RESUMO
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
Myogenic transcription factors with a basic helix-loop-helix (bHLH) such as MYOD, myogenin, MRF4, and MYF5 contribute to muscle differentiation and regulation. The MYF5 gene located on chromosome 12 encodes for myogenic factor 5 (MYF5), which has a role in skeletal and extraocular muscle development and rib formation. Variants in MYF5 were found to cause external ophthalmoplegia with rib and vertebral anomalies (EORVA), a rare recessive condition. To date, three homozygous variants in MYF5 have been reported to cause EORVA in six members of four unrelated families. Here, we present a novel homozygous MYF5 frameshift variant, c.596dupA p. (Asn199Lysfs*49), causing premature protein termination and presenting with external ophthalmoplegia, ptosis, and scoliosis in three siblings from a consanguineous family of Pakistani origin. With four MYF5 variants now discovered, genetic testing and paediatric assessment for extra-ocular features should be considered in all cases of congenital ophthalmoplegia.
Assuntos
Mutação da Fase de Leitura , Fator Regulador Miogênico 5 , Oftalmoplegia , Costelas , Criança , Feminino , Humanos , Masculino , Mutação da Fase de Leitura/genética , Homozigoto , Fator Regulador Miogênico 5/genética , Oftalmoplegia/genética , Oftalmoplegia/congênito , Linhagem , Costelas/anormalidades , Coluna Vertebral/anormalidades , Coluna Vertebral/patologiaRESUMO
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
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
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
Developmental pathways encompass transcription factors and cis-regulatory elements that interact as transcription factor-regulatory element (TF-RE) units. Independent origins of similar phenotypes likely involve changes in different parts of these units, a hypothesis promisingly tested addressing the evolution of the rib-associated lumbar (RAL) morphotype that characterizes emblematic animals such as snakes and elephants. Previous investigation in these lineages identified a polymorphism in the Homology region 1 [H1] enhancer of the Myogenic factor-5 [Myf5], which interacts with HOX10 proteins to modulate rib development. Here we address the evolution of TF-RE units focusing on independent origins of RAL morphotypes. We compiled an extensive database for H1-Myf5 and HOX10 sequences with two goals: (i) evaluate if the enhancer polymorphism is present in amphibians exhibiting the RAL morphotype and (ii) test a hypothesis of enhanced evolutionary flexibility mediated by TF-RE units, according to which independent origins of the RAL morphotype might involve changes in either component of the interaction unit. We identified the H1-Myf5 polymorphism in lineages that diverged around 340 Ma, including Lissamphibia. Independent origins of the RAL morphotype in Tetrapoda involved sequence variation in either component of the TF-RE unit, confirming that different changes may similarly affect the phenotypic outcome of a given developmental pathway.
Assuntos
Sequências Reguladoras de Ácido Nucleico , Fatores de Transcrição , Anfíbios/metabolismo , Animais , Fator Regulador Miogênico 5/genética , Fator Regulador Miogênico 5/metabolismo , Serpentes/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The vertebrate embryonic midline vasculature forms in close proximity to the developing skeletal muscle, which originates in the somites. Angioblasts migrate from bilateral positions along the ventral edge of the somites until they meet at the midline, where they sort and differentiate into the dorsal aorta and the cardinal vein. This migration occurs at the same time that myoblasts in the somites are beginning to differentiate into skeletal muscle, a process which requires the activity of the basic helix loop helix (bHLH) transcription factors Myod and Myf5. Here we examined vasculature formation in myod and myf5 mutant zebrafish. In the absence of skeletal myogenesis, angioblasts migrate normally to the midline but form only the cardinal vein and not the dorsal aorta. The phenotype is due to the failure to activate vascular endothelial growth factor ligand vegfaa expression in the somites, which in turn is required in the adjacent angioblasts for dorsal aorta specification. Myod and Myf5 cooperate with Hedgehog signaling to activate and later maintain vegfaa expression in the medial somites, which is required for angiogenic sprouting from the dorsal aorta. Our work reveals that the early embryonic skeletal musculature in teleosts evolved to organize the midline vasculature during development.
