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1.
Phytomedicine ; 80: 153393, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33120292

RESUMO

BACKGROUND: Sarcopenia progresses in chronic kidney disease (CKD) and is positively correlated with mortality in end-stage kidney disease patients. Circulating irisin, an exercise-induced myokine, gradually decreases during CKD stage progression. Irisin inhibits the progression of kidney fibrosis, which is the final common outcome of CKD. Our preliminary study with C2C12 cells showed that Dojuksan, a herbal decoction, increases the expression of PGC1α (a regulator of irisin) and FNDC5 (a precursor of irisin). HYPOTHESIS: Dojuksan may increase circulating irisin and prevent the progression of kidney fibrosis. STUDY DESIGN AND METHODS: Unilateral ureteral obstruction (UUO) was performed on seven-week-old male C57BL/6 mice to induce kidney tubulointerstitial fibrosis. Dojuksan (50, 100, or 200 mg/kg/day) or losartan (1.5 mg/kg/day), a standard clinical treatment for CKD, was administered orally one day prior to surgery and continued for seven days thereafter. To determine the role of irisin released from muscles, TGFß-stimulated murine proximal tubular epithelial cells (mProx24 cells) were treated with conditioned media (CM) from Dojuksan-treated C2C12 muscle cells transfected with FNDC5 siRNA. RESULTS: UUO mice exhibited muscle wasting along with progressive kidney injury. Similar to losartan, Dojuksan ameliorated kidney inflammation and fibrosis in UUO mice. Dojuksan, but not losartan, increased plasma irisin concentration in UUO mice. Dojuksan significantly increased basal FNDC5 expression and inhibited TNFα-induced and indoxyl sulfate-induced FNDC5 down-regulation in C2C12 cells. The TGFß-induced collagen I (COL1) up-regulation in mProx24 cells was effectively inhibited by CM from C2C12 cells after Dojuksan treatment. Moreover, irisin inhibited TGFß-induced COL1 in mProx24 cells, which was not affected by CM from C2C12 cells transfected with FNDC5 siRNA. CONCLUSION: Dojuksan ameliorates kidney fibrosis through irisin-mediated muscle-kidney crosstalk, suggesting that Dojuksan may be used as an alternative therapeutic agent against CKD.


Assuntos
Medicamentos de Ervas Chinesas/farmacologia , Fibronectinas/metabolismo , Nefropatias/tratamento farmacológico , Nefropatias/patologia , Músculo Esquelético/metabolismo , Animais , Linhagem Celular , Colágeno Tipo I/metabolismo , Fibronectinas/genética , Fibrose , Nefropatias/metabolismo , Túbulos Renais/efeitos dos fármacos , Túbulos Renais/metabolismo , Túbulos Renais/patologia , Losartan/farmacologia , Masculino , Medicina Tradicional Chinesa , Medicina Tradicional Coreana , Camundongos Endogâmicos C57BL , Músculo Esquelético/citologia , Músculo Esquelético/fisiopatologia , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Obstrução Ureteral/patologia
2.
Nat Commun ; 11(1): 6375, 2020 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-33311457

RESUMO

Syncytial skeletal muscle cells contain hundreds of nuclei in a shared cytoplasm. We investigated nuclear heterogeneity and transcriptional dynamics in the uninjured and regenerating muscle using single-nucleus RNA-sequencing (snRNAseq) of isolated nuclei from muscle fibers. This revealed distinct nuclear subtypes unrelated to fiber type diversity, previously unknown subtypes as well as the expected ones at the neuromuscular and myotendinous junctions. In fibers of the Mdx dystrophy mouse model, distinct subtypes emerged, among them nuclei expressing a repair signature that were also abundant in the muscle of dystrophy patients, and a nuclear population associated with necrotic fibers. Finally, modifications of our approach revealed the compartmentalization in the rare and specialized muscle spindle. Our data identifies nuclear compartments of the myofiber and defines a molecular roadmap for their functional analyses; the data can be freely explored on the MyoExplorer server ( https://shiny.mdc-berlin.de/MyoExplorer/ ).


