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1.
Zool Res ; 42(5): 650-659, 2021 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-34472226

RESUMO

Phosphatidylserine (PS) is distributed asymmetrically in the plasma membrane of eukaryotic cells. Phosphatidylserine flippase (P4-ATPase) transports PS from the outer leaflet of the lipid bilayer to the inner leaflet of the membrane to maintain PS asymmetry. The ß subunit TMEM30A is indispensable for transport and proper function of P4-ATPase. Previous studies have shown that the ATP11A and TMEM30A complex is the molecular switch for myotube formation. However, the role of Tmem30a in skeletal muscle regeneration remains elusive. In the current study, Tmem30a was highly expressed in the tibialis anterior (TA) muscles of dystrophin-null ( mdx) mice and BaCl 2-induced muscle injury model mice. We generated a satellite cell (SC)-specific Tmem30a conditional knockout (cKO) mouse model to investigate the role of Tmem30a in skeletal muscle regeneration. The regenerative ability of cKO mice was evaluated by analyzing the number and diameter of regenerated SCs after the TA muscles were injured by BaCl 2-injection. Compared to the control mice, the cKO mice showed decreased Pax7 + and MYH3 + SCs, indicating diminished SC proliferation, and decreased expression of muscular regulatory factors (MYOD and MYOG), suggesting impaired myoblast proliferation in skeletal muscle regeneration. Taken together, these results demonstrate the essential role of Tmem30a in skeletal muscle regeneration.


Assuntos
Proteínas de Membrana/metabolismo , Músculo Esquelético/fisiologia , Regeneração/fisiologia , Células Satélites de Músculo Esquelético/metabolismo , Animais , Proliferação de Células , Distrofina/genética , Distrofina/metabolismo , Antagonistas de Estrogênios/toxicidade , Regulação da Expressão Gênica/fisiologia , Genótipo , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos mdx , Camundongos Knockout , Músculo Esquelético/efeitos dos fármacos , Proteína MyoD/genética , Proteína MyoD/metabolismo , Miogenina/genética , Miogenina/metabolismo , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Fator de Transcrição PAX7/genética , Fator de Transcrição PAX7/metabolismo , Regeneração/genética , Tamoxifeno/toxicidade
2.
J Int Soc Sports Nutr ; 18(1): 56, 2021 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-34246303

RESUMO

BACKGROUND: The effects of low muscle glycogen on molecular markers of protein synthesis and myogenesis before and during aerobic exercise with carbohydrate ingestion is unclear. The purpose of this study was to determine the effects of initiating aerobic exercise with low muscle glycogen on mTORC1 signaling and markers of myogenesis. METHODS: Eleven men completed two cycle ergometry glycogen depletion trials separated by 7-d, followed by randomized isocaloric refeeding for 24-h to elicit low (LOW; 1.5 g/kg carbohydrate, 3.0 g/kg fat) or adequate (AD; 6.0 g/kg carbohydrate, 1.0 g/kg fat) glycogen. Participants then performed 80-min of cycle ergometry (64 ± 3% VO2peak) while ingesting 146 g carbohydrate. mTORC1 signaling (Western blotting) and gene transcription (RT-qPCR) were determined from vastus lateralis biopsies before glycogen depletion (baseline, BASE), and before (PRE) and after (POST) exercise. RESULTS: Regardless of treatment, p-mTORC1Ser2448, p-p70S6KSer424/421, and p-rpS6Ser235/236 were higher (P < 0.05) POST compared to PRE and BASE. PAX7 and MYOGENIN were lower (P < 0.05) in LOW compared to AD, regardless of time, while MYOD was lower (P < 0.05) in LOW compared to AD at PRE, but not different at POST. CONCLUSION: Initiating aerobic exercise with low muscle glycogen does not affect mTORC1 signaling, yet reductions in gene expression of myogenic regulatory factors suggest that muscle recovery from exercise may be reduced.


Assuntos
Metabolismo dos Carboidratos , Exercício Físico/fisiologia , Glicogênio/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Desenvolvimento Muscular/fisiologia , Músculo Esquelético/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Adulto , Biomarcadores/sangue , Metabolismo dos Carboidratos/genética , Estudos Cross-Over , Ergometria/métodos , Glicogênio/deficiência , Humanos , Masculino , Proteína MyoD/metabolismo , Miogenina/metabolismo , Fator de Transcrição PAX7/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/metabolismo , Fatores de Tempo , Transcrição Genética , Adulto Jovem
3.
Cell Death Dis ; 12(6): 535, 2021 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-34035232

RESUMO

Tet dioxygenases are responsible for the active DNA demethylation. The functions of Tet proteins in muscle regeneration have not been well characterized. Here we find that Tet2, but not Tet1 and Tet3, is specifically required for muscle regeneration in vivo. Loss of Tet2 leads to severe muscle regeneration defects. Further analysis indicates that Tet2 regulates myoblast differentiation and fusion. Tet2 activates transcription of the key differentiation modulator Myogenin (MyoG) by actively demethylating its enhancer region. Re-expressing of MyoG in Tet2 KO myoblasts rescues the differentiation and fusion defects. Further mechanistic analysis reveals that Tet2 enhances MyoD binding by demethylating the flanking CpG sites of E boxes to facilitate the recruitment of active histone modifications and increase chromatin accessibility and activate its transcription. These findings shed new lights on DNA methylation and pioneer transcription factor activity regulation.