Assuntos
Proteína MyoD , Fatores de Regulação Miogênica , Animais , Aorta/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Proteínas Musculares/genética , Músculo Esquelético , Proteína MyoD/genética , Proteína MyoD/metabolismo , Fator Regulador Miogênico 5/genética , Fator Regulador Miogênico 5/metabolismo , Fatores de Regulação Miogênica/genética , Fatores de Regulação Miogênica/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
Myod and Myf5 are muscle-specific basic helix-loop-helix (bHLH) transcription factors that play essential roles in regulating skeletal muscle development and growth. In order to investigate potential function of myod and myf5 of Megalobrama amblycephala, an economically important freshwater fish species, in the present study, we characterized the sequences and expression profiles of M. amblycephala myod and myf5. The open reading frame (ORF) sequences of myod and myf5 encoded 275 and 240 amino acids, respectively, possessing analogous structure with the highly conserved domains, bHLH and C-terminal helix III domains. Spatio-temporal expression patterns revealed that myod and myf5 were predominant in skeletal muscle with the highest expression in white muscle, and the highest at 10 days post-hatching (dph) and the segmentation period, respectively. Furthermore, we evaluated the effects of lipopolysaccharide (LPS) on the expression of muscle-related genes in white and red muscle, and proliferation and differentiation of satellite cells. The myod, myf5 and pax-7 expression generally increased and then decreased with increase of LPS concentration and treatment time in red muscle, while these genes showed inconsistent expression patterns in white muscle. In addition, LPS administration caused the frequency increase of satellite cells in red and white muscle especially at 3 and 7 days after LPS-injection.
Assuntos
Cipriniformes , Células Satélites de Músculo Esquelético , Animais , Diferenciação Celular , Cipriniformes/metabolismo , Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/farmacologia , Músculo Esquelético/metabolismo , Proteína MyoD/genética , Proteína MyoD/metabolismo , Fator Regulador Miogênico 5/genética , Fator Regulador Miogênico 5/metabolismo , Fatores de Regulação Miogênica/genéticaRESUMO
BACKGROUND: The turtle carapace is an evolutionary novelty resulting from changes in the processes that build ribs and their associated muscles in most tetrapod species. Turtle embryos have several unique features that might play a role in this process, including the carapacial ridge, a Myf5 gene with shorter coding region that generates an alternative splice variant lacking exon 2, and unusual expression patterns of Lbx1 and HGF. RESULTS: We investigated these turtle-specific expression differences using genetic approaches in mouse embryos. At mid-gestation, mouse embryos producing Myf5 transcripts lacking exon 2 replicated some early properties of turtle somites, but still developed into viable and fertile mice. Extending Lbx1 expression into the hypaxial dermomyotomal lip of trunk somites to mimic the turtle Lbx1 expression pattern, produced fusions in the distal part of the ribs. CONCLUSIONS: Turtle-like Myf5 activity might generate a plastic state in developing trunk somites under which they can either enter carapace morphogenetic routes, possibly triggered by signals from the carapacial ridge, or still engage in the development of a standard tetrapod ribcage in the absence of those signals. In addition, trunk Lbx1 expression might play a later role in the formation of the lateral border of the carapace.
Assuntos
Tartarugas , Exoesqueleto , Animais , Evolução Biológica , Camundongos , Fator Regulador Miogênico 5/genética , Fator Regulador Miogênico 5/metabolismo , Plásticos/metabolismo , Somitos , Tartarugas/genéticaRESUMO
MicroRNAs play an important role in myogenic differentiation, they bind to target genes and regulate muscle formation. We previously found that miR-9-5p, which is related to bone formation, was increased over time during the process of myogenic differentiation. However, the mechanism by which miR-9-5p regulates myogenic differentiation remains largely unknown. In the present study, we first examined myotube formation and miR-9-5p, myogenesis-related genes including Dlx3, Myod1, Mef2c, Desmin, MyoG and Myf5 expression under myogenic induction. Then, we detected the expression of myogenic transcription factors after overexpression or knockdown of miR-9-5p or Dlx3 in the mouse premyoblast cell line C2C12 by qPCR, western blot and myotube formation under myogenic induction. A luciferase assay was performed to confirm the regulatory relationships between not only miR-9-5p and Dlx3 but also Dlx3 and its downstream gene, Myf5, which is an essential transcription factor of myogenic differentiation. The results showed that miR-9-5p promoted myogenic differentiation by increasing myogenic transcription factor expression and promoting myotube formation, but Dlx3 exerted the opposite effect. Moreover, the luciferase assay showed that miR-9-5p bound to the 3'UTR of Dlx3 and downregulated Dlx3 expression. Dlx3 in turn suppressed Myf5 expression by binding to the Myf5 promoter, ultimately inhibiting the process of myogenic differentiation. In conclusion, the miR-9-5p/Dlx3/Myf5 axis is a novel pathway for the regulation of myogenic differentiation, and can be a potential target to treat the diseases related to muscle dysfunction.