Assuntos
Núcleo Celular/genética , Núcleo Celular/metabolismo , Músculo Esquelético/crescimento & desenvolvimento , Músculo Esquelético/metabolismo , Transcriptoma , Animais , Linhagem Celular , Citoplasma , Modelos Animais de Doenças , Regulação da Expressão Gênica no Desenvolvimento , Heterogeneidade Genética , Humanos , Camundongos , Camundongos Endogâmicos mdx , Fibras Musculares Esqueléticas , Músculo Esquelético/citologia , Distrofias Musculares/genética , Distrofias Musculares/metabolismo , RNA-Seq , Regeneração , Tendões
3.
Nat Commun ; 11(1): 5102, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-33037211

RESUMO

Skeletal muscle fibers are large syncytia but it is currently unknown whether gene expression is coordinately regulated in their numerous nuclei. Here we show by snRNA-seq and snATAC-seq that slow, fast, myotendinous and neuromuscular junction myonuclei each have different transcriptional programs, associated with distinct chromatin states and combinations of transcription factors. In adult mice, identified myofiber types predominantly express either a slow or one of the three fast isoforms of Myosin heavy chain (MYH) proteins, while a small number of hybrid fibers can express more than one MYH. By snRNA-seq and FISH, we show that the majority of myonuclei within a myofiber are synchronized, coordinately expressing only one fast Myh isoform with a preferential panel of muscle-specific genes. Importantly, this coordination of expression occurs early during post-natal development and depends on innervation. These findings highlight a previously undefined mechanism of coordination of gene expression in a syncytium.


Assuntos
Núcleo Celular/genética , Regulação da Expressão Gênica , Hibridização in Situ Fluorescente/métodos , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/fisiologia , Análise de Sequência de RNA/métodos , Animais , Feminino , Camundongos Endogâmicos C57BL , Músculo Esquelético/citologia , Músculo Esquelético/embriologia , Músculo Esquelético/crescimento & desenvolvimento , Cadeias Pesadas de Miosina/genética , Junção Neuromuscular/citologia , Análise de Célula Única , Tendões/citologia , Transcrição Genética
4.
PLoS One ; 15(9): e0239428, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32997697

RESUMO

Short chain fatty acids (SCFAs) produced endogenously in the gut by bacterial fermentation of dietary fiber have been studied as nutrients that act as signaling molecules to activate G-protein coupled receptors (GPCRs) such as GPR41 and GPR43. GPR43 functioning involves the suppression of lipid accumulation and maintaining body energy homeostasis, and is activated by acetic acid or propionic acid. Previously, we reported that the orally administered acetic acid improves lipid metabolism in liver and skeletal muscles and suppresses obesity, thus improving glucose tolerance. Acetic acid stimulates AMP-activated protein kinase (AMPK) through its metabolic pathway in skeletal muscle cells. We hypothesized that acetic acid would stimulate GPR43 in skeletal muscle cells and has function in modulating gene expression related to muscle characteristics through its signal pathway. The objective of the current study was to clarify this effect of acetic acid. The GPR43 expression, observed in the differentiated myotube cells, was increased upon acetic acid treatment. Acetic acid induced the intracellular calcium influx in the cells and this induction was significantly inhibited by the GPR43-specific siRNA treatment. The calcineurin molecule is activated by calcium/calmodulin and is associated with proliferation of slow-twitch fibers. Calcineurin was activated by acetic acid treatment and inhibited by the concomitant treatment with GPR43-siRNA. Acetic acid induced nuclear localization of myocyte enhancer factor 2A (MEF2A), peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), and nuclear factor of activated t cells c1 (NFATc1). However, these localizations were abolished by the treatment with GPR43-siRNA. It was concluded that acetic acid plays a role in the activation of GPR43 and involves the proliferation of slow-twitch fibers in L6 skeletal muscles through the calcium-signaling pathway caused by induction of intracellular calcium influx.


