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1.
Nat Commun ; 11(1): 4167, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32820177

RESUMO

Muscle regeneration depends on a robust albeit transient inflammatory response. Persistent inflammation is a feature of age-related regenerative deficits, yet the underlying mechanisms are poorly understood. Here, we find inflammatory-related CC-chemokine-receptor 2 (Ccr2) expression in non-hematopoietic myogenic progenitors (MPs) during regeneration. After injury, the expression of Ccr2 in MPs corresponds to the levels of its ligands, the chemokines Ccl2, 7, and 8. We find stimulation of Ccr2-activity inhibits MP fusion and contribution to myofibers. This occurs in association with increases in MAPKp38δ/γ signaling, MyoD phosphorylation, and repression of the terminal myogenic commitment factor Myogenin. High levels of Ccr2-chemokines are a feature of regenerating aged muscle. Correspondingly, deletion of Ccr2 in MPs is necessary for proper fusion into regenerating aged muscle. Finally, opportune Ccr2 inhibition after injury enhances aged regeneration and functional recovery. These results demonstrate that inflammatory-induced activation of Ccr2 signaling in myogenic cells contributes to aged muscle regenerative decline.


Assuntos
Mediadores da Inflamação/metabolismo , Músculo Esquelético/fisiopatologia , Receptores CCR2/metabolismo , Regeneração/fisiologia , Transdução de Sinais/fisiologia , Fatores Etários , Animais , Transplante de Células/métodos , Quimiocina CCL2/metabolismo , Quimiocina CCL7/metabolismo , Quimiocina CCL8/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Desenvolvimento Muscular/genética , Músculo Esquelético/lesões , Músculo Esquelético/metabolismo , Miogenina/genética , Miogenina/metabolismo , Receptores CCR2/genética , Regeneração/genética , Células Satélites de Músculo Esquelético/citologia , Células Satélites de Músculo Esquelético/metabolismo , Células Satélites de Músculo Esquelético/transplante , Transdução de Sinais/genética , Ferimentos e Lesões/genética , Ferimentos e Lesões/fisiopatologia , Ferimentos e Lesões/terapia
3.
Nat Commun ; 11(1): 3722, 2020 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-32709902

RESUMO

Human movement occurs through contraction of the basic unit of the muscle cell, the sarcomere. Sarcomeres have long been considered to be arranged end-to-end in series along the length of the muscle into tube-like myofibrils with many individual, parallel myofibrils comprising the bulk of the muscle cell volume. Here, we demonstrate that striated muscle cells form a continuous myofibrillar matrix linked together by frequently branching sarcomeres. We find that all muscle cells contain highly connected myofibrillar networks though the frequency of sarcomere branching goes down from early to late postnatal development and is higher in slow-twitch than fast-twitch mature muscles. Moreover, we show that the myofibrillar matrix is united across the entire width of the muscle cell both at birth and in mature muscle. We propose that striated muscle force is generated by a singular, mesh-like myofibrillar network rather than many individual, parallel myofibrils.


Assuntos
Fenômenos Mecânicos , Músculo Esquelético/fisiologia , Miofibrilas/fisiologia , Sarcômeros/fisiologia , Animais , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica , Contração Muscular/fisiologia , Desenvolvimento Muscular , Músculo Esquelético/citologia , Miofibrilas/patologia , Sarcômeros/patologia
4.
Gene ; 758: 144986, 2020 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-32711100

RESUMO

Myostatin (Mstn) inhibits muscle growth in vertebrates with endoskeleton, but it is still inconclusive that Mstn is a positive or negative regulator in crustacean with exoskeleton, and little information was available for its function on myogenesis. In this study, we identified and characterized the Mstn from Fenneropenaeus chinensis (FcMstn), and investigated its function on myogenesis and muscle growth. Two different cDNA sequences (2628 bp and 2604 bp) encoding for slightly different sizes of proteins were obtained for FcMstn, containing 86 bp of 5' untranslated regions (UTR) and 1258 bp of 3' UTR. The open reading frame of the long sequence and the short sequence contain 1284 bp and 1260 bp cDNA, encoding 427 and 419 amino acid sequence, respectively. Sequence analysis revealed that the overall protein sequence and specific functional sites of FcMstn were highly conserved with those in other crustacean species. In the early development stage, the muscle firstly appeared in nauplius stage and developed gradually until post larval, but the expression of FcMstn at mRNA and protein levels decreased from nauplius stage to post larval stage, indicating that Mstn involved in myogenesis as a negative regulator in shrimp. In the adult shrimp, the expression of FcMstn at mRNA and protein levels in muscle were significantly lower in the larger group than in the smaller group, and the diameter and number of muscle fiber of the muscle were significantly different between the two groups. Moreover, the shrimp with reduced level of FcMstn by RNAi displayed a dramatic faster growth rate compared with the control group. The present study demonstrates that FcMstn involved in myogenesis and muscle growth probably also as a negative regulator in shrimp like in vertebrates.