Assuntos
MicroRNAs , Camundongos , Animais , MicroRNAs/genética , Diferenciação Celular/genética , Linhagem Celular , Fatores de Transcrição/genética , Desenvolvimento Muscular/genética , Fator Regulador Miogênico 5/genéticaRESUMO
Splicing factor SRSF2 acts as a critical regulator for cell survival, however, it remains unknown whether SRSF2 is involved in myoblast proliferation and myogenesis. Here, knockdown of SRSF2 in myoblasts causes high rates of apoptosis and defective differentiation. Combined conditional knockout and lineage tracing approaches show that Myf5-cre mice lacking SRSF2 die immediately at birth and exhibit a complete absence of mature myofibers. Mutant Myf5-derived cells (tdtomato-positive cells) are randomly scattered in the myogenic and non-myogenic regions, indicating loss of the community effect required for skeletal muscle differentiation. Single-cell RNA-sequencing reveals high heterogeneity of myf5-derived cells and non-myogenic cells are significantly increased at the expense of skeletal muscle cells in the absence of SRSF2, reflecting altered cell fate. SRSF2 is demonstrated to regulate the entry of Myf5 cells into the myogenic program and ensures their survival by preventing precocious differentiation and apoptosis. In summary, SRSF2 functions as an essential regulator for Myf5-derived cells to respond correctly to positional cues and to adopt their myogenic fate.
Assuntos
Desenvolvimento Muscular , Músculo Esquelético , Animais , Camundongos , Camundongos Knockout , Desenvolvimento Muscular/genética , Músculo Esquelético/fisiologia , Fator Regulador Miogênico 5/genética , Análise de Sequência de RNARESUMO
Myogenic factor 5 plays active roles in the regulation of myogenesis. The aim of this study is to expose the genetic variants of the MYF5 and its association with growth performance and ultrasound traits in grassland short-tailed sheep (GSTS) in China. The combination technique of sequencing and SNaPshot revealed seven SNPs in ovine MYF5 from 533 adult individuals (male 103 and female 430), four of which are novel ones located at g.6838G > A, g.6989 G > T, g.7117 C > A in the promoter region and g.9471 T > G in the second intron, respectively. Genetic diversity indexes showed the seven SNPs in low or intermediate level, but each of them conformed HWE (p > 0.05) in genotypic frequencies. Association analysis indicated that g.6838G > A, g.7117 C > A, g.8371 T > C, g.9471 T > G, and g.10044 C > T had significant effects on growth performance and ultrasound traits. The diplotypes of H1H3 and H2H3 had higher body weight and greater body size, and haplotype H3 had better performance on meat production than the others. In addition, the dual-luciferase reporter assay showed that there are two active regions in the MYF5 promoter located at −1799~−1197 bp and −514~−241 bp, respectively, but g.6838G > A and g.7117 C > A were out of the region, suggesting these two SNPs influence the phenotype by other pathway. The results suggest that the MYF5 gene might be applied as a promising candidate of functional genetic marker in GSTS breeding.
Assuntos
Pradaria , Polimorfismo de Nucleotídeo Único , Animais , Feminino , Haplótipos , Masculino , Fator Regulador Miogênico 5/genética , Fenótipo , OvinosRESUMO
Muscle stem (satellite) cells express Pax7, a key transcription factor essential for satellite cell maintenance and adult muscle regeneration. We identify the corepressor transducin-like enhancer of split-4 (TLE4) as a Pax7 interaction partner expressed in quiescent satellite cells under homeostasis. A subset of satellite cells transiently downregulate TLE4 during early time points following muscle injury. We identify these to be activated satellite cells, and that TLE4 downregulation is required for Myf5 activation and myogenic commitment. Our results indicate that TLE4 represses Pax7-mediated Myf5 transcriptional activation by occupying the -111â kb Myf5 enhancer to maintain quiescence. Loss of TLE4 function causes Myf5 upregulation, an increase in satellite cell numbers and altered differentiation dynamics during regeneration. Thus, we have uncovered a novel mechanism to maintain satellite cell quiescence and regulate muscle differentiation mediated by the corepressor TLE4.