Assuntos
Ácido Acético/farmacologia , Cálcio/metabolismo , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Fibras Musculares Esqueléticas/citologia , Receptores Acoplados a Proteínas-G/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Animais , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Inativação Gênica , Fatores de Transcrição MEF2/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Músculo Esquelético/citologia , Fatores de Transcrição NFATC/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , RNA Mensageiro/genética , Ratos , Receptores Acoplados a Proteínas-G/deficiência , Receptores Acoplados a Proteínas-G/genética
5.
Anim Sci J ; 91(1): e13449, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32815204

RESUMO

Ectopic fats have been recognized as a new risk factor for metabolic syndrome. In obese humans, ectopic fat accumulations are affected by body fat distribution. Intramuscular adipose tissue is categorized as one of the ectopic fats. Japanese black cattle (Wagyu) are characterized by the ability to accumulate high amounts of intramuscular adipose tissue. In Japan, the marbling level is indicated by the beef marbling standard number (BMS No.), which reflects the intramuscular fat content of longissimus muscle. We hypothesized that the intramuscular fat accumulation is affected by the body fat distribution in Wagyu cattle. In this study, we showed that the BMS No. was not correlated with the subcutaneous and visceral adipocyte diameter. In contrast, the BMS No. was positively correlated with intramuscular adipocyte diameter. These results indicate that the intramuscular adipocyte diameter of Wagyu is hypertrophied with an increase in the intramuscular fat accumulation. In addition, we showed that the BMS No. was positively correlated with the subcutaneous fat percentage. In contrast, the BMS No. was negatively correlated with the visceral fat percentage. These results indicate that highly marbled Wagyu cattle have a higher percentage of subcutaneous fat and a lower percentage of visceral fat.


Assuntos
Adipócitos/patologia , Adipogenia , Distribuição da Gordura Corporal , Bovinos/metabolismo , Qualidade dos Alimentos , Gordura Intra-Abdominal/metabolismo , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Carne Vermelha/normas , Gordura Subcutânea/metabolismo , Animais , Masculino
6.
Nat Commun ; 11(1): 3953, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32769974

RESUMO

Many important cell types in adult vertebrates have a mesenchymal origin, including fibroblasts and vascular mural cells. Although their biological importance is undisputed, the level of mesenchymal cell heterogeneity within and between organs, while appreciated, has not been analyzed in detail. Here, we compare single-cell transcriptional profiles of fibroblasts and vascular mural cells across four murine muscular organs: heart, skeletal muscle, intestine and bladder. We reveal gene expression signatures that demarcate fibroblasts from mural cells and provide molecular signatures for cell subtype identification. We observe striking inter- and intra-organ heterogeneity amongst the fibroblasts, primarily reflecting differences in the expression of extracellular matrix components. Fibroblast subtypes localize to discrete anatomical positions offering novel predictions about physiological function(s) and regulatory signaling circuits. Our data shed new light on the diversity of poorly defined classes of cells and provide a foundation for improved understanding of their roles in physiological and pathological processes.


Assuntos
Diferenciação Celular , Fibroblastos/fisiologia , Células-Tronco Mesenquimais/fisiologia , Miócitos de Músculo Liso/fisiologia , Pericitos/fisiologia , Animais , Separação Celular , Vasos Coronários/citologia , Matriz Extracelular/metabolismo , Fibroblastos/citologia , Citometria de Fluxo , Intestinos/irrigação sanguínea , Intestinos/citologia , Masculino , Camundongos , Músculo Esquelético/irrigação sanguínea , Músculo Esquelético/citologia , Músculo Liso Vascular/citologia , Miocárdio/citologia , Miócitos de Músculo Liso/citologia , Pericitos/citologia , RNA-Seq , Análise de Célula Única , Bexiga Urinária/irrigação sanguínea , Bexiga Urinária/citologia
7.
PLoS One ; 15(8): e0236781, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32776961

RESUMO

It has been reported that Bach1-deficient mice show reduced tissue injuries in diverse disease models due to increased expression of heme oxygenase-1 (HO-1)that possesses an antioxidant function. In contrast, we found that Bach1 deficiency in mice exacerbated skeletal muscle injury induced by cardiotoxin. Inhibition of Bach1 expression in C2C12 myoblast cells using RNA interference resulted in reduced proliferation, myotube formation, and myogenin expression compared with control cells. While the expression of HO-1 was increased by Bach1 silencing in C2C12 cells, the reduced myotube formation was not rescued by HO-1 inhibition. Up-regulations of Smad2, Smad3 and FoxO1, known inhibitors of muscle cell differentiation, were observed in Bach1-deficient mice and Bach1-silenced C2C12 cells. Therefore, Bach1 may promote regeneration of muscle by increasing proliferation and differentiation of myoblasts.


Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Diferenciação Celular , Músculo Esquelético/fisiologia , Mioblastos/citologia , Regeneração , Proteínas Smad/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina Básica/deficiência , Fatores de Transcrição de Zíper de Leucina Básica/genética , Linhagem Celular , Técnicas de Silenciamento de Genes , Inativação Gênica , Camundongos , Músculo Esquelético/citologia , Transcriptoma/genética
8.
Life Sci ; 259: 118187, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32781061

RESUMO

AIMS: Voluntary exercise training has cardioprotective effects in humans, but the underlying mechanism is unknown. This research was done to estimate the effect of voluntary exercise training to attenuate middle-aged maturity-induced cardiac apoptosis. MATERIALS AND METHODS: The study was designed to divide 64 male mice randomly into four groups, consisting of a 9-month sedentary pre-middle-aged group (9M), 15-month sedentary middle-aged group (15M), and two exercise groups using a voluntary wheel running respectively (9M+EX, 15M+EX). After 3 months, the condition of cardiac apoptosis in different groups was measured by HE dying, TUNEL and DAPI staining, and Western Blot analysis. KEY FINDINGS: TUNEL-positive cells were increased in 15M group compared with 9M group, while decreased in 9M+EX and 15M+EX groups compared with their control groups respectively. Protein levels of AIF, Endo G, TNF-α, TNFR1, TRAF2, TRADD, Fas, FasL, FADD, activated caspase 8, 3, 9, Bax/Bcl2, Bak/BclxL, and tBid were decreased in 9M+EX and 15M+EX groups compared with their control groups respectively. The protein levels of pBad/Bad, 14-3-3, IGF1, IGFR1, pPI3K/PI3K, and pAKT/AKT were more activated in the 9M+EX and 15M+EX groups than those in their control groups respectively. Significant differences were found between 9M group and 15M group for the protein levels of TRAF2, FADD, Bax/Bcl2, tBid and pAKT/AKT. SIGNIFICANCE: Voluntary exercise training as an important lifestyle modification may prevent cardiac widely dispersed apoptosis and enhance cardiac survival at middle-aged maturity.


Assuntos
Envelhecimento/fisiologia , Apoptose/fisiologia , Coração/fisiologia , Condicionamento Físico Animal/fisiologia , Animais , Marcação In Situ das Extremidades Cortadas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias Cardíacas/fisiologia , Músculo Esquelético/citologia , Músculo Esquelético/fisiologia , Corrida/fisiologia , Comportamento Sedentário
9.
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
10.
Am J Physiol Endocrinol Metab ; 319(2): E447-E454, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32691630

RESUMO

The aim of the present study was to determine if the training status decreases inflammation, slows down senescence, and preserves telomere health in skeletal muscle in older compared with younger subjects, with a specific focus on satellite cells. Analyses were conducted on skeletal muscle and cultured satellite cells from vastus lateralis biopsies (n = 34) of male volunteers divided into four groups: young sedentary (YS), young trained cyclists (YT), old sedentary (OS), and old trained cyclists (OT). The senescence state and inflammatory profile were evaluated by telomere dysfunction-induced foci (TIF) quantification, senescence-associated ß-galactosidase (SA-ß-Gal) staining, and quantitative (q)RT-PCR. Independently of the endurance training status, TIF levels (+35%, P < 0.001) and the percentage of SA-ß-Gal-positive cells (+30%, P < 0.05) were higher in cultured satellite cells of older compared with younger subjects. p16 (4- to 5-fold) and p21 (2-fold) mRNA levels in skeletal muscle were higher with age but unchanged by the training status. Aging induced higher CD68 mRNA levels in human skeletal muscle (+102%, P = 0.009). Independently of age, both trained groups had lower IL-8 mRNA levels (-70%, P = 0.011) and tended to have lower TNF-α mRNA levels (-40%, P = 0.10) compared with the sedentary subjects. All together, we found that the endurance training status did not slow down senescence in skeletal muscle and satellite cells in older compared with younger subjects despite reduced inflammation in skeletal muscle. These findings highlight that the link between senescence and inflammation can be disrupted in skeletal muscle.


