscular/fisiologia , Desenvolvimento Muscular/genética , Linhagem Celular , Miogenina/genética , Miogenina/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Técnicas de Silenciamento de Genes , Fator de Transcrição PAX7/metabolismo , Fator de Transcrição PAX7/genética , Regulação da Expressão Gênica , SestrinasRESUMO
Muscle stem cells (MuSCs) play an indispensable role in postnatal muscle growth and hypertrophy in adults. MuSCs also retain a highly regenerative capacity and are therefore considered a promising stem cell source for regenerative therapy for muscle diseases. In this study, we identify tumor-suppressor protein Tob1 as a Pax7 target protein that negatively controls the population expansion of MuSCs. Tob1 protein is undetectable in the quiescent state but is upregulated during activation in MuSCs. Tob1 ablation in mice accelerates MuSC population expansion and boosts muscle regeneration. Moreover, inactivation of Tob1 in MuSCs ameliorates the efficiency of MuSC transplantation in a murine muscular dystrophy model. Collectively, selective targeting of Tob1 might be a therapeutic option for the treatment of muscular diseases, including muscular dystrophy and age-related sarcopenia.
Assuntos
Músculo Esquelético , Fator de Transcrição PAX7 , Regeneração , Células-Tronco , Animais , Camundongos , Músculo Esquelético/metabolismo , Fator de Transcrição PAX7/metabolismo , Fator de Transcrição PAX7/genética , Células-Tronco/metabolismo , Células-Tronco/citologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proliferação de Células , Camundongos Endogâmicos C57BLRESUMO
Although studies have identified characteristics of quiescent satellite cells (SCs), their isolation has been hampered by the fact that the isolation procedures result in the activation of these cells into their rapidly proliferating progeny (myoblasts). Thus, the use of myoblasts for therapeutic (regenerative medicine) or industrial applications (cellular agriculture) has been impeded by the limited proliferative and differentiative capacity of these myogenic progenitors. Here we identify a subpopulation of satellite cells isolated from mouse skeletal muscle using flow cytometry that is highly Pax7-positive, exhibit a very slow proliferation rate (7.7 ± 1.2 days/doubling), and are capable of being maintained in culture for at least 3 mo without a change in phenotype. These cells can be activated from quiescence using a p38 inhibitor or by exposure to freeze-thaw cycles. Once activated, these cells proliferate rapidly (22.7 ± 0.2 h/doubling), have reduced Pax7 expression (threefold decrease in Pax7 fluorescence vs. quiescence), and differentiate into myotubes with a high efficiency. Furthermore, these cells withstand freeze-thawing readily without a significant loss of viability (83.1 ± 2.1% live). The results presented here provide researchers with a method to isolate quiescent satellite cells, allowing for more detailed examinations of the factors affecting satellite cell quiescence/activation and providing a cell source that has a unique potential in the regenerative medicine and cellular agriculture fields.NEW & NOTEWORTHY We provide a method to isolate quiescent satellite cells from skeletal muscle. These cells are highly Pax7-positive, exhibit a very slow proliferation rate, and are capable of being maintained in culture for months without a change in phenotype. The use of these cells by muscle researchers will allow for more detailed examinations of the factors affecting satellite cell quiescence/activation and provide a novel cell source for the regenerative medicine and cellular agriculture fields.
Assuntos
Diferenciação Celular , Proliferação de Células , Fator de Transcrição PAX7 , Células Satélites de Músculo Esquelético , Animais , Células Satélites de Músculo Esquelético/metabolismo , Células Satélites de Músculo Esquelético/citologia , Fator de Transcrição PAX7/metabolismo , Fator de Transcrição PAX7/genética , Camundongos , Diferenciação Celular/fisiologia , Células Cultivadas , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Camundongos Endogâmicos C57BL , Separação Celular/métodos , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/citologia , Desenvolvimento Muscular/fisiologia , MasculinoRESUMO
Increasing evidence suggests that the muscle stem cell (MuSC) pool is heterogeneous. In particular, a rare subset of PAX7-positive MuSCs that has never expressed the myogenic regulatory factor MYF5 displays unique self-renewal and engraftment characteristics. However, the scarcity and limited availability of protein markers make the characterization of these cells challenging. Here, we describe the generation of StemRep reporter mice enabling the monitoring of PAX7 and MYF5 proteins based on equimolar levels of dual nuclear fluorescence. High levels of PAX7 protein and low levels of MYF5 delineate a deeply quiescent MuSC subpopulation with an increased capacity for asymmetric division and distinct dynamics of activation, proliferation, and commitment. Aging primarily reduces the MYF5Low MuSCs and skews the stem cell pool toward MYF5High cells with lower quiescence and self-renewal potential. Altogether, we establish the StemRep model as a versatile tool to study MuSC heterogeneity and broaden our understanding of mechanisms regulating MuSC quiescence and self-renewal in homeostatic, regenerating, and aged muscles.
