RESUMO
Duchenne Muscular Dystrophy (DMD) is characterized by muscle extracellular matrix disorganization due to the increased collagen deposition leading to fibrosis that significantly exacerbates disease progression. Fractal dimension analysis is a method that quantifies tissue/cellular disorganization and characterizes complex structures. The first objective of the present study was use fractal analysis to evaluate extracellular matrix disorganization in mdx mice soleus muscle. Next, we mimic a hyper-proliferation of fibrogenic cells by co-culturing NIH3T3 fibroblasts and C2C12 myoblasts to test whether fibroblasts induce disorganization in myoblast arrangement. Here, we show mdx presented high skeletal muscle disorganization as revealed by fractal analysis. Similarly, this method revealed that myoblasts co-cultured with fibroblast also presented cellular arrangement disorganization. We also reanalyzed skeletal muscle microarrays transcriptomic data from mdx and DMD patients that revealed transcripts related to extracellular matrix organization. This analysis also identified Osteoglycin, which was validated as a potential regulator of ECM organization in mdx dystrophic muscles. Our results demonstrate that fractal dimension is useful tool for the analysis of skeletal muscle disorganization in DMD and also reveal a fibroblast-myoblast cross-talk that contributes to "in vitro" myoblast disarrangement.
Assuntos
Fractais , Músculo Esquelético/patologia , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patologia , Animais , Proliferação de Células , Técnicas de Cocultura , Modelos Animais de Doenças , Matriz Extracelular/genética , Matriz Extracelular/patologia , Fibroblastos/patologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Músculo Esquelético/metabolismo , Mioblastos Esqueléticos/metabolismo , Mioblastos Esqueléticos/patologia , Células NIH 3T3 , Regulação para CimaRESUMO
Cell lines with high passage numbers exhibit alterations in cell morphology and functions. In the present work, C2C12 skeletal muscle cells with either low (<20) or high (>60) passage numbers (identified as l-C2C12 or h-C2C12, respectively) were used to investigate the apoptotic response to H2O2 as a function of culture age h-C2C12. We found that older cultures (h-C2C12 group) were depleted of mitochondrial DNA (mtDNA). When we analyzed the behavior of Bad, Bax, caspase-3 and mitochondrial transmembrane potential, we observed that cells in the h-C2C12 group were resistant to H2O2 induction of apoptosis. We propose serially cultured C2C12 cells as a refractory model to H2O2-induced apoptosis. In addition, the data obtained in this work suggest that mtDNA is required for apoptotic cell death in skeletal muscle C2C12 cells.
Assuntos
Animais , Camundongos , Apoptose/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Mitocôndrias/patologia , Mioblastos Esqueléticos/patologia , Oxidantes/farmacologia , Western Blotting , Técnicas de Cultura de Células , Células Cultivadas , /metabolismo , Divisão Celular/efeitos dos fármacos , Imunoprecipitação , Microscopia de Fluorescência , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mioblastos Esqueléticos/efeitos dos fármacos , Mioblastos Esqueléticos/metabolismo , /metabolismoRESUMO
Cell lines with high passage numbers exhibit alterations in cell morphology and functions. In the present work, C2C12 skeletal muscle cells with either low (<20) or high (>60) passage numbers (identified as l-C2C12 or h-C2C12, respectively) were used to investigate the apoptotic response to H2O2 as a function of culture age h-C2C12. We found that older cultures (h-C2C12 group) were depleted of mitochondrial DNA (mtDNA). When we analyzed the behavior of Bad, Bax, caspase-3 and mitochondrial transmembrane potential, we observed that cells in the h-C2C12 group were resistant to H2O2 induction of apoptosis. We propose serially cultured C2C12 cells as a refractory model to H2O2-induced apoptosis. In addition, the data obtained in this work suggest that mtDNA is required for apoptotic cell death in skeletal muscle C2C12 cells.(AU)
Assuntos
Animais , Camundongos , Apoptose/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Mitocôndrias/patologia , Mioblastos Esqueléticos/patologia , Oxidantes/farmacologia , Western Blotting , Caspase 3/metabolismo , Técnicas de Cultura de Células , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Imunoprecipitação , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Microscopia de Fluorescência , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mioblastos Esqueléticos/efeitos dos fármacos , Mioblastos Esqueléticos/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
Cell lines with high passage numbers exhibit alterations in cell morphology and functions. In the present work, C2C12 skeletal muscle cells with either low (< 20) or high (> 60) passage numbers (identified as 1-C2C12 or h-C2C12, respectively) were used to investigate the apoptotic response to H2O2 as a function of culture age h-C2C12. We found that older cultures (h-C2C12 group) were depleted of mitochondrial DNA (mtDNA). When we analyzed the behavior of Bad, Bax, caspase-3 and mitochondrial transmembrane potential, we observed that cells in the h-C2C12 group were resistant to H2O2 induction of apoptosis. We propose serially cultured C2Cl2 cells as a refractory model to H2O2-induced apoptosis. In addition, the data obtained in this work suggest that mtDNA is required for apoptotic cell death in skeletal muscle C2C12 cells.