Assuntos
Envelhecimento/fisiologia , Treino Aeróbico , Inflamação/prevenção & controle , Músculo Esquelético/fisiologia , Resistência Física/fisiologia , Homeostase do Telômero/fisiologia , Idoso , Senescência Celular/genética , Senescência Celular/fisiologia , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Humanos , Masculino , Músculo Esquelético/química , Músculo Esquelético/citologia , RNA Mensageiro/análise , Células Satélites de Músculo Esquelético/fisiologia , Células Satélites de Músculo Esquelético/ultraestrutura , Telômero/fisiologia , Telômero/ultraestrutura , Adulto Jovem , beta-Galactosidase/análise
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.
Nat Commun ; 11(1): 2796, 2020 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-32493965

RESUMO

Cell fate decisions involved in vascular and hematopoietic embryonic development are still poorly understood. An ETS transcription factor Etv2 functions as an evolutionarily conserved master regulator of vasculogenesis. Here we report a single-cell transcriptomic analysis of hematovascular development in wild-type and etv2 mutant zebrafish embryos. Distinct transcriptional signatures of different types of hematopoietic and vascular progenitors are identified using an etv2ci32Gt gene trap line, in which the Gal4 transcriptional activator is integrated into the etv2 gene locus. We observe a cell population with a skeletal muscle signature in etv2-deficient embryos. We demonstrate that multiple etv2ci32Gt; UAS:GFP cells differentiate as skeletal muscle cells instead of contributing to vasculature in etv2-deficient embryos. Wnt and FGF signaling promote the differentiation of these putative multipotent etv2 progenitor cells into skeletal muscle cells. We conclude that etv2 actively represses muscle differentiation in vascular progenitors, thus restricting these cells to a vascular endothelial fate.


Assuntos
Vasos Sanguíneos/citologia , Perfilação da Expressão Gênica , Músculo Esquelético/citologia , Análise de Célula Única , Células-Tronco/metabolismo , Proteínas de Peixe-Zebra/deficiência , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Biomarcadores/metabolismo , Diferenciação Celular/genética , Movimento Celular , Embrião não Mamífero/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Resposta ao Choque Térmico , Modelos Biológicos , Mutação/genética , Somitos/metabolismo , Transcrição Genética , Via de Sinalização Wnt , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
13.
J Vis Exp ; (159)2020 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-32478721

RESUMO

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the isolation of these vesicles, including ultracentrifugation, ultrafiltration, and size exclusion chromatography. However, not all are suitable for large scale exosome purification and characterization. Outlined here is a protocol for establishing cultures of primary fibroblasts isolated from adult mouse skeletal muscles, followed by purification and characterization of exosomes from the culture media of these cells. The method is based on the use of sequential centrifugation steps followed by sucrose density gradients. Purity of the exosomal preparations is then validated by western blot analyses using a battery of canonical markers (i.e., Alix, CD9, and CD81). The protocol describes how to isolate and concentrate bioactive exosomes for electron microscopy, mass spectrometry, and uptake experiments for functional studies. It can easily be scaled up or down and adapted for exosome isolation from different cell types, tissues, and biological fluids.


Assuntos
Exossomos/ultraestrutura , Fibroblastos , Músculo Esquelético/citologia , Animais , Células Cultivadas , Camundongos
14.
Plast Reconstr Surg ; 146(1): 43e-53e, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32590650

RESUMO

BACKGROUND: Skeletal muscle trauma can produce grave functional deficits, but therapeutic options remain limited. The authors studied whether a decellularized skeletal muscle scaffold would provide benefits in inducing skeletal muscle regeneration over acellular dermal matrices. METHODS: Eighty-two rat muscle defects were surgically created and assigned to no intervention or implantation of AlloDerm, Strattice, decellularized rat muscle, or decellularized rat dermis to 30 or 60 days. Decellularized rat muscle and dermis were prepared using a negative pressure-assisted protocol. Assessment for cellularity, neovascularization, myogenesis, inflammation and fibrosis were done histologically and by polymerase chain reaction. RESULTS: Histology showed relative hypercellularity of AlloDerm (p < 0.003); Strattice appeared encapsulated. Immunofluorescence for CD31 and myosin heavy chain in decellularized rat muscle revealed dense microvasculature and peripheral islands of myogenesis. MyoD expression in muscle scaffolds was 23-fold higher than in controls (p < 0.01). Decellularized rat muscle showed no up-regulation of COX-2 (p < 0.05), with less expression than decellularized rat dermis and Strattice (p < 0.002). Decellularized rat muscle scaffolds expressed tumor necrosis factor-α less than Strattice, AlloDerm, and decellularized rat dermis (p < 0.01); collagen-1a less than decellularized rat dermis and Strattice (p < 0.04); α-smooth muscle actin 7-fold less than AlloDerm (p = 0.04); and connective tissue growth factor less than Strattice, AlloDerm, and decellularized rat dermis (p < 0.02). CONCLUSION: Decellularized muscle matrix appears to reduce inflammation and fibrosis in an animal muscle defect as compared with dermal matrices and promotes greater expression of myocyte differentiation-inducing genes.