Assuntos
Envelhecimento , Genes Reporter , Fator Regulador Miogênico 5 , Fator de Transcrição PAX7 , Regeneração , Animais , Fator de Transcrição PAX7/metabolismo , Fator de Transcrição PAX7/genética , Fator Regulador Miogênico 5/metabolismo , Fator Regulador Miogênico 5/genética , Camundongos , Envelhecimento/metabolismo , Células-Tronco/metabolismo , Células-Tronco/citologia , Proliferação de Células , Músculo Esquelético/metabolismo , Músculo Esquelético/citologia , Diferenciação Celular , Camundongos Transgênicos , Autorrenovação CelularRESUMO
BACKGROUND: Adult muscle-resident myogenic stem cells, satellite cells (SCs), that play non-redundant role in muscle regeneration, are intrinsically impaired in Duchenne muscular dystrophy (DMD). Previously we revealed that dystrophic SCs express low level of anti-inflammatory and anti-oxidative heme oxygenase-1 (HO-1, HMOX1). Here we assess whether targeted induction of HMOX1 affect SC function and alleviates hallmark symptoms of DMD. METHODS: We generated double-transgenic mouse model (mdx;HMOX1Pax7Ind) that allows tamoxifen (TX)-inducible HMOX1 expression in Pax7 positive cells of dystrophic muscles. Mdx;HMOX1Pax7Ind and control mdx mice were subjected to 5-day TX injections (75 mg/kg b.w.) followed by acute exercise protocol with high-speed treadmill (12 m/min, 45 min) and downhill running to worsen skeletal muscle phenotype and reveal immediate effects of HO-1 on muscle pathology and SC function. RESULTS: HMOX1 induction caused a drop in SC pool in mdx;HMOX1Pax7Ind mice (vs. mdx counterparts), while not exaggerating the effect of physical exercise. Upon physical exercise, the proliferation of SCs and activated CD34- SC subpopulation, was impaired in mdx mice, an effect that was reversed in mdx;HMOX1Pax7Ind mice, however, both in vehicle- and TX-treated animals. This corresponded to the pattern of HO-1 expression in skeletal muscles. At the tissue level, necrotic events of selective skeletal muscles of mdx mice and associated increase in circulating levels of muscle damage markers were blunted in HO-1 transgenic animals which showed also anti-inflammatory cytokine profile (vs. mdx). CONCLUSIONS: Targeted expression of HMOX1 plays protective role in DMD and alleviates dystrophic muscle pathology.
Assuntos
Heme Oxigenase-1 , Camundongos Endogâmicos mdx , Camundongos Transgênicos , Músculo Esquelético , Distrofia Muscular de Duchenne , Células Satélites de Músculo Esquelético , Animais , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Células Satélites de Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patologia , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Camundongos , Fator de Transcrição PAX7/genética , Fator de Transcrição PAX7/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Condicionamento Físico Animal , Proteínas de MembranaRESUMO
Gene regulatory networks that act upstream of skeletal muscle fate determinants are distinct in different anatomical locations. Despite recent efforts, a clear understanding of the cascade of events underlying the emergence and maintenance of the stem cell pool in specific muscle groups remains unresolved and debated. Here, we invalidated Pitx2 with multiple Cre-driver mice prenatally, postnatally, and during lineage progression. We showed that this gene becomes progressively dispensable for specification and maintenance of the muscle stem (MuSC) cell pool in extraocular muscles (EOMs) despite being, together with Myf5, a major upstream regulator during early development. Moreover, constitutive inactivation of Pax7 postnatally led to a greater loss of MuSCs in the EOMs compared to the limb. Thus, we propose a relay between Pitx2, Myf5 and Pax7 for EOM stem cell maintenance. We demonstrate also that MuSCs in the EOMs adopt a quiescent state earlier that those in limb muscles and do not spontaneously proliferate in the adult, yet EOMs have a significantly higher content of Pax7+ MuSCs per area pre- and post-natally. Finally, while limb MuSCs proliferate in the mdx mouse model for Duchenne muscular dystrophy, significantly less MuSCs were present in the EOMs of the mdx mouse model compared to controls, and they were not proliferative. Overall, our study provides a comprehensive in vivo characterisation of MuSC heterogeneity along the body axis and brings further insights into the unusual sparing of EOMs during muscular dystrophy.