Assuntos
Desenvolvimento Muscular/genética , Músculos/metabolismo , Miostatina/genética , Miostatina/metabolismo , Penaeidae/genética , Regiões 3' não Traduzidas/genética , Regiões 5' não Traduzidas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Perfilação da Expressão Gênica , Fases de Leitura Aberta/genética , Filogenia , Interferência de RNA , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Alinhamento de Sequência
5.
PLoS One ; 15(7): e0235433, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32726316

RESUMO

ADP-ribosylhydrolase-like 1 (Adprhl1) is a pseudoenzyme expressed in the developing heart myocardium of all vertebrates. In the amphibian Xenopus laevis, knockdown of the two cardiac Adprhl1 protein species (40 and 23 kDa) causes failure of chamber outgrowth but this has only been demonstrated using antisense morpholinos that interfere with RNA-splicing. Transgenic production of 40 kDa Adprhl1 provides only part rescue of these defects. CRISPR/Cas9 technology now enables targeted mutation of the adprhl1 gene in G0-generation embryos with routine cleavage of all alleles. Testing multiple gRNAs distributed across the locus reveals exonic locations that encode critical amino acids for Adprhl1 function. The gRNA recording the highest frequency of a specific ventricle outgrowth phenotype directs Cas9 cleavage of an exon 6 sequence, where microhomology mediated end-joining biases subsequent DNA repairs towards three small in-frame deletions. Mutant alleles encode discrete loss of 1, 3 or 4 amino acids from a di-arginine (Arg271-Arg272) containing peptide loop at the centre of the ancestral ADP-ribosylhydrolase site. Thus despite lacking catalytic activity, it is the modified (adenosine-ribose) substrate binding cleft of Adprhl1 that fulfils an essential role during heart formation. Mutation results in striking loss of myofibril assembly in ventricle cardiomyocytes. The defects suggest Adprhl1 participation from the earliest stage of cardiac myofibrillogenesis and are consistent with previous MO results and Adprhl1 protein localization to actin filament Z-disc boundaries. A single nucleotide change to the gRNA sequence renders it inactive. Mice lacking Adprhl1 exons 3-4 are normal but production of the smaller ADPRHL1 species is unaffected, providing further evidence that cardiac activity is concentrated at the C-terminal protein portion.


Assuntos
Ventrículos do Coração/crescimento & desenvolvimento , Coração/crescimento & desenvolvimento , Desenvolvimento Muscular/genética , N-Glicosil Hidrolases/genética , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/crescimento & desenvolvimento , Catálise , Domínio Catalítico/genética , Coração/fisiopatologia , Ventrículos do Coração/patologia , Humanos , Camundongos , Camundongos Knockout , Morfolinos/genética , Oligodesoxirribonucleotídeos Antissenso/genética , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Organogênese/genética , Xenopus laevis/genética , Xenopus laevis/crescimento & desenvolvimento
6.
PLoS One ; 15(7): e0234792, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32614850

RESUMO

The Myo/Nog cell lineage was discovered in the chick embryo and is also present in adult mammalian tissues. The cells are named for their expression of mRNA for the skeletal muscle specific transcription factor MyoD and bone morphogenetic protein inhibitor Noggin. A third marker for Myo/Nog cells is the cell surface molecule recognized by the G8 monoclonal antibody (mAb). G8 has been used to detect, track, isolate and kill Myo/Nog cells. In this study, we screened a membrane proteome array for the target of the G8 mAb. The array consisted of >5,000 molecules, each synthesized in their native confirmation with appropriate post-translational modifications in a single clone of HEK-293T cells. G8 mAb binding to the clone expressing brain-specific angiogenesis inhibitor 1 (BAI1) was detected by flow cytometry, re-verified by sequencing and validated by transfection with the plasmid construct for BAI1. Further validation of the G8 target was provided by enzyme-linked immunosorbent assay. The G8 epitope was identified by screening a high-throughput, site directed mutagenesis library designed to cover 95-100% of the 954 amino acids of the extracellular domain of the BAI1 protein. The G8 mAb binds within the third thrombospondin repeat of the extracellular domain of human BAI1. Immunofluorescence localization experiments revealed that G8 and a commercially available BAI1 mAb co-localize to the subpopulation of Myo/Nog cells in the skin, eyes and brain. Expression of the multi-functional BAI1 protein in Myo/Nog cells introduces new possibilities for the roles of Myo/Nog cells in normal and diseased tissues.