Assuntos
Dioxigenases/fisiologia , Músculos/fisiologia , Regeneração/genética , Animais , Diferenciação Celular/genética , Células Cultivadas , Dioxigenases/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mioblastos/metabolismo , Mioblastos/fisiologia , Miogenina/genética , Miogenina/metabolismo
4.
Mol Med Rep ; 24(1)2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33955507

RESUMO

The protein extracted from red algae Pyropia yezoensis has various biological activities, including anti­inflammatory, anticancer, antioxidant, and antiobesity properties. However, the effects of P. yezoensis protein (PYCP) on tumor necrosis factor­α (TNF­α)­induced muscle atrophy are unknown. Therefore, the present study investigated the protective effects and related mechanisms of PYCP against TNF­α­induced myotube atrophy in C2C12 myotubes. Treatment with TNF­α (20 ng/ml) for 48 h significantly reduced myotube viability and diameter and increased intracellular reactive oxygen species levels; these effects were significantly reversed in a dose­dependent manner following treatment with 25­100 µg/ml PYCP. PYCP inhibited the expression of TNF receptor­1 in TNF­α­induced myotubes. In addition, PYCP markedly downregulated the nuclear translocation of nuclear factor­κB (NF­κB) by inhibiting the phosphorylation of inhibitor of κB. Furthermore, PYCP treatment suppressed 20S proteasome activity, IL­6 production, and the expression of the E3 ubiquitin ligases, atrogin­1/muscle atrophy F­box and muscle RING­finger protein­1. Finally, PYCP treatment increased the protein expression levels of myoblast determination protein 1 and myogenin in TNF­α­induced myotubes. The present findings indicate that PYCP may protect against TNF­α­induced myotube atrophy by inhibiting the proinflammatory NF­κB pathway.


Assuntos
Proteínas de Algas/farmacologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Atrofia Muscular/tratamento farmacológico , Substâncias Protetoras/farmacologia , Rodófitas/química , Transdução de Sinais/efeitos dos fármacos , Animais , Sobrevivência Celular/efeitos dos fármacos , Interleucina-6/metabolismo , Camundongos , Desenvolvimento Muscular/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Proteínas Musculares/metabolismo , Atrofia Muscular/induzido quimicamente , Atrofia Muscular/metabolismo , Proteína MyoD/metabolismo , Miogenina/metabolismo , NF-kappa B/metabolismo , Cultura Primária de Células , Complexo de Endopeptidases do Proteassoma/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Proteínas Ligases SKP Culina F-Box/metabolismo , Proteínas com Motivo Tripartido/metabolismo , Fator de Necrose Tumoral alfa/toxicidade , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
5.
Life Sci ; 277: 119520, 2021 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-33887345

RESUMO

AIMS: Mouse bone marrow mesenchymal stem cells (BMSCs) are pluripotent cells with self-renewal and differentiation abilities. Since the effects of senescent BMSCs on C2C12 cells are not fully clear, the present study aimed to elucidate these effects. MAIN METHODS: Senescence-associated ß-galactosidase staining and western blotting were performed to confirm the senescence of BMSCs. Immunofluorescence and western blotting were used to assess myoblast differentiation in each group. The role of the AKT/P70 signaling pathway and forkhead box O3 (FOXO3) nuclear translocation was explored by western blotting. BMSC-derived exosomes were injected into the tibialis anterior of mice, and RT-qPCR was used to assess the role of exosomes in promoting muscle differentiation. KEY FINDINGS: Conditioned medium (CM) from early-senescent BMSCs promoted myogenic differentiation in vitro, which was detected as enhanced expression of myosin heavy chain (MHC), myogenin (MYOG), and myogenic differentiation 1 (MyoD). The AKT signaling pathway was found to be regulated by CM, which inhibited FOXO3 nuclear translocation. RT-qPCR analysis results showed that MHC, MyoD, and MYOG mRNA expression increased in the tibialis anterior of mice after exosome injection. SIGNIFICANCE: The present study demonstrated that early-senescent BMSCs accelerated C2C12 cell myogenic differentiation, and the transcription factor, FOXO3, was the target of senescent cells. Collectively, our results suggest that the AKT/P70 signaling pathway mediates the effect of BMSCs on neighboring cells.


