Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 3.513
Filtrar
1.
Cells ; 11(13)2022 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-35805114

RESUMO

Actin cytoskeleton dynamics are essential regulatory processes in muscle development, growth, and regeneration due to their modulation of mechanotransduction, cell proliferation, differentiation, and morphological changes. Although the KN motif and ankyrin repeat domain-containing protein 1 (Kank1) plays a significant role in cell adhesion dynamics, actin polymerization, and cell proliferation in various cells, the functional significance of Kank1 during the myogenic differentiation of progenitor cells has not been explored. Here, we report that Kank1 acts as a critical regulator of the proliferation and differentiation of muscle progenitor cells. Kank1 was found to be expressed at a relatively high level in C2C12 myoblasts, and its expression was modulated during the differentiation. Depletion of Kank1 by siRNA (siKank1) increased the accumulation of filamentous actin (F-actin). Furthermore, it facilitated the nuclear localization of Yes-associated protein 1 (YAP1) by diminishing YAP1 phosphorylation in the cytoplasm, which activated the transcriptions of YAP1 target genes and promoted proliferation and cell cycle progression in myoblasts. Notably, depletion of Kank1 suppressed the protein expression of myogenic regulatory factors (i.e., MyoD and MyoG) and dramatically inhibited myoblast differentiation and myotube formation. Our results show that Kank1 is an essential regulator of actin dynamics, YAP1 activation, and cell proliferation and that its depletion impairs the myogenic differentiation of progenitor cells by promoting myoblast proliferation triggered by the F-actin-induced nuclear translocation of YAP1.


Assuntos
Actinas , Mecanotransdução Celular , Actinas/metabolismo , Proliferação de Células , Desenvolvimento Muscular/genética , Mioblastos/metabolismo
2.
Methods Cell Biol ; 170: 117-125, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35811095

RESUMO

Skeletal muscle is a highly regenerative tissue that can efficiently recover from various damages caused by injuries and excessive exercises. In adult muscle, stem cells termed satellite cells are mitotically quiescent but activated upon muscle damages to enter the cell cycle as myogenic precursor cells or myoblasts. After several rounds of cell cycles, they exist the cycle and fuse to each other to form multinucleated myotubes, and eventually mature to become contractile myofibers. Satellite cells can be readily isolated from mouse skeletal muscle with enzymatic digestion and magnetic separation with antibodies against specific surface markers. C2C12 cells are an immortalized mouse myoblast cell line that is commercially available and more readily expandable than primary myoblasts. Both primary myoblasts and C2C12 cells have been extensively used as useful in vitro models for myogenic differentiation. Proper examination of this process requires monitoring specific protein expression in subcellular compartments, which can be accomplished through immunofluorescence staining. This chapter describes the workflow for the isolation of satellite cells from mouse skeletal muscle and subsequent immunofluorescence staining to assess the proliferation and differentiation of primary myoblasts and C2C12 cells.


Assuntos
Desenvolvimento Muscular , Mioblastos , Animais , Diferenciação Celular/fisiologia , Imunofluorescência , Camundongos , Músculo Esquelético , Mioblastos/metabolismo , Coloração e Rotulagem
3.
FASEB J ; 36(8): e22441, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35816155

RESUMO

Vesicle-mediated transport is necessary for maintaining cellular homeostasis and proper signaling. The synaptosome-associated protein 23 (SNAP23) is a member of the SNARE protein family and mediates the vesicle docking and membrane fusion steps of secretion during exocytosis. Skeletal muscle has been established as a secretory organ; however, the role of SNAP23 in the context of skeletal muscle development is still unknown. Here, we show that depletion of SNAP23 in C2C12 mouse myoblasts reduces their ability to differentiate into myotubes as a result of premature cell cycle exit and early activation of the myogenic transcriptional program. This effect is rescued when cells are seeded at a high density or when cultured in conditioned medium from wild type cells. Proteomic analysis of collected medium indicates that SNAP23 depletion leads to a misregulation of exocytosis, including decreased secretion of the insulin-like growth factor 1 (IGF1), a critical protein for muscle growth, development, and function. We further demonstrate that treatment of SNAP23-depleted cells with exogenous IGF1 rescues their myogenic capacity. We propose that SNAP23 mediates the secretion of specific proteins, such as IGF1, that are important for achieving proper differentiation of skeletal muscle cells during myogenesis. This work highlights the underappreciated role of skeletal muscle as a secretory organ and contributes to the understanding of factors necessary for myogenesis.