Assuntos
Proteínas Angiogênicas/biossíntese , Miofibroblastos/metabolismo , Receptores Acoplados a Proteínas-G/biossíntese , Substituição de Aminoácidos , Proteínas Angiogênicas/química , Proteínas Angiogênicas/genética , Proteínas Angiogênicas/imunologia , Animais , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Reações Antígeno-Anticorpo , Encéfalo/citologia , Proteínas de Transporte/análise , Linhagem da Célula , Epitopos/imunologia , Proteínas do Olho/biossíntese , Proteínas do Olho/química , Proteínas do Olho/genética , Proteínas do Olho/imunologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Desenvolvimento Muscular , Proteína MyoD/análise , Especificidade de Órgãos , Conformação Proteica , Domínios Proteicos , Coelhos , Ratos Sprague-Dawley , Receptores Acoplados a Proteínas-G/química , Receptores Acoplados a Proteínas-G/genética , Receptores Acoplados a Proteínas-G/imunologia , Sequências Repetitivas de Aminoácidos , Pele/citologia , Especificidade da Espécie , Tatuagem , Adulto Jovem
7.
Gene ; 757: 144943, 2020 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-32652105

RESUMO

The growth of animal skeletal muscle is mainly determined by the synthesis processes of total proteins in skeletal muscle cells, which has a significant impact on the postnatal growth of young animals. An increasing number of studies are focusing on the functions of Tuberous sclerosis complex 2 (TSC2) during the process of cell protein synthesis and growth. However, it is still unclear the effect of whether and how TSC2 on goat myoblasts proliferation and differentiation. Here, we found that TSC2 gene has opposite expression patterns in proliferation and differentiation of myoblasts. An expression vector containing goat TSC2 cDNA sequences linked with pcDNA3.1 plasmid was constructed. Myoblasts proliferation activity was significantly inhibited and cell cycle transition slowed down after the transfection of pcDNA3.1-TSC2 plasmid into goat primary myoblasts by EdU staining, CCK-8 and flow cytometry. Mechanically, we further confirmed that the overexpression TSC2 was able to down-regulate the mRNA and protein expression of mechanistic target of rapamycin (mTOR), p70 ribosomal S6 kinase 1 (p70S6K) and some cell cycle related genes. In addition, the expression of myogenic genes and myotube formation were attenuated. Collectively, all our results of the experiment demonstrate that TSC2 could regulate myoblasts cells proliferation and differentiation via the activation of the mTOR/p70S6K signaling pathway.


Assuntos
Diferenciação Celular , Proliferação de Células , Desenvolvimento Muscular , Mioblastos/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa/genética , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Cabras , Mioblastos/citologia , Mioblastos/fisiologia , Proteínas Quinases S6 Ribossômicas 70-kDa/genética , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa/metabolismo , Regulação para Cima
8.
Mol Med ; 26(1): 69, 2020 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-32641037

RESUMO

BACKGROUND: We previously showed that the autophagy inhibitor chloroquine (CQ) increases inflammatory cleaved caspase-1 activity in myocytes, and that caspase-1/11 is protective in sterile liver injury. However, the role of caspase-1/11 in the recovery of muscle from ischemia caused by peripheral arterial disease is unknown. We hypothesized that caspase-1/11 mediates recovery in muscle via effects on autophagy and this is modulated by CQ. METHODS: C57Bl/6 J (WT) and caspase-1/11 double-knockout (KO) mice underwent femoral artery ligation (a model of hind-limb ischemia) with or without CQ (50 mg/kg IP every 2nd day). CQ effects on autophagosome formation, microtubule associated protein 1A/1B-light chain 3 (LC3), and caspase-1 expression was measured using electron microscopy and immunofluorescence. Laser Doppler perfusion imaging documented perfusion every 7 days. After 21 days, in situ physiologic testing in tibialis anterior muscle assessed peak force contraction, and myocyte size and fibrosis was also measured. Muscle satellite cell (MuSC) oxygen consumption rate (OCR) and extracellular acidification rate was measured. Caspase-1 and glycolytic enzyme expression was detected by Western blot. RESULTS: CQ increased autophagosomes, LC3 consolidation, total caspase-1 expression and cleaved caspase-1 in muscle. Perfusion, fibrosis, myofiber regeneration, muscle contraction, MuSC fusion, OCR, ECAR and glycolytic enzyme expression was variably affected by CQ depending on presence of caspase-1/11. CQ decreased perfusion recovery, fibrosis and myofiber size in WT but not caspase-1/11KO mice. CQ diminished peak force in whole muscle, and myocyte fusion in MuSC and these effects were exacerbated in caspase-1/11KO mice. CQ reductions in maximal respiration and ATP production were reduced in caspase-1/11KO mice. Caspase-1/11KO MuSC had significant increases in protein kinase isoforms and aldolase with decreased ECAR. CONCLUSION: Caspase-1/11 signaling affects the response to ischemia in muscle and effects are variably modulated by CQ. This may be critically important for disease treated with CQ and its derivatives, including novel viral diseases (e.g. COVID-19) that are expected to affect patients with comorbidities like cardiovascular disease.