Assuntos
Diferenciação Celular/fisiologia , Proteína Forkhead Box O3/metabolismo , Células-Tronco Mesenquimais/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Células da Medula Óssea/citologia , Linhagem Celular , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Células-Tronco Mesenquimais/fisiologia , Camundongos , Desenvolvimento Muscular/fisiologia , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , Miogenina/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo
6.
Photochem Photobiol Sci ; 20(4): 571-583, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33895984

RESUMO

Envenoming caused by snakebites is a very important neglected tropical disease worldwide. The myotoxic phospholipases present in the bothropic venom disrupt the sarcolemma and compromise the mechanisms of energy production, leading to myonecrosis. Photobiomodulation therapy (PBMT) has been used as an effective tool to treat diverse cases of injuries, such as snake venom-induced myonecrosis. Based on that, the aim of this study was to analyze the effects of PBMT through low-level laser irradiation (904 nm) on the muscle regeneration after the myonecrosis induced by Bothrops jararacussu snake venom (Bjssu) injection, focusing on myogenic regulatory factors expression, such as Pax7, MyoD, and Myogenin (MyoG). Male Swiss mice (Mus musculus), 6-8-week-old, weighing 22 ± 3 g were used. Single sub-lethal Bjssu dose or saline was injected into the right mice gastrocnemius muscle. At 3, 24, 48, and 72 h after injections, mice were submitted to PBMT treatment. When finished the periods of 48 and 72 h, mice were euthanized and the right gastrocnemius were collected for analyses. We observed extensive inflammatory infiltrate in all the groups submitted to Bjssu injections. PBMT was able to reduce the myonecrotic area at 48 and 72 h after envenomation. There was a significant increase of MyoG mRNA expression at 72 h after venom injection. The data suggest that beyond the protective effect promoted by PBMT against Bjssu-induced myonecrosis, the low-level laser irradiation was able to stimulate the satellite cells, thus enhancing the muscle repair by improving myogenic differentiation.


Assuntos
Bothrops , Venenos de Crotalídeos/toxicidade , Regulação da Expressão Gênica/efeitos da radiação , Terapia a Laser , Miogenina/metabolismo , Necrose/terapia , Animais , Diferenciação Celular , Terapia com Luz de Baixa Intensidade , Masculino , Camundongos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/efeitos da radiação , Miogenina/genética
7.
Am J Respir Cell Mol Biol ; 64(5): 617-628, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33689672

RESUMO

Skeletal muscle dysfunction is one of the important comorbidities of chronic obstructive pulmonary disease (COPD); however, the underlying mechanisms remain largely unknown. RANKL (receptor activator of nuclear factor κB ligand), a key mediator in osteoclast differentiation, was also found to play a role in skeletal muscle pathogenesis. Whether RANKL is involved in COPD-related skeletal muscle dysfunction is as-of-yet unknown. We examined the expression of RANKL/RANK in skeletal muscles from mice exposed to cigarette smoke (CS) for 24 weeks. Grip strength and exercise capacity as well as muscular morphology were evaluated in CS-exposed mice with or without anti-RANKL treatment. The expressions of protein synthesis- or muscle growth-related molecules (IGF-1, myogenin, and myostatin), muscle-specific ubiquitin E3 ligases (MuRF1 and atrogin-1), and the NF-κb inflammatory pathway were also evaluated in skeletal muscles. The effect of CS extract on RANKL/RANK expression and that of exogenous RANKL on the ubiquitin-proteasome pathway in C2C12 myotubes were investigated in vitro. Long-term CS exposure induced skeletal muscle dysfunction and atrophy together with upregulation of RANKL/RANK expression in a well-established mouse model of COPD. RANKL neutralization prevented skeletal muscle dysfunction and atrophy. RANKL inhibition decreased expressions of myostatin and MuRF1/Atrogin1 and suppressed the NF-κb pathway in skeletal muscles from CS-exposed mice. In in vitro experiments with C2C12 myotubes, CS extract induced expression of RANKL/RANK, and exogenous RANKL induced activation of the ubiquitin-proteasome pathway and NF-κb pathway via RANK. Our results revealed an important role of the RANKL/RANK pathway in muscle atrophy induced by CS exposure, suggesting that RANKL may be a potential therapeutic target in COPD-related skeletal muscle dysfunction.


Assuntos
Atrofia Muscular/genética , NF-kappa B/genética , Doença Pulmonar Obstrutiva Crônica/genética , Ligante RANK/genética , Receptor Ativador de Fator Nuclear kappa-B/genética , Animais , Anticorpos Neutralizantes/farmacologia , Linhagem Celular , Fumar Cigarros/efeitos adversos , Misturas Complexas/antagonistas & inibidores , Misturas Complexas/farmacologia , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica , Força da Mão/fisiologia , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Força Muscular/efeitos dos fármacos , Força Muscular/genética , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Atrofia Muscular/metabolismo , Atrofia Muscular/patologia , Atrofia Muscular/prevenção & controle , Miogenina/genética , Miogenina/metabolismo , Miostatina/genética , Miostatina/metabolismo , NF-kappa B/metabolismo , Doença Pulmonar Obstrutiva Crônica/induzido quimicamente , Doença Pulmonar Obstrutiva Crônica/metabolismo , Doença Pulmonar Obstrutiva Crônica/prevenção & controle , Ligante RANK/antagonistas & inibidores , Ligante RANK/metabolismo , Receptor Ativador de Fator Nuclear kappa-B/metabolismo , Proteínas Ligases SKP Culina F-Box/genética , Proteínas Ligases SKP Culina F-Box/metabolismo , Transdução de Sinais , Proteínas com Motivo Tripartido/genética , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
8.
Biochem Biophys Res Commun ; 552: 84-90, 2021 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-33743352