Assuntos
Diferenciação Celular , Desenvolvimento Muscular , Músculo Esquelético , Proteínas Nucleares , Proteínas Repressoras , Diferenciação Celular/genética , Humanos , Desenvolvimento Muscular/genética , Músculo Esquelético/citologia , Músculo Esquelético/lesões , Doenças Musculares/fisiopatologia , Fator Regulador Miogênico 5/genética , Fator Regulador Miogênico 5/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fator de Transcrição PAX7/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Células Satélites de Músculo Esquelético/citologiaRESUMO
This study was conducted to ivnestigate the associations of GH-AluI, STAT5A-AvaI and MYF5-TaqI gene polymorphisms with milk somatic cell count (SCC), electrical conductivity (EC) and pH levels in Holstein dairy cows. For this purpose, 167 blood and 1670 milk samples of 167 Holstein cows in their 2nd lactation were used. There were significant relationships between GH-AluI genotypes and milk EC (p < 0.001) and between STAT5A-AvaI genotypes and milk EC (p = 0.007), but there were not any significant relationships between MYF5 gene polymorphism and the investigated traits (p > 0.05). The greatest EC values were observed in GH-AluI-LV and STAT5A-AvaI-TT-genotyped individuals. Just because of association of EC with mastitis, it was concluded that present GH-AluI and STAT5A-AvaI polymorphisms could be used in further studies to be conducted to improve mastitis resistance and milk quality traits of Holstein dairy cows.
Assuntos
Doenças dos Bovinos , Hormônio do Crescimento/genética , Mastite Bovina , Fator Regulador Miogênico 5/genética , Fator de Transcrição STAT5/genética , Animais , Bovinos/genética , Contagem de Células , Condutividade Elétrica , Feminino , Concentração de Íons de Hidrogênio , Lactação/genética , Leite , Polimorfismo Genético/genéticaRESUMO
[Figure: see text].
Assuntos
Células Endoteliais/metabolismo , Endotélio Vascular/citologia , Morfogênese , Mioblastos/metabolismo , Proteína Proto-Oncogênica c-fli-1/metabolismo , Animais , Diferenciação Celular , Células Cultivadas , Células Endoteliais/citologia , Endotélio Vascular/embriologia , Endotélio Vascular/metabolismo , Mesoderma/citologia , Mesoderma/embriologia , Mesoderma/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteína MyoD/genética , Proteína MyoD/metabolismo , Mioblastos/citologia , Fator Regulador Miogênico 5/genética , Fator Regulador Miogênico 5/metabolismo , Proteína Proto-Oncogênica c-fli-1/genéticaRESUMO
Inductors of myogenic stem cell differentiation attract attention, as they can be used to treat myodystrophies and post-traumatic injuries. Functionalization of fullerenes makes it possible to obtain water-soluble derivatives with targeted biochemical activity. This study examined the effects of the phosphonate C60 fullerene derivatives on the expression of myogenic transcription factors and myogenic differentiation of human mesenchymal stem cells (MSCs). Uptake of the phosphonate C60 fullerene derivatives in human MSCs, intracellular ROS visualization, superoxide scavenging potential, and the expression of myogenic, adipogenic, and osteogenic differentiation genes were studied. The prolonged MSC incubation (within 7-14 days) with the C60 pentaphoshonate potassium salt promoted their differentiation towards the myogenic lineage. The transcription factors and gene expressions determining myogenic differentiation (MYOD1, MYOG, MYF5, and MRF4) increased, while the expression of osteogenic differentiation factors (BMP2, BMP4, RUNX2, SPP1, and OCN) and adipogenic differentiation factors (CEBPB, LPL, and AP2 (FABP4)) was reduced or did not change. The stimulation of autophagy may be one of the factors contributing to the increased expression of myogenic differentiation genes in MSCs. Autophagy may be caused by intracellular alkalosis and/or short-term intracellular oxidative stress.
Assuntos
Fulerenos/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Desenvolvimento Muscular , Autofagia , Diferenciação Celular , Células Cultivadas , Regulação da Expressão Gênica , Humanos , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/fisiologia , Proteína MyoD/genética , Fator Regulador Miogênico 5/genética , Miogenina/genética , Espécies Reativas de Oxigênio/metabolismoRESUMO
Induced pluripotent stem (iPS) cells constitute a perfect tool to study human embryo development processes such as myogenesis, thanks to their ability to differentiate into three germ layers. Currently, many protocols to obtain myogenic cells have been described in the literature. They differ in many aspects, such as media components, including signaling modulators, feeder layer constituents, and duration of culture. In our study, we compared three different myogenic differentiation protocols to verify, side by side, their efficiency. Protocol I was based on embryonic bodies differentiation induction, ITS addition, and selection with adhesion to collagen I type. Protocol II was based on strong myogenic induction at the embryonic bodies step with BIO, forskolin, and bFGF, whereas cells in Protocol III were cultured in monolayers in three special media, leading to WNT activation and TGF-ß and BMP signaling inhibition. Myogenic induction was confirmed by the hierarchical expression of myogenic regulatory factors MYF5, MYOD, MYF6 and MYOG, as well as the expression of myotubes markers MYH3 and MYH2, in each protocol. Our results revealed that Protocol III is the most efficient in obtaining myogenic cells. Furthermore, our results indicated that CD56 is not a specific marker for the evaluation of myogenic differentiation.