Assuntos
Caspase 1/metabolismo , Caspases Iniciadoras/metabolismo , Cloroquina/farmacologia , Infecções por Coronavirus/patologia , Isquemia/patologia , Músculo Esquelético/patologia , Pneumonia Viral/patologia , Animais , Autofagossomos/metabolismo , Autofagia/efeitos dos fármacos , Betacoronavirus , Infecções por Coronavirus/tratamento farmacológico , Glicólise/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/metabolismo , Células Musculares/metabolismo , Desenvolvimento Muscular , Músculo Esquelético/metabolismo , Neovascularização Fisiológica , Fosforilação Oxidativa , Pandemias , Doença Arterial Periférica/patologia , Pneumonia Viral/tratamento farmacológico , Regeneração , Transdução de Sinais
9.
Cell Prolif ; 53(7): e12857, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32578911

RESUMO

Exosomes are membrane-bound extracellular vesicles that are produced in the endosomal compartment of most mammalian cell types and then released. Exosomes are effective carriers for the intercellular material transfer of material that can influence a series of physiological and pathological processes in recipient cells. Among loaded cargoes, non-coding RNAs (ncRNAs) vary for the exosome-producing cell and its homeostatic state, and characterization of the biogenesis and secretion of exosomal ncRNAs and the functions of these ncRNAs in skeletal muscle myogenesis remain preliminary. In this review, we will describe what is currently known of exosome biogenesis, release and uptake of exosomal ncRNAs, as well as the varied functions of exosomal miRNAs in skeletal muscle myogenesis.


Assuntos
Exossomos/metabolismo , MicroRNAs/metabolismo , Desenvolvimento Muscular/fisiologia , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiologia , Animais , Humanos
10.
Am J Clin Nutr ; 112(1): 84-95, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32520344

RESUMO

BACKGROUND: Protein intake recommendations advise ≥0.8 g/kg body weight (BW)/d, whereas experts propose a higher intake for older adults (1.0-1.2 g/kg BW/d). It is unknown whether optimal protein intake differs by sex or race. OBJECTIVES: We examined the shape of sex- and race-specific associations of dietary protein intake with 3- and 6-y changes in appendicular lean mass (aLM) and gait speed and also 6-y incidence of mobility limitation in community-dwelling older men and women. METHODS: We used data on men (n = 1163) and women (n = 1237) aged 70-81 y of the Health, Aging, and Body Composition Study. Protein intake was assessed using an FFQ (1998-1999). aLM and gait speed were measured at baseline and at 3 and 6 y. Difficulty walking one-quarter mile or climbing stairs was measured every 6 mo over 6 y. Prospective associations were evaluated with linear and Cox regression models, comparing fit of models with and without spline functions. All analyses were stratified by sex and additionally by race. RESULTS: Mean ± SD protein intake was 0.94 ± 0.36 g/kg adjusted body weight (aBW)/d in men and 0.95 ± 0.36 g/kg aBW/d in women. There were no strong indications of nonlinear associations. In women, higher protein intake was associated with less aLM loss over 3 y (adjusted B per 0.1 g/kg aBW/d: 39.4; 95% CI: 11.6, 67.2), specifically in black women, but not over 6 y or with gait speed decline. In men, protein intake was not associated with changes in aLM and gait speed. Higher protein intake was associated with a lower risk of mobility limitation in men (adjusted HR per 1.0 g/kg aBW/d: 0.55; 95% CI: 0.34, 0.91) and women (adjusted HR: 0.56; 95% CI: 0.33, 0.94), specifically white women. CONCLUSIONS: Associations between protein intake and physical outcomes may vary by sex and race. Therefore, it is important to consider sex and race in future studies regarding protein needs in older adults.


Assuntos
Envelhecimento/metabolismo , Proteínas na Dieta/metabolismo , Idoso , Idoso de 80 Anos ou mais , Biomassa , Composição Corporal , Peso Corporal , Feminino , Humanos , Vida Independente , Masculino , Desenvolvimento Muscular , Força Muscular , Músculos/fisiologia , Estudos Prospectivos , Fatores Sexuais
11.
Gene ; 754: 144849, 2020 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-32512157