RESUMO

BACKGROUND: Angiotensin II (Ang II), an important component of the renin-angiotensin system (RAS), plays a critical role in the pathogenesis of cardiovascular disorders. In addition, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been considered as a promising platform for studying personalized medicine for heart diseases. However, whether Ang II can induce the apoptosis of hiPSC-CMs is not known. METHODS: In this study, we treated hiPSC-CMs with different concentrations of Ang II [0 nM (vehicle as a control), 1 nM, 10 nM, 100 nM, 1 µM, 10 µM, 100 µM, and 1 mM] for various time periods (24 h, 48 h, 6 days, and 10 days) and analyzed the viability and apoptosis of hiPSC-CMs. RESULTS: We found that treatment with 1 mM Ang II for 10 days reduced the viability of hiPSC-CMs by 41% (p = 2.073E-08) and increased apoptosis by 2.74-fold, compared to the control group (p = 6.248E-12). MYOG, which encodes the muscle-specific transcription factor myogenin, was also identified as an apoptosis-suppressor gene in Ang II-treated hiPSC-CMs. Ectopic MYOG expression decreased the apoptosis and increased the viability of Ang II-treated hiPSC-CMs. Further analysis of the RNA sequencing (RNA-seq) data illustrated that myogenin ameliorated Ang II-induced apoptosis of hiPSC-CMs by downregulating the expression of proinflammatory genes. CONCLUSION: Our findings suggest that Ang II induces the apoptosis of hiPSC-CMs and that myogenin attenuates Ang II-induced apoptosis.


Assuntos
Angiotensina II/farmacologia , Apoptose/efeitos dos fármacos , Regulação da Expressão Gênica , Células-Tronco Pluripotentes Induzidas/metabolismo , Miócitos Cardíacos/metabolismo , Miogenina/genética , Apoptose/genética , Células Cultivadas , Perfilação da Expressão Gênica/métodos , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia , Miogenina/metabolismo , Fatores de Tempo
9.
Biosci Biotechnol Biochem ; 85(5): 1227-1234, 2021 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-33704409

RESUMO

Among many factors of controlling stem cell differentiation, the key transcription factor upregulation via physical force is a good strategy on the lineage-specific differentiation of stem cells. The study aimed to compare growth and myogenic potentials between the parental cells (PCs) and the 1-day-old C2C12 spheroid-derived cells (SDCs) in two-dimensional (2D) and three-dimensional (3D) culture conditions through examination of the cell proliferation and the expression of myogenic genes. The data showed that 1-day-old spheroids had more intense expression of MyoD gene with respect to the PCs. The proliferation of the SDCs is significantly higher than the PCs in a time-dependent manner. The SDCs had also significantly higher myogenic potential than the PCs in 2D and 3D culture conditions. The results suggest that MyoD gene upregulation through cell-cell contacts is the good approach for preparation of seed cells in muscle tissue engineering.


Assuntos
Técnicas de Cultura de Células , Células Musculares/metabolismo , Desenvolvimento Muscular/genética , Proteína MyoD/genética , Mioblastos/metabolismo , Esferoides Celulares/metabolismo , Actinina/genética , Actinina/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Colágeno/química , Colágeno/farmacologia , Regulação da Expressão Gênica , Camundongos , Células Musculares/citologia , Células Musculares/efeitos dos fármacos , Desenvolvimento Muscular/efeitos dos fármacos , Proteína MyoD/antagonistas & inibidores , Proteína MyoD/metabolismo , Mioblastos/citologia , Mioblastos/efeitos dos fármacos , Miogenina/genética , Miogenina/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Esferoides Celulares/citologia , Esferoides Celulares/efeitos dos fármacos , Engenharia Tecidual/métodos
10.
Biomed Res Int ; 2021: 4218086, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33628781

RESUMO

The capability of regeneration for skeletal muscle after injury depends on the differentiation and proliferation ability of the resident stem cells called satellite cells. It has been reported that electrical stimulation was widely used in clinical conditions to facilitate muscle regeneration after injury, but the characterization of satellite cell responses to the context of low-frequency electrical stimulation in early-phase muscle strain conditions has not been fully clarified. In this study, we aim to investigate the effects of low-frequency electrical stimulation (frequency: 20 Hz; duration: 30 minutes, twice daily) on satellite cell activities in a rat model for the early phase of muscle strain. Firstly, we adopted our previously developed rat model to mimic the early phase of muscle strain in human. After then, we examined the effects of low-frequency electrical stimulation on histopathological changes of the muscle fiber by hematoxylin and eosin (H&E) staining. Finally, we investigated the effects of low-frequency electrical stimulation on satellite cell proliferation and differentiation by quantification of the expression level of the specific proteins using western blot analyses. The muscle strain in biceps femoris muscles of rats can be induced by high-speed rotation from knee flexion 50° to full knee extension at 960°·s-1 angular velocity during its tetany by activating the sciatic nerve, as evidenced by a widening of the interstitial space between fibers, and more edema or necrosis fibers were detected in the model rats without treatment than in control rats. After treatment with low-frequency electrical stimulation (frequency: 20 Hz; duration: 30 minutes, twice daily), the acute strained biceps femoris muscles of rats showed obvious improvement of histomorphology as indicated by more mature muscle fibers with well-ordered formation with clear boundaries. Consistently, the expression levels of the MyoD and myogenin were marked higher than those in the rats in the animal model group, indicating increased satellite cell proliferating and differentiating activities by low-frequency electrical stimulation. This study shows that low-frequency electrical stimulation provides an effective stimulus to upregulate the protein expression of MyoD/myogenin and accelerate the restoration of structure during the early phase of muscle strain. This may have significance for clinical practice. Optimization of low-frequency electrical stimulation parameters may enhance the therapeutic outcome in patients.