Assuntos
Técnicas de Cultura de Células , Meios de Cultura/farmacologia , Corpos Embrioides/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Desenvolvimento Muscular/efeitos dos fármacos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Colforsina/farmacologia , Colágeno Tipo I/farmacologia , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Corpos Embrioides/citologia , Corpos Embrioides/metabolismo , Fator 2 de Crescimento de Fibroblastos/farmacologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Expressão Gênica , Humanos , Indóis/farmacologia , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Insulina/farmacologia , Desenvolvimento Muscular/genética , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/metabolismo , Proteína MyoD/genética , Proteína MyoD/metabolismo , Fator Regulador Miogênico 5/genética , Fator Regulador Miogênico 5/metabolismo , Fatores de Regulação Miogênica/genética , Fatores de Regulação Miogênica/metabolismo , Miogenina/genética , Miogenina/metabolismo , Oximas/farmacologia , Selênio/farmacologia , Transferrina/farmacologiaRESUMO
Mechanical stimulation has benefits for muscle mass and function. Passive stretching is widely performed in clinical rehabilitation medicine. However, the hypertrophic effects of passive repetitive stretching on senescent skeletal muscles against muscle atrophy remain unknown. We used senescence-accelerated model SAM-P8 mice. The gastrocnemius muscle was passively repetitive stretched by manual ankle dorsiflexion for 15 min, 5 days a week for 2 weeks under deep anesthesia. We examined the effects of passive stretching on muscle mass, myofiber cross-sectional area, muscle fiber type composition, satellite cell and myonuclei content, signaling pathways involved in muscle protein synthesis, and myogenic regulatory factors. The gastrocnemius muscle weight and fiber cross-sectional area of the stretched side was found greater compared with that of the unstretched side. Passive repetitive stretching increased the mRNA expression level of Akt, p70S6K, 4E-BP1, Myf5, myogenin, MuRF1.The phosphorylation level of p70S6K significantly increased in the stretched muscles, whereas of Akt and 4E-BP1 remained unchanged, compared to the unstretched side. The Pax7+ cells and myonuclei content did not differ between the stretched and unstretched muscles. These findings suggest that the hypertrophic or suppressed atrophic observation in the stretched muscles are mainly attributable to the protein turnover provoked by stretching. These findings are applicable to clinical muscle strengthening and sarcopenia prevention.
Assuntos
Exercícios de Alongamento Muscular , Músculo Esquelético/patologia , Sarcopenia/patologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas de Ciclo Celular/genética , Camundongos , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Proteínas Musculares/genética , Músculo Esquelético/enzimologia , Músculo Esquelético/metabolismo , Fator Regulador Miogênico 5/genética , Tamanho do Órgão , Proteínas Proto-Oncogênicas c-akt/genética , RNA Mensageiro/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/genética , Sarcopenia/enzimologia , Sarcopenia/metabolismo , Proteínas com Motivo Tripartido/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
Because post-mortem human skeletal muscle is not viable, autologous muscle grafts are typically required in tissue reconstruction after muscle loss due to disease or injury. However, the use of autologous tissue often leads to donor-site morbidity. Here, we show that intraspecies and interspecies chimaeric pig embryos lacking native skeletal muscle can be produced by deleting the MYF5, MYOD and MYF6 genes in the embryos via CRISPR, followed by somatic-cell nuclear transfer and the delivery of exogenous cells (porcine blastomeres or human induced pluripotent stem cells) via blastocyst complementation. The generated intraspecies chimaeras were viable and displayed normal histology, morphology and function. Human:pig chimaeras generated with TP53-null human induced pluripotent stem cells led to higher chimaerism efficiency, with embryos collected at embryonic days 20 and 27 containing humanized muscle, as confirmed by immunohistochemical and molecular analyses. Human:pig chimaeras may facilitate the production of exogenic organs for research and xenotransplantation.