RESUMO

Skeletal muscles constitute a high proportion of the cellular mass that is essential for the growth traits in cattle. Resveratrol (RSV) is a natural polyphenol compound involved in pleiotropic biological activities of muscle. Therefore, the aim of our study was to investigate the transcriptome-level effects of RSV on bovine primary myoblast to reveal differentially expressed genes (DEGs). We treated three replicates of primary myoblasts with 20 µM mother solution containing RSV, whereas three other replicates without RSV were used as control group. Then, we conducted genome-wide transcriptome analysis for the two groups. The results of expression analysis identified 3856 DEGs of which 1805 genes were up-regulated and 2051 genes were down-regulated (adjusted P < 0.05). In addition, qRT-PCR analysis of 19 selected DEGs were consistent with the expression levels observed in the transcriptome data. Gene Ontology (GO) and pathway enrichment analysis showed 72 and 66 significant GO terms and KEGG pathways, respectively (adjusted P < 0.05). The most significant GO term was actin cytoskeleton organization (GO:0030036). The top significant KEGG pathway was focal adhesion (bta04510). Predicted protein-protein interactions (PPIs) showed that CDKN1A encoding cyclindependent kinase inhibitor 1A connects several larger protein complexes. In conclusion, our results found a list of DEGs, significant GO terms and pathways, and provided an improved and expanded understanding of the impact of RSV on cattle muscle cells at the transcriptomic level. The study elucidates the potential of using the genes enriched in pathways mediating resveratrol effects as targets in genomic selection for muscle development and growth in beef cattle.


Assuntos
Biomarcadores/análise , Regulação da Expressão Gênica/efeitos dos fármacos , Desenvolvimento Muscular/genética , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , Resveratrol/farmacologia , Transcriptoma/efeitos dos fármacos , Animais , Antioxidantes/farmacologia , Bovinos , Redes Reguladoras de Genes , Sequenciamento de Nucleotídeos em Larga Escala , Desenvolvimento Muscular/efeitos dos fármacos , Músculo Esquelético/citologia , Músculo Esquelético/efeitos dos fármacos , Mioblastos/citologia , Mioblastos/efeitos dos fármacos , Mapas de Interação de Proteínas
12.
PLoS Genet ; 16(5): e1008586, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32463816

RESUMO

The strength of bone depends on bone quantity and quality. Osteocalcin (Ocn) is the most abundant noncollagenous protein in bone and is produced by osteoblasts. It has been previously claimed that Ocn inhibits bone formation and also functions as a hormone to regulate insulin secretion in the pancreas, testosterone synthesis in the testes, and muscle mass. We generated Ocn-deficient (Ocn-/-) mice by deleting Bglap and Bglap2. Analysis of Ocn-/-mice revealed that Ocn is not involved in the regulation of bone quantity, glucose metabolism, testosterone synthesis, or muscle mass. The orientation degree of collagen fibrils and size of biological apatite (BAp) crystallites in the c-axis were normal in the Ocn-/-bone. However, the crystallographic orientation of the BAp c-axis, which is normally parallel to collagen fibrils, was severely disrupted, resulting in reduced bone strength. These results demonstrate that Ocn is required for bone quality and strength by adjusting the alignment of BAp crystallites parallel to collagen fibrils; but it does not function as a hormone.


Assuntos
Apatitas/metabolismo , Calcificação Fisiológica/genética , Metabolismo dos Carboidratos/genética , Glucose/metabolismo , Músculo Esquelético/crescimento & desenvolvimento , Osteocalcina/fisiologia , Testosterona/biossíntese , Animais , Apatitas/química , Osso e Ossos/metabolismo , Colágeno/metabolismo , Cristalização , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Desenvolvimento Muscular/genética , Músculo Esquelético/metabolismo , Tamanho do Órgão/genética , Osteoblastos/metabolismo , Osteocalcina/genética , Osteogênese/genética , Testículo/crescimento & desenvolvimento , Testículo/metabolismo
13.
PLoS One ; 15(5): e0232081, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32374763

RESUMO

The reproduction of reliable in vitro models of human skeletal muscle is made harder by the intrinsic 3D structural complexity of this tissue. Here we coupled engineered hydrogel with 3D structural cues and specific mechanical properties to derive human 3D muscle constructs ("myobundles") at the scale of single fibers, by using primary myoblasts or myoblasts derived from embryonic stem cells. To this aim, cell culture was performed in confined, laminin-coated micrometric channels obtained inside a 3D hydrogel characterized by the optimal stiffness for skeletal muscle myogenesis. Primary myoblasts cultured in our 3D culture system were able to undergo myotube differentiation and maturation, as demonstrated by the proper expression and localization of key components of the sarcomere and sarcolemma. Such approach allowed the generation of human myobundles of ~10 mm in length and ~120 µm in diameter, showing spontaneous contraction 7 days after cell seeding. Transcriptome analyses showed higher similarity between 3D myobundles and skeletal signature, compared to that found between 2D myotubes and skeletal muscle, mainly resulting from expression in 3D myobundles of categories of genes involved in skeletal muscle maturation, including extracellular matrix organization. Moreover, imaging analyses confirmed that structured 3D culture system was conducive to differentiation/maturation also when using myoblasts derived from embryonic stem cells. In conclusion, our structured 3D model is a promising tool for modelling human skeletal muscle in healthy and diseases conditions.