Assuntos
Proteína Homeobox PITX2 , Proteínas de Homeodomínio , Fator Regulador Miogênico 5 , Músculos Oculomotores , Fator de Transcrição PAX7 , Fatores de Transcrição , Animais , Humanos , Camundongos , Diferenciação Celular/genética , Linhagem da Célula/genética , Proliferação de Células/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Camundongos Endogâmicos mdx , Desenvolvimento Muscular/genética , Músculo Esquelético/metabolismo , Músculo Esquelético/crescimento & desenvolvimento , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patologia , Fator Regulador Miogênico 5/genética , Fator Regulador Miogênico 5/metabolismo , Músculos Oculomotores/metabolismo , Fator de Transcrição PAX7/metabolismo , Fator de Transcrição PAX7/genética , Células-Tronco/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Myogenic regeneration relies on the proliferation and differentiation of satellite cells. TECRL (trans-2,3-enoyl-CoA reductase like) is an endoplasmic reticulum protein only expressed in cardiac and skeletal muscle. However, its role in myogenesis remains unknown. We show that TECRL expression is increased in response to injury. Satellite cell-specific deletion of TECRL enhances muscle repair by increasing the expression of EGR2 through the activation of the ERK1/2 signaling pathway, which in turn promotes the expression of PAX7. We further show that TECRL deletion led to the upregulation of the histone acetyltransferase general control nonderepressible 5, which enhances the transcription of EGR2 through acetylation. Importantly, we showed that AAV9-mediated TECRL silencing improved muscle repair in mice. These findings shed light on myogenic regeneration and muscle repair.
Assuntos
Proteína 2 de Resposta de Crescimento Precoce , Desenvolvimento Muscular , Músculo Esquelético , Regeneração , Animais , Camundongos , Músculo Esquelético/metabolismo , Proteína 2 de Resposta de Crescimento Precoce/metabolismo , Proteína 2 de Resposta de Crescimento Precoce/genética , Desenvolvimento Muscular/genética , Regeneração/genética , Regulação para Cima , Células Satélites de Músculo Esquelético/metabolismo , Fator de Transcrição PAX7/metabolismo , Fator de Transcrição PAX7/genética , Sistema de Sinalização das MAP Quinases , Camundongos Knockout , Diferenciação CelularRESUMO
Lines with few or no pigment cells have been established in fishes, and these lines are useful for bioimaging. The transparent goldfish (tra) line previously established by N-ethyl-N-nitrosourea (ENU) mutagenesis is also suitable for such experiments. However, in the case of tra, leucophores form in the adult fish, making it difficult to observe the organs inside body from outside the body. In this study, we attempted to create a knockout line of the pax7a and pax7b genes, which are thought to be involved in the formation of leucophores, to further improve the transparency of tra strain.Mutations were introduced by microinjection of the CRISPR/Cas9 mixture into single-cell embryos, mutant individuals were found in F0, and the next generation was generated to confirm the mutation patterns. As a result, multiple mutation patterns, including knockout, were obtained. The same pattern of knockout F1 with pax7a and pax7b mutations was crossed to generate a homozygous knockout in F2.In the resulting pax7b-/- (tra) fish but not in pax7a-/- (tra) fish, the number of leucophores was reduced compared to that in tra, and the transparency of the body was improved. It was suggested that pax7b plays an important role in leucophore formation in goldfish. The established transparent pax7b-/- (tra) goldfish line will be a useful model for bioimaging of the body interior.