Assuntos
Derme Acelular , Músculo Esquelético , Engenharia Tecidual/métodos , Tecidos Suporte , Animais , Modelos Animais de Doenças , Masculino , Músculo Esquelético/citologia , Músculo Esquelético/lesões , Ratos , Ratos Sprague-Dawley , Cicatrização
15.
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
16.
PLoS One ; 15(5): e0233372, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32428048

RESUMO

Intramuscular fat content (IMF) is a complex trait influencing the technological and sensorial features of meat products and determining pork quality. Thus, we aimed at analyzing through RNA-sequencing the Semimembranosus muscle transcriptome of Italian Large White pigs to study the gene networks associated with IMF deposition. Two groups of samples were used; each one was composed of six unrelated pigs with extreme and divergent IMF content (0.67 ± 0.09% in low IMF vs. 6.81 ± 1.17% in high IMF groups) that were chosen from 950 purebred individuals. Paired-end RNA sequences were aligned to Sus scrofa genome assembly 11.1 and gene counts were analyzed using WGCNA and DeSeq2 packages in R environment. Interestingly, among the 58 differentially expressed genes (DEGs), several were related to primary cilia organelles (such as Lebercilin 5 gene), in addition to the genes involved in the regulation of cell differentiation, in the control of RNA-processing, and G-protein and ERK signaling pathways. Together with cilia-related genes, we also found in high IMF pigs an over-expression of the Fibroblast Growth Factor 2 (FGF2) gene, which in other animal species was found to be a regulator of ciliogenesis. Four WGCNA gene modules resulted significantly associated with IMF deposition: grey60 (P = 0.003), darkturquoise (P = 0.022), skyblue1 (P = 0.022), and lavenderblush3 (P = 0.030). The genes in the significant modules confirmed the results obtained for the DEGs, and the analysis with "cytoHubba" indicated genes controlling RNA splicing and cell differentiation as hub genes. Among the complex molecular processes affecting muscle fat depots, genes involved in primary cilia may have an important role, and the transcriptional reprogramming observed in high IMF pigs may be related to an FGF-related molecular cascade and to ciliogenesis, which in the literature have been associated with fibro-adipogenic precursor differentiation.


Assuntos
Tecido Adiposo/crescimento & desenvolvimento , Cílios/genética , Perfilação da Expressão Gênica , Músculo Esquelético/citologia , Animais , Composição Corporal , Diferenciação Celular/genética , Fator 2 de Crescimento de Fibroblastos/genética , Qualidade dos Alimentos , Redes Reguladoras de Genes , Carne de Porco , Processamento de RNA/genética , Suínos
17.
PLoS One ; 15(4): e0231600, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32294113

RESUMO

Charcot-Marie-Tooth (CMT) disease is an inherited peripheral motor and sensory neuropathy. The disease is divided into demyelinating (CMT1) and axonal (CMT2) neuropathies, and although we have gained molecular information into the details of CMT1 pathology, much less is known about CMT2. Due to its clinical and genetic heterogeneity, coupled with a lack of animal models, common underlying mechanisms remain elusive. In order to gain an understanding of the normal function of genes associated with CMT2, and to draw direct comparisons between them, we have studied the behavioural, cellular and molecular consequences of mutating nine different genes in the nematode Caenorhabditis elegans (lin-41/TRIM2, dyn-1/DNM2, unc-116/KIF5A, fzo-1/MFN2, osm-9/TRPV4, cua-1/ATP7A, hsp-25/HSPB1, hint-1/HINT1, nep-2/MME). We show that C. elegans defective for these genes display debilitated movement in crawling and swimming assays. Severe morphological defects in cholinergic motors neurons are also evident in two of the mutants (dyn-1 and unc-116). Furthermore, we establish methods for quantifying muscle morphology and use these to demonstrate that loss of muscle structure occurs in the majority of mutants studied. Finally, using electrophysiological recordings of neuromuscular junction (NMJ) activity, we uncover reductions in spontaneous postsynaptic current frequency in lin-41, dyn-1, unc-116 and fzo-1 mutants. By comparing the consequences of mutating numerous CMT2-related genes, this study reveals common deficits in muscle structure and function, as well as NMJ signalling when these genes are disrupted.