Assuntos
Técnicas de Cultura de Células , Fibras Musculares Esqueléticas/citologia , Músculo Esquelético/citologia , Engenharia Tecidual , Tecidos Suporte/química , Animais , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Células Cultivadas , Dimetilpolisiloxanos/química , Humanos , Hidrogéis/química , Teste de Materiais , Camundongos , Modelos Biológicos , Conformação Molecular , Desenvolvimento Muscular , Músculo Esquelético/fisiologia , Mioblastos/citologia , Mioblastos/fisiologia , Análise de Célula Única/instrumentação , Análise de Célula Única/métodos , Engenharia Tecidual/instrumentação , Engenharia Tecidual/métodos
14.
Gene ; 751: 144706, 2020 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-32387386

RESUMO

Skeletal muscle, the most abundant and plasticity tissue in mammals, is essential for various functions such as movement, breathing, maintaining posture and metabolism. Myogenesis is a complex and precise process, which is regulated by the sequential expression of multiple transcription factors, and accumulating evidence have confirmed that multiple lncRNAs are involved in muscle development as the important transcriptional regulator. In this study, a novel lncRNA, named lnc403 was obtained, with a full-length 2689 bp, which had poor coding ability and was mainly expressed in the nucleus of myoblasts and myotubes. The expression of lnc403 was significantly different in the proliferation and differentiation stages of muscle cells. Then we successfully constructed lnc403 loss/gain-function cell models by transfecting silnc403 and pCDNA3.1-EGFP-lnc403 into satellite cells, respectively; and found that lnc403 inhibited skeletal muscle satellite cell differentiation but had no significant effect on cell proliferation, either in the case of lnc403 knockdown or overexpression. In order to further screen the target factors regulated by lncRNA in the process of myogenic differentiation, the RNA-pull down, mass spectrometry and bioinformatics analysis were performed. The results showed that lnc403 negatively regulated the expression of the adjacent gene Myf6 and positively regulated interaction proteins KRAS expression. The above results indicate that lnc403 affects skeletal muscle cell differentiation by affecting the expression of nearby genes and interacting proteins, implying lnc403 might participate in the bovine myoblasts differentiation through multi-pathway network regulation mode. This study provides a new perspective for further understanding of the regulation mechanism of lncRNAs on bovine myogenic process.


Assuntos
Desenvolvimento Muscular/genética , Mioblastos Esqueléticos/metabolismo , Fatores de Regulação Miogênica/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , RNA Longo não Codificante/metabolismo , Animais , Bovinos , Proliferação de Células , Células Cultivadas , Regulação da Expressão Gênica , Fatores de Regulação Miogênica/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Células Satélites de Músculo Esquelético/citologia
15.
Nucleic Acids Res ; 48(12): 6563-6582, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32459350

RESUMO

Functional crosstalk between histone modifications and chromatin remodeling has emerged as a key regulatory mode of transcriptional control during cell fate decisions, but the underlying mechanisms are not fully understood. Here we discover an HRP2-DPF3a-BAF epigenetic pathway that coordinates methylated histone H3 lysine 36 (H3K36me) and ATP-dependent chromatin remodeling to regulate chromatin dynamics and gene transcription during myogenic differentiation. Using siRNA screening targeting epigenetic modifiers, we identify hepatoma-derived growth factor-related protein 2 (HRP2) as a key regulator of myogenesis. Knockout of HRP2 in mice leads to impaired muscle regeneration. Mechanistically, through its HIV integrase binding domain (IBD), HRP2 associates with the BRG1/BRM-associated factor (BAF) chromatin remodeling complex by interacting directly with the BAF45c (DPF3a) subunit. Through its Pro-Trp-Trp-Pro (PWWP) domain, HRP2 preferentially binds to H3K36me2. Consistent with the biochemical studies, ChIP-seq analyses show that HRP2 colocalizes with DPF3a across the genome and that the recruitment of HRP2/DPF3a to chromatin is dependent on H3K36me2. Integrative transcriptomic and cistromic analyses, coupled with ATAC-seq, reveal that HRP2 and DPF3a activate myogenic genes by increasing chromatin accessibility through recruitment of BRG1, the ATPase subunit of the BAF complex. Taken together, these results illuminate a key role for the HRP2-DPF3a-BAF complex in the epigenetic coordination of gene transcription during myogenic differentiation.