Assuntos
Técnicas de Inativação de Genes , Carpa Dourada , Fator de Transcrição PAX7 , Animais , Carpa Dourada/genética , Técnicas de Inativação de Genes/métodos , Fator de Transcrição PAX7/genética , Fator de Transcrição PAX7/metabolismo , Sistemas CRISPR-Cas , Mutação , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismoRESUMO
BACKGROUND: Photobiomodulation has exhibited promise in mitigating the local effects induced by Bothrops snakebite envenoming; however, the mechanisms underlying this protection are not yet fully understood. Herein, the effectiveness of photobiomodulation effects on regenerative response of C2C12 myoblast cells following exposure to Bothrops jararacussu venom (BjsuV), as well as the mechanisms involved was investigated. METHODOLOGY/PRINCIPAL FINDINGS: C2C12 myoblast cells were exposed to BjsuV (12.5 µg/mL) and irradiated once for 10 seconds with laser light of 660 nm (14.08 mW; 0.04 cm2; 352 mW/cm2) or 780 nm (17.6 mW; 0.04 cm2; 440 mW/ cm2) to provide energy densities of 3.52 and 4.4 J/cm2, and total energies of 0.1408 and 0.176 J, respectively. Cell migration was assessed through a wound-healing assay. The expression of MAPK p38-α, NF-Ðß, Myf5, Pax-7, MyoD, and myogenin proteins were assessed by western blotting analysis. In addition, interleukin IL1-ß, IL-6, TNF-alfa and IL-10 levels were measured in the supernatant by ELISA. The PBM applied to C2C12 cells exposed to BjsuV promoted cell migration, increase the expression of myogenic factors (Pax7, MyF5, MyoD and myogenin), reduced the levels of proinflammatory cytokines, IL1-ß, IL-6, TNF-alfa, and increased the levels of anti-inflammatory cytokine IL-10. In addition, PBM downregulates the expression of NF-kB, and had no effect on p38 MAKP. CONCLUSION/SIGNIFICANCE: These data demonstrated that protection of the muscle cell by PBM seems to be related to the increase of myogenic factors as well as the modulation of inflammatory mediators. PBM therapy may offer a new therapeutic strategy to address the local effects of snakebite envenoming by promoting muscle regeneration and reducing the inflammatory process.
Assuntos
Bothrops , Venenos de Crotalídeos , Citocinas , Terapia com Luz de Baixa Intensidade , Mioblastos , Miogenina , Animais , Mioblastos/efeitos dos fármacos , Mioblastos/efeitos da radiação , Mioblastos/metabolismo , Camundongos , Terapia com Luz de Baixa Intensidade/métodos , Citocinas/metabolismo , Linhagem Celular , Venenos de Crotalídeos/toxicidade , Miogenina/metabolismo , Miogenina/genética , Fator de Transcrição PAX7/metabolismo , Fator de Transcrição PAX7/genética , NF-kappa B/metabolismo , Proteína MyoD/metabolismo , Proteína MyoD/genética , Movimento Celular/efeitos dos fármacos , Movimento Celular/efeitos da radiação , Fator Regulador Miogênico 5/metabolismo , Fator Regulador Miogênico 5/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Mordeduras de Serpentes/radioterapia , Serpentes PeçonhentasRESUMO
The most common form of facioscapulohumeral dystrophy (FSHD1) is caused by a partial loss of the D4Z4 macrosatellite repeat array in the subtelomeric region of chromosome 4. Patients with FSHD1 typically carry 1 to 10 D4Z4 repeats, whereas nonaffected individuals have 11 to 150 repeats. The ~150-kilobyte subtelomeric region of the chromosome 10q exhibits a ~99% sequence identity to the 4q, including the D4Z4 array. Nevertheless, contractions of the chr10 array do not cause FSHD or any known disease, as in most people D4Z4 array on chr10 is flanked by the nonfunctional polyadenylation signal, not permitting the DUX4 expression. Here, we attempted to correct the FSHD genotype by a CRISPR-Cas9-induced exchange of the chr4 and chr10 subtelomeric regions. We demonstrated that the induced t(4;10) translocation can generate recombinant genotypes translated into improved FSHD phenotype. FSHD myoblasts with the t(4;10) exhibited reduced expression of the DUX4 targets, restored PAX7 target expression, reduced sensitivity to oxidative stress, and improved differentiation capacity.
Assuntos
Cromossomos Humanos Par 10 , Cromossomos Humanos Par 4 , Genótipo , Proteínas de Homeodomínio , Distrofia Muscular Facioescapuloumeral , Fenótipo , Telômero , Humanos , Cromossomos Humanos Par 10/genética , Cromossomos Humanos Par 4/genética , Sistemas CRISPR-Cas , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Distrofia Muscular Facioescapuloumeral/genética , Mioblastos/metabolismo , Fator de Transcrição PAX7/genética , Fator de Transcrição PAX7/metabolismo , Telômero/genética , Telômero/metabolismo , Translocação GenéticaRESUMO
Essential for muscle fiber formation and hypertrophy, muscle stem cells, also called satellite cells, reside beneath the basal lamina of the muscle fiber. Satellite cells have been commonly identified by the expression of the Paired box 7 (Pax7) due to its specificity and the availability of antibodies in tetrapods. In fish, the identification of satellite cells remains difficult due to the lack of specific antibodies in most species. Based on the development of a highly sensitive in situ hybridization (RNAScope®) for pax7, we showed that pax7+ cells were detected in the undifferentiated myogenic epithelium corresponding to the dermomyotome at day 14 post-fertilization in rainbow trout. Then, from day 24, pax7+ cells gradually migrated into the deep myotome and were localized along the muscle fibers and reach their niche in satellite position of the fibres after hatching. Our results showed that 18 days after muscle injury, a large number of pax7+ cells accumulated at the wound site compared to the uninjured area. During the in vitro differentiation of satellite cells, the percentage of pax7+ cells decreased from 44% to 18% on day 7, and some differentiated cells still expressed pax7. Taken together, these results show the dynamic expression of pax7 genes and the follow-up of these muscle stem cells during the different situations of muscle fiber formation in trout.