Assuntos
Apoptose/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Mitocôndrias/patologia , Mioblastos Esqueléticos/patologia , Oxidantes/farmacologia , Animais , Western Blotting , Caspase 3/metabolismo , Técnicas de Cultura de Células , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Imunoprecipitação , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Microscopia de Fluorescência , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mioblastos Esqueléticos/efeitos dos fármacos , Mioblastos Esqueléticos/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
Cell lines with high passage numbers exhibit alterations in cell morphology and functions. In the present work, C2C12 skeletal muscle cells with either low (< 20) or high (> 60) passage numbers (identified as 1-C2C12 or h-C2C12, respectively) were used to investigate the apoptotic response to H2O2 as a function of culture age h-C2C12. We found that older cultures (h-C2C12 group) were depleted of mitochondrial DNA (mtDNA). When we analyzed the behavior of Bad, Bax, caspase-3 and mitochondrial transmembrane potential, we observed that cells in the h-C2C12 group were resistant to H2O2 induction of apoptosis. We propose serially cultured C2Cl2 cells as a refractory model to H2O2-induced apoptosis. In addition, the data obtained in this work suggest that mtDNA is required for apoptotic cell death in skeletal muscle C2C12 cells.
Assuntos
Apoptose/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Mitocôndrias/patologia , Mioblastos Esqueléticos/patologia , Oxidantes/farmacologia , Animais , Western Blotting , Caspase 3/metabolismo , Técnicas de Cultura de Células , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Imunoprecipitação , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Microscopia de Fluorescência , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mioblastos Esqueléticos/efeitos dos fármacos , Mioblastos Esqueléticos/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
This paper reports the biochemical and pharmacological characterization of a new myotoxic PLA(2) (EC 3.1.1.4) called PhTX-I, purified from Porthidium hyoprora venom by one step analytical chromatography reverse phase HPLC. The homogeneity of the PhTX-I fraction and its molecular mass were initially evaluated by SDS-PAGE and confirmed by MALDI-TOF spectrometry, indicating a molecular mass of 14.249Da and constituted of a single polipeptidic chain. Amino acid sequence was determined by "de novo sequencing," in tandem mass spectrometry, belonging to D49-PLA(2) enzyme class and exhibiting high identity (44-90%) with other myotoxics PLA(2) from snake venoms. The enzymatic investigation showed maximal activity at pH 8 and 35-45°C. This activity was dependent on Ca(2+), other cations (Mg(2+), Mn(2+), Cd(2+) and Zn(2+)) reduced notably the enzymatic activity, suggesting that the arrangement of the catalytic site presents an exclusive structure for Ca(2+). Ex vivo, whole venom and PhTX-I PLA(2) caused blockade of the neuromuscular transmission in young chick biventer cervicis preparations similar to other isolated snake venom toxins from the Bothrops genus. In vivo, both induced local myotoxicity and systemic interleukin-6 response upon intramuscular injection, additionally, induced moderate footpad edema. In vitro, both induced low cytotoxicity in skeletal muscle myoblasts, however PhTX-I PLA(2) was able to lyse myotubes.