Assuntos
Montagem e Desmontagem da Cromatina , Proteínas de Ligação a DNA/metabolismo , Código das Histonas , Mioblastos/metabolismo , Fatores de Transcrição/metabolismo , Animais , Sítios de Ligação , Diferenciação Celular , Proteínas de Ligação a DNA/genética , Células HEK293 , Humanos , Masculino , Camundongos , Desenvolvimento Muscular , Mioblastos/citologia , Ligação Proteica , Fatores de Transcrição/genética
16.
Surg Technol Int ; 36: 41-47, 2020 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-32243565

RESUMO

Skeletal muscle represents the largest mass of tissue in the body and is essential for motion and posture. Traumatic injury, tumor ablation, prolonged denervation or genetic defects lead to skeletal myopathies. The loss of muscle function or its regenerative properties often results in pain, deformity, and joint malfunction. The regenerative capacity of skeletal muscles depends on adult muscle stem cells, the so-called satellite cells; however, the population of these myogenic precursors, and thus their potential to restore large muscle tissue defects, is strongly limited. On the other hand, surgical treatment of skeletal muscle loss is hampered by the scarcity of functional replacement tissue. Only a few options currently exist to provide functional and aesthetic restoration of lost muscle tissues, other than free muscle flap transfer. While this reconstructive technique is a common practice, it involves the risk of significant donor-site morbidity. Therefore, alternative cells with the potential to regenerate muscle tissue need to be examined. Recently, many surgeons have studied the potential clinical application of mesenchymal stem cells (MSCs), which are an adult stem cell population that can undergo differentiation along the mesodermal lineage and secrete growth factors that can enhance tissue regeneration processes by promoting neovascularization. The regenerative potential of MSCs has been widely studied in vitro and in vivo in animal models. MSCs from adipose tissue as well as bone marrow have been shown to bear myogenic potential, which makes them ideal candidate stem cells for skeletal muscle tissue engineering applications. When compared to reconstructive procedures using autograft tissues, MSC therapy offers the potential of reducing or even eliminating donor-site morbidity. This review gives a comprehensive overview of the use of MSCs in in vitro muscle generation and in vivo muscle regeneration.


Assuntos
Células-Tronco Mesenquimais , Animais , Diferenciação Celular , Desenvolvimento Muscular , Músculo Esquelético , Regeneração , Engenharia Tecidual
17.
J Biosci Bioeng ; 130(1): 98-105, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32278672

RESUMO

Although various types of artificial skeletal muscle tissue have been reported, the contractile forces generated by tissue-engineered artificial skeletal muscles remain to be improved for biological model and clinical applications. In this study, we investigated the effects of extracellular matrix (ECM) and supplementation of a small molecule, which has been reported to enhance α7ß1 integrin expression (SU9516), on cell migration speed, cell fusion rate, myoblast (mouse C2C12 cells) differentiation and contractile force generation of tissue-engineered artificial skeletal muscles. When cells were cultured on varying ECM coated-surfaces, we observed significant enhancement in the migration speed, while the myotube formation (differentiation ratio) decreased in all except for cells cultured on Matrigel coated-surfaces. In contrast, SU9516 supplementation resulted in an increase in both the myotube width and differentiation ratio. Following combined culture with a Matrigel-coated surface and SU9516 supplementation, myotube width was further increased. Additionally, contractile forces produced by the tissue-engineered artificial skeletal muscles was augmented following combined culture. These findings indicate that regulation of the cell-ECM interaction is a promising approach to improve the function of tissue-engineered artificial skeletal muscles.


Assuntos
Matriz Extracelular/metabolismo , Desenvolvimento Muscular , Músculo Esquelético/citologia , Animais , Diferenciação Celular/efeitos dos fármacos , Fusão Celular , Linhagem Celular , Colágeno/metabolismo , Combinação de Medicamentos , Integrinas/genética , Integrinas/metabolismo , Laminina/metabolismo , Camundongos , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Mioblastos/citologia , Mioblastos/metabolismo , Proteoglicanas/metabolismo
18.
PLoS One ; 15(4): e0231265, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32267872

RESUMO

Transcriptional co-activator with PDZ-binding motif (TAZ) plays versatile roles in the regulation of cell proliferation and differentiation. TAZ activity changes in response to the cellular environment such as mechanic and nutritional stimuli, osmolarity, and hypoxia. To understand the physiological roles of TAZ, chemical compounds that activate TAZ in cells are useful as experimental reagents. Kaempferol, TM-25659, and ethacridine are reported as TAZ activators. However, as each TAZ activator has a distinct property in cellular functions, additional TAZ activators are awaiting. We screened for TAZ activators and previously reported IB008738 as a TAZ activator that promotes myogenesis in C2C12 cells. In this study, we have characterized IBS004735 that was obtained in the same screening. IBS004735 also promotes myogenesis in C2C12 cells, but is not similar to IBS008738 in the structure. IBS004735 activates TAZ via Akt and has no effect on TAZ phosphorylation, which is the well-described key modification to regulate TAZ activity. Thus, we introduce IBS004735 as a novel TAZ activator that regulates TAZ in a yet unidentified mechanism.


Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Imidazóis/farmacologia , Desenvolvimento Muscular/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tetrazóis/farmacologia , Transativadores/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Camundongos , Mioblastos Esqueléticos/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/genética , Transativadores/genética , Transfecção
19.
Am J Chin Med ; 48(3): 631-650, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32329640

RESUMO

The loss of skeletal muscle mass and function is a serious consequence of chronic diseases and aging. BST204 is a purified ginseng (the root of Panax ginseng) extract that has been processed using ginsenoside-ß-glucosidase and acid hydrolysis to enrich ginsenosides Rg3 and Rh2 from the crude ginseng. BST204 has a broad range of health benefits, but its effects and mechanism on muscle atrophy are currently unknown. In this study, we have examined the effects and underlying mechanisms of BST204 on myotube formation and myotube atrophy induced by tumor necrosis factor-α (TNF-α). BST204 promotes myogenic differentiation and multinucleated myotube formation through Akt activation. BST204 prevents myotube atrophy induced by TNF-α through the activation of Akt/mTOR signaling and down-regulation of muscle-specific ubiquitin ligases, MuRF1, and Atrogin-1. Furthermore, BST204 treatment in atrophic myotubes suppresses mitochondrial reactive oxygen species (ROS) production and regulates mitochondrial transcription factors such as NRF1 and Tfam, through enhancing the activity and expression of peroxisome proliferator-activated receptor-γ coactivator1α (PGC1α). Collectively, our findings indicate that BST204 improves myotube formation and PGC1α-mediated mitochondrial function, suggesting that BST204 is a potential therapeutic or neutraceutical remedy to intervene muscle weakness and atrophy.


Assuntos
Desenvolvimento Muscular/efeitos dos fármacos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Panax/química , Fitoterapia , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Animais , Atrofia/induzido quimicamente , Atrofia/tratamento farmacológico , Humanos , Mitocôndrias Musculares/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Fibras Musculares Esqueléticas/fisiologia , Fator 1 Nuclear Respiratório/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Extratos Vegetais/isolamento & purificação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Estimulação Química , Serina-Treonina Quinases TOR/metabolismo , Fator de Necrose Tumoral alfa
20.
Am J Physiol Cell Physiol ; 318(6): C1154-C1165, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32233950

RESUMO

Homeobox A9 (HOXA9), the expression of which is promoted by mixed lineage leukemia 1 (MLL1) and WD-40 repeat protein 5 (WDR5), is a homeodomain-containing transcription factor that plays an essential role in regulating stem cell activity. HOXA9 has been found to inhibit skeletal muscle regeneration and delay recovery after muscle wounding in aged mice, but little is known about its role in denervated/reinnervated muscles. We performed detailed time-dependent expression analyses of HOXA9 and its promoters, MLL1 and WDR5, in rat gastrocnemius muscles after the following three types of sciatic nerve surgeries: nerve transection (denervation), end-to-end repair (repair), and sham operation (sham). Then, the specific mechanisms of HOXA9 were detected in vitro by transfecting primary satellite cells with empty pIRES2-DsRed2, pIRES2-DsRed2-HOXA9, empty pPLK/GFP-Puro, and pPLK/GFP-Puro-HOXA9 small hairpin RNA (shRNA) plasmids. We found, for the first time, that HOXA9 protein expression simultaneously increased with increasing denervated muscle atrophy severity and that upregulated MLL1 and WDR5 expression was partly associated with denervation. Indeed, in vitro experiments revealed that HOXA9 inhibited myogenic differentiation, affected the best known atrophic signaling pathways, and promoted apoptosis but did not eliminate the differentiation potential of primary satellite cells. HOXA9 may promote denervated muscle atrophy by regulating the activity of satellite cells.


Assuntos
Proteínas de Homeodomínio/metabolismo , Desenvolvimento Muscular , Músculo Esquelético/inervação , Atrofia Muscular/metabolismo , Regeneração Nervosa , Células Satélites de Músculo Esquelético/metabolismo , Nervo Isquiático/cirurgia , Animais , Animais Recém-Nascidos , Apoptose , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Modelos Animais de Doenças , Regulação da Expressão Gênica , Membro Posterior , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Proteínas de Homeodomínio/genética , Masculino , Denervação Muscular , Atrofia Muscular/genética , Atrofia Muscular/patologia , Atrofia Muscular/fisiopatologia , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Ratos Sprague-Dawley , Células Satélites de Músculo Esquelético/patologia , Nervo Isquiático/metabolismo , Nervo Isquiático/patologia , Transdução de Sinais , Fatores de Tempo
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