Assuntos
Oncorhynchus mykiss , Fator de Transcrição PAX7 , Regeneração , Células Satélites de Músculo Esquelético , Animais , Diferenciação Celular , Regulação da Expressão Gênica no Desenvolvimento , Desenvolvimento Muscular/genética , Oncorhynchus mykiss/metabolismo , Oncorhynchus mykiss/genética , Fator de Transcrição PAX7/metabolismo , Fator de Transcrição PAX7/genética , Regeneração/genética , Células Satélites de Músculo Esquelético/metabolismo , Células Satélites de Músculo Esquelético/citologiaRESUMO
Cell culture meat is based on the scaled-up expansion of seed cells. The biological differences between seed cells from large yellow croakers in the two-dimensional (2D) and three-dimensional (3D) culture systems have not been explored. Here, satellite cells (SCs) from large yellow croakers (Larimichthys crocea) were grown on cell climbing slices, hydrogels, and microcarriers for five days to analyze the biological differences of SCs on different cell scaffolds. The results exhibited that SCs had different cell morphologies in 2D and 3D cultures. Cell adhesion receptors (Itgb1andsdc4) and adhesion spot markervclof the 3D cultures were markedly expressed. Furthermore, myogenic decision markers (Pax7andmyod) were significantly enhanced. However, the expression of myogenic differentiation marker (desmin) was significantly increased in the microcarrier group. Combined with the transcriptome data, this suggests that cell adhesion of SCs in 3D culture was related to the integrin signaling pathway. In contrast, the slight spontaneous differentiation of SCs on microcarriers was associated with rapid cell proliferation. This study is the first to report the biological differences between SCs in 2D and 3D cultures, providing new perspectives for the rapid expansion of cell culture meat-seeded cells and the development of customized scaffolds.
Assuntos
Adesão Celular , Técnicas de Cultura de Células , Diferenciação Celular , Proliferação de Células , Hidrogéis , Células Satélites de Músculo Esquelético , Alicerces Teciduais , Animais , Células Satélites de Músculo Esquelético/metabolismo , Células Satélites de Músculo Esquelético/citologia , Hidrogéis/química , Alicerces Teciduais/química , Técnicas de Cultura de Células em Três Dimensões/métodos , Células Cultivadas , Desmina/metabolismo , Fator de Transcrição PAX7/metabolismo , Fator de Transcrição PAX7/genética , Desenvolvimento MuscularRESUMO
Embryonic skeletal muscle growth is contingent upon a population of somite derived satellite cells, however, the contribution of these cells to early postnatal skeletal muscle growth remains relatively high. As prepubertal postnatal development proceeds, the activity and contribution of satellite cells to skeletal muscle growth diminishes. Eventually, at around puberty, a population of satellite cells escapes terminal commitment, continues to express the paired box transcription factor Pax7, and reside in a quiescent state orbiting the myofiber periphery adjacent to the basal lamina. After adolescence, some satellite cell contributions to muscle maintenance and adaptation occur, however, their necessity is reduced relative to embryonic, early postnatal, and prepubertal growth.