Assuntos
Comportamento Animal/fisiologia , Proteínas de Caenorhabditis elegans/genética , Doença de Charcot-Marie-Tooth/genética , Atividade Motora/genética , Junção Neuromuscular/patologia , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans , Doença de Charcot-Marie-Tooth/patologia , Doença de Charcot-Marie-Tooth/fisiopatologia , Neurônios Colinérgicos/patologia , Modelos Animais de Doenças , Heterogeneidade Genética , Humanos , Neurônios Motores/patologia , Músculo Esquelético/citologia , Músculo Esquelético/inervação , Músculo Esquelético/fisiopatologia , Mutação , Técnicas de Patch-Clamp , Potenciais Sinápticos/fisiologia
18.
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
19.
Nucleic Acids Res ; 48(9): 4601-4613, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32266374

RESUMO

While the histone variant H2A.Z is known to be required for mitosis, it is also enriched in nucleosomes surrounding the transcription start site of active promoters, implicating H2A.Z in transcription. However, evidence obtained so far mainly rely on correlational data generated in actively dividing cells. We have exploited a paradigm in which transcription is uncoupled from the cell cycle by developing an in vivo system to inactivate H2A.Z in terminally differentiated post-mitotic muscle cells. ChIP-seq, RNA-seq and ATAC-seq experiments performed on H2A.Z KO post-mitotic muscle cells show that this histone variant is neither required to maintain nor to activate transcription. Altogether, this study provides in vivo evidence that in the absence of mitosis H2A.Z is dispensable for transcription and that the enrichment of H2A.Z on active promoters is a marker but not an active driver of transcription.


Assuntos
Histonas/fisiologia , Músculo Esquelético/metabolismo , Transcrição Genética , Ativação Transcricional , Animais , Diferenciação Celular , Células Cultivadas , Cromatina , Sequenciamento de Cromatina por Imunoprecipitação , Histonas/genética , Histonas/metabolismo , Camundongos , Fibras Musculares Esqueléticas , Músculo Esquelético/citologia , RNA-Seq , Sequências Repetitivas de Ácido Nucleico , Sítio de Iniciação de Transcrição
20.
Anim Sci J ; 91(1): e13368, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32285501

RESUMO

Accumulation of intramuscular adipose tissue (IMAT) and development of fibrous tissues due to accumulation of collagen both affect meat quality such as tenderness, texture, and flavor. Thus, it is important for the production of high-quality meat to regulate the amount of adipose and fibrous tissues in skeletal muscle. IMAT is comprised of adipocytes, while collagens included in fibrous tissues are mainly produced by activated fibroblasts. Both adipocytes and fibroblasts are differentiated from their common ancestors, called mesenchymal progenitor cells (MPC). We previously established rat MPC clone, 2G11 cells. As several reports implicated the plasticity of fibroblast differentiation, in the present study, using 2G11 cells, we asked whether myofibroblasts differentiated from MPC are capable of re-gaining adipogenic potential in vitro. By treating with bFGF, their αSMA expression was reduced and adipogenic potential was restored partially. Furthermore, by lowering cell density together with bFGF treatment, 2G11 cell-derived myofibroblasts lost αSMA expression and showed the highest adipogenic potential, and this was along with their morphological change from flattened- to spindle-like shape, which is typically observed with MPC. These results indicated that MPC-derived myofibroblasts could re-acquire adipogenic potential, possibly mediated through returning to an undifferentiated MPC-like state.


Assuntos
Diferenciação Celular , Células-Tronco Mesenquimais/fisiologia , Músculo Esquelético/citologia , Miofibroblastos , Adipócitos/metabolismo , Tecido Adiposo/metabolismo , Animais , Células Cultivadas , Colágeno/metabolismo , Desenvolvimento Muscular/fisiologia , Músculo Esquelético/metabolismo , Miofibroblastos/metabolismo
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