Assuntos
Estimulação Elétrica , Fibras Musculares Esqueléticas , Regeneração/efeitos da radiação , Células Satélites de Músculo Esquelético , Animais , Masculino , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/patologia , Fibras Musculares Esqueléticas/fisiologia , Fibras Musculares Esqueléticas/efeitos da radiação , Proteína MyoD/metabolismo , Miogenina/metabolismo , Ratos , Ratos Sprague-Dawley , Células Satélites de Músculo Esquelético/citologia , Células Satélites de Músculo Esquelético/fisiologia , Células Satélites de Músculo Esquelético/efeitos da radiação
11.
Development ; 148(3)2021 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-33462116

RESUMO

SMAD2 is a transcription factor, the activity of which is regulated by members of the transforming growth factor ß (TGFß) superfamily. Although activation of SMAD2 and SMAD3 downstream of TGFß or myostatin signaling is known to inhibit myogenesis, we found that SMAD2 in the absence of TGFß signaling promotes terminal myogenic differentiation. We found that, during myogenic differentiation, SMAD2 expression is induced. Knockout of SMAD2 expression in primary myoblasts did not affect the efficiency of myogenic differentiation but produced smaller myotubes with reduced expression of the terminal differentiation marker myogenin. Conversely, overexpression of SMAD2 stimulated myogenin expression, and enhanced both differentiation and fusion, and these effects were independent of classical activation by the TGFß receptor complex. Loss of Smad2 in muscle satellite cells in vivo resulted in decreased muscle fiber caliber and impaired regeneration after acute injury. Taken together, we demonstrate that SMAD2 is an important positive regulator of myogenic differentiation, in part through the regulation of Myog.


Assuntos
Diferenciação Celular/fisiologia , Desenvolvimento Muscular/fisiologia , Miogenina/metabolismo , Proteína Smad2/metabolismo , Animais , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Técnicas de Inativação de Genes , Camundongos , Camundongos Knockout , Desenvolvimento Muscular/genética , Fibras Musculares Esqueléticas/metabolismo , Mioblastos/metabolismo , Miogenina/genética , Miostatina , Transdução de Sinais , Proteína Smad2/genética , Proteína Smad3 , Fator de Crescimento Transformador beta/metabolismo
12.
Nat Commun ; 12(1): 192, 2021 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-33420019

RESUMO

Rhabdomyosarcoma (RMS) is an aggressive pediatric malignancy of the muscle, that includes Fusion Positive (FP)-RMS harboring PAX3/7-FOXO1 and Fusion Negative (FN)-RMS commonly with RAS pathway mutations. RMS express myogenic master transcription factors MYOD and MYOG yet are unable to terminally differentiate. Here, we report that SNAI2 is highly expressed in FN-RMS, is oncogenic, blocks myogenic differentiation, and promotes growth. MYOD activates SNAI2 transcription via super enhancers with striped 3D contact architecture. Genome wide chromatin binding analysis demonstrates that SNAI2 preferentially binds enhancer elements and competes with MYOD at a subset of myogenic enhancers required for terminal differentiation. SNAI2 also suppresses expression of a muscle differentiation program modulated by MYOG, MEF2, and CDKN1A. Further, RAS/MEK-signaling modulates SNAI2 levels and binding to chromatin, suggesting that the differentiation blockade by oncogenic RAS is mediated in part by SNAI2. Thus, an interplay between SNAI2, MYOD, and RAS prevents myogenic differentiation and promotes tumorigenesis.


Assuntos
Carcinogênese/metabolismo , Diferenciação Celular , Proteína MyoD/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Rabdomiossarcoma/genética , Rabdomiossarcoma/metabolismo , Fatores de Transcrição da Família Snail/metabolismo , Animais , Carcinogênese/genética , Diferenciação Celular/genética , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Xenoenxertos , Humanos , Fatores de Transcrição MEF2/metabolismo , Masculino , Camundongos , Camundongos SCID , Desenvolvimento Muscular/genética , Proteína MyoD/genética , Miogenina/metabolismo , Proteínas de Fusão Oncogênica/genética , Oncogenes , Rabdomiossarcoma/patologia , Rabdomiossarcoma Alveolar/genética , Rabdomiossarcoma Embrionário/genética , Fatores de Transcrição da Família Snail/genética , Transcriptoma
13.
PLoS One ; 16(1): e0245618, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33465133