Assuntos
Desenvolvimento Muscular , Músculo Esquelético , Células Satélites de Músculo Esquelético , Células Satélites de Músculo Esquelético/citologia , Células Satélites de Músculo Esquelético/metabolismo , Células Satélites de Músculo Esquelético/fisiologia , Animais , Músculo Esquelético/crescimento & desenvolvimento , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Humanos , Fator de Transcrição PAX7/metabolismo , Fator de Transcrição PAX7/genética , Diferenciação CelularRESUMO
During embryogenesis, basic fibroblast growth factor (bFGF) is released from neural tube and myotome to promote myogenic fate in the somite, and is routinely used for the culture of adult skeletal muscle (SKM) stem cells (MuSC, called satellite cells). However, the mechanism employed by bFGF to promote SKM lineage and MuSC proliferation has not been analyzed in detail. Furthermore, the question of if the post-translational modification (PTM) of bFGF is important to its stemness-promoting effect has not been answered. In this study, GST-bFGF was expressed and purified from E.coli, which lacks the PTM system in eukaryotes. We found that both GST-bFGF and commercially available bFGF activated the Akt-Erk pathway and had strong cell proliferation effect on C2C12 myoblasts and MuSC. GST-bFGF reversibly compromised the myogenesis of C2C12 myoblasts and MuSC, and it increased the expression of Myf5, Pax3/7, and Cyclin D1 but strongly repressed that of MyoD, suggesting the maintenance of myogenic stemness amid repressed MyoD expression. The proliferation effect of GST-bFGF was conserved in C2C12 over-expressed with MyoD (C2C12-tTA-MyoD), implying its independence of the down-regulation of MyoD. In addition, the repressive effect of GST-bFGF on myogenic differentiation was almost totally rescued by the over-expression of MyoD. Together, these evidences suggest that (1) GST-bFGF and bFGF have similar effects on myogenic cell proliferation and differentiation, and (2) GST-bFGF can promote MuSC stemness and proliferation by differentially regulating MRFs and Pax3/7, (3) MyoD repression by GST-bFGF is reversible and independent of the proliferation effect, and (4) GST-bFGF can be a good substitute for bFGF in sustaining MuSC stemness and proliferation.
Assuntos
Proliferação de Células , Fator 2 de Crescimento de Fibroblastos , Desenvolvimento Muscular , Proteína MyoD , Mioblastos , Desenvolvimento Muscular/genética , Animais , Camundongos , Proteína MyoD/metabolismo , Proteína MyoD/genética , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fator 2 de Crescimento de Fibroblastos/farmacologia , Fator 2 de Crescimento de Fibroblastos/genética , Mioblastos/metabolismo , Mioblastos/citologia , Linhagem Celular , Fator de Transcrição PAX7/metabolismo , Fator de Transcrição PAX7/genética , Fator de Transcrição PAX3/metabolismo , Fator de Transcrição PAX3/genética , Fator Regulador Miogênico 5/metabolismo , Fator Regulador Miogênico 5/genética , Ciclina D1/metabolismo , Ciclina D1/genética , Células Satélites de Músculo Esquelético/metabolismo , Células Satélites de Músculo Esquelético/citologia , Diferenciação Celular , Proteínas Proto-Oncogênicas c-akt/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/citologiaRESUMO
Previous studies have shown that vitamin C (VC), an essential vitamin for the human body, can promote the differentiation of muscle satellite cells (MuSCs) in vitro and play an important role in skeletal muscle post-injury regeneration. However, the molecular mechanism of VC regulating MuSC proliferation has not been elucidated. In this study, the role of VC in promoting MuSC proliferation and its molecular mechanism were explored using cell molecular biology and animal experiments. The results showed that VC accelerates the progress of skeletal muscle post-injury regeneration by promoting MuSC proliferation in vivo. VC can also promote skeletal muscle regeneration in the case of atrophy. Using the C2C12 myoblast murine cell line, we observed that VC also stimulated cell proliferation. In addition, after an in vitro study establishing the occurrence of a physical interaction between VC and Pax7, we observed that VC also upregulated the total and nuclear Pax7 protein levels. This mechanism increased the expression of Myf5 (Myogenic Factor 5), a Pax7 target gene. This study establishes a theoretical foundation for understanding the regulatory mechanisms underlying VC-mediated MuSC proliferation and skeletal muscle regeneration. Moreover, it develops the application of VC in animal muscle nutritional supplements and treatment of skeletal muscle-related diseases.
