RESUMO
AIM: We investigated the expression pattern of Notch-1 in normal and hypertrophied plantaris muscle of mice. MAIN METHODS: We performed immunofluorescence of both Notch-1 and the Notch-1-linking molecules. KEY FINDINGS: Immunofluorescence labeling revealed Notch-1 protein in Pax7-positive satellite cells during days 2-6. We observed clear co-localization between Notch-1 and myogenin (4.9+/-1.3%) in the hypertrophied muscle at 4days. Several mononuclei (possibly satellite cells) possessed both Notch-1 and Foxo1 in the plantaris muscle subjected to mechanical overloading (4.1+/-1.2%). SIGNIFICANCE: Notch-1 may play an important role in the maintenance of quiescent satellite cells.
Assuntos
Regulação da Expressão Gênica , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Receptor Notch1/análise , Receptor Notch1/genética , Suporte de Carga , Animais , Diferenciação Celular , Proliferação de Células , Imunofluorescência , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/análise , Fatores de Transcrição Forkhead/metabolismo , Hipertrofia/genética , Hipertrofia/metabolismo , Macrófagos/citologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Miogenina/análise , Miogenina/metabolismo , Fator de Transcrição PAX7/análise , Fator de Transcrição PAX7/metabolismo , Receptor Notch1/metabolismo , Células Satélites de Músculo Esquelético/citologiaRESUMO
The molecular signaling pathways linking the atrophy of skeletal muscle during aging have not been identified. Using reverse transcription (RT)-PCR, Western blotting, and immunofluorescence microscopy, we investigated whether the amounts of RhoA, RhoGDI, SRF, MRTF-A, and MyoD in the triceps brachii and quadriceps muscles change with aging in mice. Young adult (3 mo) and aged (24 mo) C57BL/6J mice were used. Senescent mice possessed many fibers with central nuclei in the quadriceps muscle. Western blotting using a homogenate of whole muscle or the cytosolic fraction clearly showed that the amount of SRF protein was significantly decreased in the aged skeletal muscles. Immunofluorescence labeling indicated more SRF-positive muscle fibers in young mice. Both young and old mice possessed SRF immunoreactivity in some satellite cells expressing Pax7. MRTF-A and STARS mRNA levels significantly declined with aging in the triceps brachii and quadriceps muscles. The amount of MRTF-A protein was markedly reduced in the nuclear fraction of aged muscle of mice. The amounts of RhoA and RhoGDI in the crude homogenate or the cytosolic and membrane fractions were greater in the aged muscle. Senescent mice possessed significantly higher levels of MyoD protein in the cytosol and nucleus. Decreased SRF and MRTF expression may induce the atrophy of skeletal muscle with aging.
Assuntos
Envelhecimento/genética , Músculo Esquelético/metabolismo , Fator de Resposta Sérica/genética , Transativadores/genética , Envelhecimento/metabolismo , Envelhecimento/patologia , Animais , Sequência de Bases , Primers do DNA/genética , Expressão Gênica , Inibidores de Dissociação do Nucleotídeo Guanina/metabolismo , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Músculo Esquelético/patologia , Atrofia Muscular/genética , Atrofia Muscular/metabolismo , Atrofia Muscular/patologia , Proteína MyoD/metabolismo , Fator de Transcrição PAX7/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Resposta Sérica/metabolismo , Transdução de Sinais , Transativadores/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Inibidores da Dissociação do Nucleotídeo Guanina rho-Específico , Proteína rhoA de Ligação ao GTPRESUMO
The molecular signaling pathway linked to hypertrophy of the anti-gravity/postural soleus muscle after mechanical overloading has not been identified. Using reverse transcription-polymerase chain reaction (RT-PCR), Western blot, and immunohistochemical analyses, we investigated whether the amounts of myocyte enhancer factor (MEF)2C, MEF2D, and myogenin change in the mechanically overloaded soleus muscle after treatment with the calcineurin inhibitor cyclosporine A (CsA). Adult male ICR mice were subjected to a surgical ablation of the gastrocnemius muscle and treated with either CsA (25 mg/kg) or vehicle, once daily. They were killed at 2, 4, 7, 10, and 14 days post-injury. Mechanical overloading resulted in a significant increase in the wet weight and the cross-sectional area of slow and fast fibers of the soleus muscle in placebo-treated mice but not CsA-treated mice. RT-PCR analysis did not show a marked difference in MEF2C and MEF2D mRNA levels in the overloaded soleus muscle in placebo- or CsA-administered mice. After 2 days of mechanical overloading, we observed co-localization of MEF2C and myogenin in several mononuclear cells under both conditions. These MEF2C-positive mononuclear cells also possessed immunoreactivity for c-Met, a satellite cell marker. At 4 days, mechanical overloading induced marked expression of MEF2C but not MEF2D in the subsarcolemmal region in a group of myotubes and/or myofibers. Such a MEF2C-positive region emerged less often in the hypertrophied soleus muscle subjected to the treatment with CsA. At 7 days, we observed many mononuclear cells possessing both MEF2C and myogenin protein in mice treated with CsA, but not the placebo. Our results demonstrated that CsA treatment modulates the amount and cellular localization of MEF2C protein. The modulation of MEF2C by CsA treatment may inhibit the hypertrophic process in the soleus muscle after mechanical overloading.