RESUMO

Skeletal muscle gene expression is governed by the myogenic regulatory family (MRF) which includes MyoD (MYOD1) and myogenin (MYOG). MYOD1 and MYOG are known to regulate an overlapping set of muscle genes, but MYOD1 cannot compensate for the absence of MYOG in vivo. In vitro, late muscle genes have been shown to be bound by both factors, but require MYOG for activation. The molecular basis for this requirement was unclear. We show here that MYOG is required for the recruitment of TBP and RNAPII to muscle gene promoters, indicating that MYOG is essential in assembling the transcription machinery. Genes regulated by MYOD1 and MYOG include genes required for muscle fusion, myomaker and myomerger, and we show that myomaker is fully dependent on activation by MYOG. We also sought to determine the role of MYOD1 in MYOG dependent gene activation and unexpectedly found that MYOG is required to maintain Myod1 expression. However, we also found that exogenous MYOD1 was unable to compensate for the loss of Myog and activate muscle gene expression. Thus, our results show that MYOD1 and MYOG act in a feed forward loop to maintain each other's expression and also show that it is MYOG, and not MYOD1, that is required to load TBP and activate gene expression on late muscle gene promoters bound by both factors.


Assuntos
Desenvolvimento Muscular/genética , Músculo Esquelético/metabolismo , Proteína MyoD/metabolismo , Mioblastos/metabolismo , Miogenina/metabolismo , Animais , Linhagem Celular , Imunoprecipitação da Cromatina , Fibroblastos/metabolismo , Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/genética , Técnicas de Inativação de Genes , Camundongos , Proteína MyoD/genética , Miogenina/genética , Organofosfatos/metabolismo , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , RNA Interferente Pequeno , Reação em Cadeia da Polimerase em Tempo Real , Ativação Transcricional
14.
Biochim Biophys Acta Mol Cell Res ; 1868(2): 118917, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33259860

RESUMO

Duchenne's muscular dystrophy (DMD) is a severe muscle wasting disorder characterized by the loss of dystrophin expression, muscle necrosis, inflammation and fibrosis. Ongoing muscle regeneration is impaired by persistent cytokine stress, further decreasing muscle function. Patients with DMD rarely survive beyond their early 20s, with cardiac and respiratory dysfunction being the primary cause of death. Despite an increase in our understanding of disease progression as well as promising preclinical animal models for therapeutic intervention, treatment options for muscular dystrophy remain limited and novel therapeutic targets are required. Many reports suggest that the TGFß signalling pathway is activated in dystrophic muscle and contributes to the pathology of DMD in part by impairing the differentiation of myoblasts into mature myofibers. Here, we show that in vitro knockdown of the Ste20-like kinase, SLK, can partially restore myoblast differentiation downstream of TGFß in a Smad2/3 independent manner. In an mdx model, we demonstrate that SLK is expressed at high levels in regenerating myofibers. Muscle-specific deletion of SLK reduced leukocyte infiltration, increased myogenin and utrophin expression and enhanced differentiation. This was accompanied by resistance to eccentric contraction-induced injury in slow fiber type-enriched soleus muscles. Finally, we found that these effects were partially dependent on the upregulation of p38 signalling. Collectively, these results demonstrate that SLK downregulation can restore some aspects of disease progression in DMD.


Assuntos
Técnicas de Inativação de Genes , Sistema de Sinalização das MAP Quinases/genética , Desenvolvimento Muscular/genética , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Células Cultivadas , Modelos Animais de Doenças , Cães , Camundongos , Camundongos Endogâmicos mdx , Camundongos Knockout , Distrofia Muscular de Duchenne/patologia , Mioblastos/metabolismo , Miogenina/metabolismo , Proteínas Serina-Treonina Quinases/genética , Fator de Crescimento Transformador beta/metabolismo
15.
Int J Dermatol ; 60(1): 93-98, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32530054

RESUMO

BACKGROUND: Cutaneous carcinosarcoma is a rare biphasic tumor comprising malignant epithelial and heterologous mesenchymal elements. Data on the clinical and histopathologic characteristics of this tumor are scarce. The objective of this study was to describe the clinicopathologic and immunohistochemical features of cutaneous carcinosarcoma. METHODS: A descriptive retrospective study was conducted in a tertiary care hospital from Spain. We reviewed the records of eight patients with cutaneous carcinosarcoma who were diagnosed from 2009 to 2019. RESULTS: The mean patient age at diagnosis was 72.13 years (range 44-91 years), and there was a male predilection (6 cases). The most common site of cutaneous carcinosarcoma was the head and neck (5 cases). Carcinosarcomas demonstrated variable histopathological and immunohistochemical features. Follow-up was available for 7-8 patients. There were two cases of local recurrence and one case of metastasis. Two patients died from the tumor during the entire follow-up. CONCLUSIONS: Although the number of cases in this study was limited, our results provide valuable insight into the clinical, histopathologic, and immunohistochemical characteristics of primary cutaneous carcinosarcoma.