Assuntos
Ácido Ascórbico , Proliferação de Células , Músculo Esquelético , Mioblastos , Fator de Transcrição PAX7 , Regeneração , Animais , Masculino , Camundongos , Ácido Ascórbico/farmacologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Mioblastos/efeitos dos fármacos , Mioblastos/metabolismo , Fator Regulador Miogênico 5/metabolismo , Fator Regulador Miogênico 5/genética , Fator de Transcrição PAX7/metabolismo , Fator de Transcrição PAX7/genética , Regeneração/efeitos dos fármacos , Células Satélites de Músculo Esquelético/metabolismo , Células Satélites de Músculo Esquelético/efeitos dos fármacosRESUMO
Cordyceps sinensis is a parasitic fungus known to induce immune responses. The impact of Cordyceps supplementation on stem cell homing and expansion to human skeletal muscle after exercise remains unexplored. In this study, we examined how pre-exercise Cordyceps supplementation influences cell infiltration, CD34+ cell recruitment, and Pax7+ cell expansion in human skeletal muscle after high-intensity interval exercise (HIIE) on a cycloergometer. A randomized, double-blind, placebo-controlled crossover study was conducted with 14 young adults (age: 24 ± 0.8 years). A placebo (1 g cornstarch) and Cordyceps (1 g Cordyceps sinensis) were administered before exercise (at 120% maximal aerobic power). Multiple biopsies were taken from the vastus lateralis for muscle tissue analysis before and after HIIE. This exercise regimen doubled the VEGF mRNA in the muscle at 3 h post-exercise (P = 0.006). A significant necrotic cell infiltration (+284%, P = 0.05) was observed 3 h after HIIE and resolved within 24 h. This response was substantially attenuated by Cordyceps supplementation. Moreover, we observed increases in CD34+ cells at 24 h post-exercise, notably accelerated by Cordyceps supplementation to 3 h (+51%, P = 0.002). This earlier response contributed to a four-fold expansion in Pax7+ cell count, as demonstrated by immunofluorescence double staining (CD34+/Pax7+) (P = 0.01). In conclusion, our results provide the first human evidence demonstrating the accelerated resolution of exercise-induced muscle damage by Cordyceps supplementation. This effect is associated with earlier stem cell recruitment into the damaged sites for muscle regeneration.
Assuntos
Cordyceps , Estudos Cross-Over , Exercício Físico , Músculo Esquelético , Humanos , Cordyceps/química , Adulto Jovem , Masculino , Exercício Físico/fisiologia , Adulto , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Método Duplo-Cego , Células-Tronco/efeitos dos fármacos , Antígenos CD34/metabolismo , Feminino , Fator de Transcrição PAX7/metabolismo , Fator de Transcrição PAX7/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Obestatin is derived from the same gene as that of ghrelin and their functions were perceived to be antagonistic. Recent developments have shown that although they are known to have contradictory functions, effect of obestatin on skeletal muscle regeneration is similar to that of ghrelin. Obestatin works through a receptor called GPR39, a ghrelin and motilin family receptor and transduces signals in skeletal muscle similar to that of ghrelin. Not only there is a similarity in the receptor family, but also obestatin targets similar proteins and transcription factors as that of ghrelin (for example, FoxO family members) for salvaging skeletal muscle atrophy. Moreover, like ghrelin, obestatin also works by inducing the transcription of Pax7 which is required for muscle stem cell mobilisation. Hence, there are quite some evidences which points to the fact that obestatin can be purposed as a peptide intervention to prevent skeletal muscle wasting and induce myogenesis. This review elaborates these aspects of obestatin which can be further exploited and addressed to bring obestatin as a clinical intervention towards preventing skeletal muscle atrophy and sarcopenia.
Assuntos
Grelina , Músculo Esquelético , Atrofia Muscular , Regeneração , Humanos , Músculo Esquelético/metabolismo , Músculo Esquelético/efeitos dos fármacos , Regeneração/efeitos dos fármacos , Animais , Atrofia Muscular/metabolismo , Atrofia Muscular/prevenção & controle , Atrofia Muscular/tratamento farmacológico , Grelina/farmacologia , Grelina/metabolismo , Grelina/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Desenvolvimento Muscular/efeitos dos fármacos , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , Fator de Transcrição PAX7/metabolismo , Fator de Transcrição PAX7/genéticaRESUMO
Skeletal muscle stem and progenitor cells including those derived from human pluripotent stem cells (hPSCs) offer an avenue towards personalized therapies and readily fuse to form human-mouse myofibres in vivo. However, skeletal muscle progenitor cells (SMPCs) inefficiently colonize chimeric stem cell niches and instead associate with human myofibres resembling foetal niches. We hypothesized competition with mouse satellite cells (SCs) prevented SMPC engraftment into the SC niche and thus generated an SC ablation mouse compatible with human engraftment. Single-nucleus RNA sequencing of SC-ablated mice identified the absence of a transient myofibre subtype during regeneration expressing Actc1. Similarly, ACTC1+ human myofibres supporting PAX7+ SMPCs increased in SC-ablated mice, and after re-injury we found SMPCs could now repopulate into chimeric niches. To demonstrate ACTC1+ myofibres are essential to supporting PAX7 SMPCs, we generated caspase-inducible ACTC1 depletion human pluripotent stem cells, and upon SMPC engraftment we found a 90% reduction in ACTC1+ myofibres and a 100-fold decrease in PAX7 cell numbers compared with non-induced controls. We used spatial RNA sequencing to identify key factors driving emerging human niche formation between ACTC1+ myofibres and PAX7+ SMPCs in vivo. This revealed that transient regenerating human myofibres are essential for emerging niche formation in vivo to support PAX7 SMPCs.