Assuntos
Carcinossarcoma/metabolismo , Carcinossarcoma/patologia , Recidiva Local de Neoplasia/patologia , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Actinas/metabolismo , Adulto , Idoso de 80 Anos ou mais , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Carcinossarcoma/secundário , Carcinossarcoma/cirurgia , Desmina/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Queratina-1/metabolismo , Queratina-3/metabolismo , Masculino , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Proteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Miogenina/metabolismo , Neprilisina/metabolismo , Estudos Retrospectivos , Neoplasias Cutâneas/cirurgia , Fatores de Transcrição/metabolismo , alfa 1-Antitripsina/metabolismo
16.
FASEB J ; 35(1): e21154, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33140469

RESUMO

Myogenesis includes sequential stages of progenitor cell proliferation, myogenic commitment and differentiation, myocyte fusion, and myotube maturation. Different stages of myogenesis are orchestrated and regulated by myogenic regulatory factors and various downstream cellular signaling. Here we identify phosphatase orphan 1 (Phospho1) as a new player in myogenesis. During activation, proliferation, and differentiation of quiescent satellite cells, the expression of Phospho1 gradually increases. Overexpression of Phospho1 inhibits myoblast proliferation but promotes their differentiation and fusion. Conversely, knockdown of Phospho1 accelerates myoblast proliferation but impairs myotube formation. Moreover, knockdown of Phospho1 decreases the OXPHO protein levels and mitochondria density, whereas overexpression of Phospho1 upregulates OXPHO protein levels and promotes mitochondrial oxygen consumption. Finally, we show that Phospho1 expression is controlled by myogenin, which binds to the promoter of Phospho1 to regulate its transcription. These results indicate a key role of Phospho1 in regulating myogenic differentiation and mitochondrial function.


Assuntos
Diferenciação Celular , Proliferação de Células , Regulação Enzimológica da Expressão Gênica , Desenvolvimento Muscular , Mioblastos Esqueléticos/enzimologia , Monoéster Fosfórico Hidrolases/biossíntese , Animais , Camundongos , Mitocôndrias Musculares/genética , Mitocôndrias Musculares/metabolismo , Miogenina/genética , Miogenina/metabolismo , Monoéster Fosfórico Hidrolases/genética
17.
Muscle Nerve ; 63(2): 239-249, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33125736

RESUMO

INTRODUCTION: We conducted an open-label study to examine the effects of the flavonoid (-)-epicatechin in seven ambulatory adult patients with Becker muscular dystrophy (BMD). METHODS: Seven participants received (-)-epicatechin 50 mg twice per day for 8 weeks. Pre- and postprocedures included biceps brachii biopsy to assess muscle structure and growth-relevant endpoints by western blotting, mitochondria volume measurement, and cristae abundance by electron microscopy, graded exercise testing, and muscle strength and function tests. RESULTS: Western blotting showed significantly increased levels of enzymes modulating cellular bioenergetics (liver kinase B1 and 5'-adenosine monophosphate-activated protein kinase). Peroxisome proliferator-activated receptor gamma coactivator-1alpha, a transcriptional coactivator of genes involved in mitochondrial biogenesis and cristae-associated mitofilin levels, increased as did cristae abundance. Muscle and plasma follistatin increased significantly while myostatin decreased. Markers of skeletal muscle regeneration myogenin, myogenic regulatory factor-5, myoblast determination protein 1, myocyte enhancer factor-2, and structure-associated proteins, including dysferlin, utrophin, and intracellular creatine kinase, also increased. Exercise testing demonstrated decreased heart rate, maximal oxygen consumption per kilogram, and plasma lactate levels at defined workloads. Tissue saturation index improved in resting and postexercise states. DISCUSSION: (-)-Epicatechin, an exercise mimetic, appears to have short-term positive effects on tissue biomarkers indicative of mitochondrial biogenesis and muscle regeneration, and produced improvements in graded exercise testing parameters in patients with BMD.


Assuntos
Catequina/uso terapêutico , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/tratamento farmacológico , Adulto , Biópsia , Western Blotting , Creatina Quinase/metabolismo , Disferlina/metabolismo , Teste de Esforço , Folistatina/metabolismo , Frequência Cardíaca , Humanos , Ácido Láctico/sangue , Fatores de Transcrição MEF2/metabolismo , Masculino , Microscopia Eletrônica , Pessoa de Meia-Idade , Mitocôndrias/ultraestrutura , Proteínas Mitocondriais/metabolismo , Tamanho Mitocondrial , Proteínas Musculares/metabolismo , Força Muscular , Músculo Esquelético/fisiopatologia , Músculo Esquelético/ultraestrutura , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patologia , Distrofia Muscular de Duchenne/fisiopatologia , Proteína MyoD/metabolismo , Fator Regulador Miogênico 5/metabolismo , Miogenina/metabolismo , Miostatina/metabolismo , Biogênese de Organelas , Consumo de Oxigênio , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Regeneração , Utrofina/metabolismo
18.
Eur Rev Med Pharmacol Sci ; 24(23): 12041-12049, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33336722

RESUMO

OBJECTIVE: We investigated the effect of electrical stimulation (ES) of varying pulse frequency on differentiation and proliferation of canine myloglossus satellite cells in vitro. MATERIALS AND METHODS: Cellular viability and proliferation were assayed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay and flow cytometry fluorescence-activated cell sorting analysis. Cellular differentiation and expression of mark molecule were assayed by Real Time-PCR and Western blot. RESULTS: With increasing frequency ES, we found a significant increase in Myod (r=0.988, p<0.0001), myogenin (r=0.988, p<0.0001), MyHC-slow (r=0.988, p<0.0001), MyHC-fast (r=0.875, p<0.0001) protein expression, and Pax7 mRNA expression (r=0.712, p=0.001). CONCLUSIONS: Pax7 mRNA expression and MyoD, myogenin, and MyHC protein expression were increased with increment of electrical stimulation frequency in myloglossus muscle satellite. Higher frequency ES enhanced myloglossus satellite cell differentiation, not proliferation and viability.


Assuntos
Estimulação Elétrica , Células Satélites de Músculo Esquelético/metabolismo , Regulação para Cima , Animais , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Cães , Feminino , Proteína MyoD/genética , Proteína MyoD/metabolismo , Miogenina/genética , Miogenina/metabolismo , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Fator de Transcrição PAX7/genética , Fator de Transcrição PAX7/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células Satélites de Músculo Esquelético/citologia
19.
Int J Mol Sci ; 21(24)2020 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-33322515

RESUMO

Skeletal myogenesis is a multi-stage process that includes the cell cycle exit, myogenic transcriptional activation, and morphological changes to form multinucleated myofibers. Recent studies have shown that saturated fatty acids (SFA) and miRNAs play crucial roles in myogenesis and muscle homeostasis. Nevertheless, the target molecules and myogenic regulatory mechanisms of miRNAs are largely unknown, particularly when myogenesis is dysregulated by SFA deposition. This study investigated the critical role played by miR-96-5p on the myogenic differentiation in C2C12 myoblasts. Long-chain SFA palmitic acid (PA) significantly reduced FHL1 expression and inhibited the myogenic differentiation of C2C12 myoblasts but induced miR-96-5p expression. The knockdown of FHL1 by siRNA stimulated cell proliferation and inhibited myogenic differentiation of myoblasts. Interestingly, miR-96-5p suppressed FHL1 expression by directly targeting the 3'UTR of FHL1 mRNA. The transfection of an miR-96-5p mimic upregulated the expressions of cell cycle-related genes, such as PCNA, CCNB1, and CCND1, and increased myoblast proliferation. Moreover, the miR-96-5p mimic inhibited the expressions of myogenic factors, such as myoblast determination protein (MyoD), myogenin (MyoG), myocyte enhancer factor 2C (MEF2C), and myosin heavy chain (MyHC), and dramatically impeded differentiation and fusion of myoblasts. Overall, this study highlights the role of miR-96-5p in myogenesis via FHL1 suppression and suggests a novel regulatory mechanism for myogenesis mediated by miRNA in a background of obesity.


Assuntos
Ácido Palmítico/farmacologia , Regiões 3' não Traduzidas/genética , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Imunofluorescência , Immunoblotting , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas com Domínio LIM/genética , Proteínas com Domínio LIM/metabolismo , Fatores de Transcrição MEF2/genética , Fatores de Transcrição MEF2/metabolismo , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Desenvolvimento Muscular/efeitos dos fármacos , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Proteína MyoD/genética , Proteína MyoD/metabolismo , Miogenina/genética , Miogenina/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
20.
Aging (Albany NY) ; 12(21): 21446-21468, 2020 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-33136552

RESUMO

Skeletal muscle wasting represents both a common phenotype of aging and a feature of pathological conditions such as chronic kidney disease (CKD). Although both clinical data and genetic experiments in mice suggest that hyperphosphatemia accelerates muscle wasting, the underlying mechanism remains unclear. Here, we showed that inorganic phosphate (Pi) dose-dependently decreases myotube size, fusion index, and myogenin expression in mouse C2C12 skeletal muscle cells. These changes were accompanied by increases in reactive oxygen species (ROS) production and Nrf2 and p62 expression, and reductions in mitochondrial membrane potential (MMP) and Keap1 expression. Inhibition of Pi entry, cytosolic ROS production, or Nrf2 activation reversed the effects of high Pi on Nrf2, p62, and myogenin expression. Overexpression of Nrf2 respectively increased and decreased the promoter activity of p62-Luc and myogenin-Luc reporters. Analysis of nuclear extracts from gastrocnemius muscles from mice fed a high-Pi (2% Pi) diet showed increased Nrf2 phosphorylation in sham-operated and 5/6 nephrectomized (CKD) mice, and both increased p62 phosphorylation and decreased myogenin expression in CKD mice. These data suggest that high Pi suppresses myogenic differentiation in vitro and promotes muscle atrophy in vivo through oxidative stress-mediated protein degradation and both canonical (ROS-mediated) and non-canonical (p62-mediated) activation of Nrf2 signaling.


Assuntos
Diferenciação Celular , Hiperfosfatemia/complicações , Desenvolvimento Muscular , Atrofia Muscular/etiologia , Mioblastos Esqueléticos/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , Animais , Linhagem Celular , Modelos Animais de Doenças , Hiperfosfatemia/induzido quimicamente , Hiperfosfatemia/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Atrofia Muscular/metabolismo , Atrofia Muscular/patologia , Mioblastos Esqueléticos/patologia , Miogenina/genética , Miogenina/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fosfatos , Fosforilação , Insuficiência Renal Crônica/complicações , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismo , Transdução de Sinais
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