Assuntos
Músculo Esquelético , Fator de Transcrição PAX7 , Regeneração , Células Satélites de Músculo Esquelético , Animais , Humanos , Camundongos , Músculo Esquelético/fisiologia , Fator de Transcrição PAX7/genética , Fator de Transcrição PAX7/metabolismo , Células-Tronco Pluripotentes , Células Satélites de Músculo Esquelético/fisiologiaRESUMO
Ever since its introduction as a genetic tool, the Cre-lox system has been widely used for molecular genetic studies in vivo in the context of health and disease, as it allows time- and cell-specific gene modifications. However, insertion of the Cre-recombinase cassette in the gene of interest can alter transcription, protein expression, or function, either directly, by modifying the landscape of the locus, or indirectly, due to the lack of genetic compensation or by indirect impairment of the non-targeted allele. This is sometimes the case when Cre-lox is used for muscle stem cell studies. Muscle stem cells are required for skeletal muscle growth, regeneration and to delay muscle disease progression, hence providing an attractive model for stem cell research. Since the transcription factor Pax7 is specifically expressed in all muscle stem cells, tamoxifen-inducible Cre cassettes (CreERT2) have been inserted into this locus by different groups to allow targeted gene recombination. Here we compare the two Pax7-CreERT2 mouse lines that are mainly used to evaluate muscle regeneration and development of pathological features upon deletion of specific factors or pathways. We applied diverse commonly used tamoxifen schemes of CreERT2 activation, and we analyzed muscle repair after cardiotoxin-induced injury. We show that consistently the Pax7-CreERT2 allele targeted into the Pax7 coding sequence (knock-in/knock-out allele) produces an inherent defect in regeneration, manifested as delayed post-injury repair and reduction in muscle stem cell numbers. In genetic ablation studies lacking proper controls, this inherent defect could be misinterpreted as being provoked by the deletion of the factor of interest. Instead, using an alternative Pax7-CreERT2 allele that maintains bi-allelic Pax7 expression or including appropriate controls can prevent misinterpretation of experimental data. The findings presented here can guide researchers establish appropriate experimental design for muscle stem cell genetic studies.
Assuntos
Haploinsuficiência , Células Satélites de Músculo Esquelético , Camundongos , Animais , Fator de Transcrição PAX7/genética , Fator de Transcrição PAX7/metabolismo , Haploinsuficiência/genética , Tamoxifeno/farmacologia , Integrases/genética , Integrases/metabolismo , Células-Tronco/metabolismo , Músculos , Células Satélites de Músculo Esquelético/metabolismo , Músculo Esquelético/metabolismoRESUMO
Muscular dystrophy is a heterogenous group of hereditary muscle disorders caused by mutations in the genes responsible for muscle development, and is generally defined by a disastrous progression of muscle wasting and massive loss in muscle regeneration. Pax7 is closely associated with myogenesis, which is governed by various signaling pathways throughout a lifetime and is frequently used as an indicator in muscle research. In this review, an extensive literature search adhering to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines was performed to identify research that examined signaling pathways in living models, while quantifying Pax7 expression in myogenesis. A total of 247 articles were retrieved from the Web of Science (WoS), PubMed and Scopus databases and were thoroughly examined and evaluated, resulting in 19 articles which met the inclusion criteria. Admittedly, we were only able to discuss the quantification of Pax7 carried out in research affecting various type of genes and signaling pathways, rather than the expression of Pax7 itself, due to the massive differences in approach, factor molecules and signaling pathways analyzed across the research. However, we highlighted the thorough evidence for the alteration of the muscle stem cell precursor Pax7 in multiple signaling pathways described in different living models, with an emphasis on the novel approach that could be taken in manipulating Pax7 expression itself in dystrophic muscle, towards the discovery of an effective treatment for muscular dystrophy. Therefore, we believe that this could be applied to the potential gap in muscle research that could be filled by tuning the well-established marker expression to improve dystrophic muscle.