Assuntos
Proteômica , Sinaptossomos , Animais , Diferenciação Celular , Camundongos , Desenvolvimento Muscular , Mioblastos/metabolismo , Proteínas Qb-SNARE/genética , Proteínas Qc-SNARE/genética , Proteínas SNARE/metabolismo , Sinaptossomos/metabolismo
4.
Am J Physiol Cell Physiol ; 323(2): C595-C605, 2022 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-35848618

RESUMO

Satellite cells are required for muscle regeneration, remodeling, and repair through their activation, proliferation, and differentiation; however, how dietary factors regulate this process remains poorly understood. The L-type amino acid transporter 1 (LAT1) transports amino acids, such as leucine, into mature myofibers, which then stimulate protein synthesis and anabolic signaling. However, whether LAT1 is expressed on myoblasts and is involved in regulating myogenesis is unknown. The aim of this study was to characterize the expressional and functional relevance of LAT1 during different stages of myogenesis and in response to growth and atrophic conditions in vitro. We determined that LAT1 is expressed by C2C12 and human primary myoblasts, and its gene expression is lower during differentiation (P < 0.05). Pharmacological inhibition and genetic knockdown of LAT1 impaired myoblast viability, differentiation, and fusion (all P < 0.05). LAT1 protein content in C2C12 myoblasts was not significantly altered in response to different leucine concentrations in cell culture media or in two in vitro atrophy models. However, LAT1 content was decreased in myotubes under atrophic conditions in vitro (P < 0.05). These findings indicate that LAT1 is stable throughout myogenesis and in response to several in vitro conditions that induce muscle remodeling. Further, amino acid transport through LAT1 is required for normal myogenesis in vitro.


Assuntos
Transportador 1 de Aminoácidos Neutros Grandes , Desenvolvimento Muscular , Aminoácidos/metabolismo , Humanos , Transportador 1 de Aminoácidos Neutros Grandes/genética , Transportador 1 de Aminoácidos Neutros Grandes/metabolismo , Leucina/metabolismo , Mioblastos/metabolismo
5.
Cells ; 11(14)2022 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-35883670

RESUMO

The production of goat meat is determined by the growth speed of muscle fibers, and the autophagy and apoptosis of myoblast cells is a crucial process in the growth of muscle fibers. The rapid growth of muscle fibers occurs from one month old to nine months old in goats; however, the mechanisms of myoblast cells' autophagy and apoptosis in this process are still unknown. To identify candidate genes and signaling pathway mechanisms involved in myoblast apoptosis and autophagy, we compared the expression characteristics of longissimus dorsi tissues from Wu'an goats-a native goat breed of China-at 1 month old (mon1 group) and 9 months old (mon9 group). Herein, a total of 182 differentially expressed mRNAs (DEGs) in the mon1 vs. mon9 comparison, along with the KEGG enrichments, showed that the PI3K-Akt pathway associated with autophagy and apoptosis was significantly enriched. Among these DEGs, expression of vacuole membrane protein 1 (VMP1)-a key gene for the PI3K-Akt pathway-was significantly upregulated in the older goats relative to the 1-month-old goats. We demonstrated that VMP1 promotes the proliferation and autophagy of myoblasts, and inhibits their apoptosis. The integration analysis of miRNA-mRNA showed that miR-124a was a regulator of VMP1 in muscle tissue, and overexpression and inhibition of miR-124a suppressed the proliferation and autophagy of myoblasts. The PI3K/Akt/mTOR pathway was an important pathway for cell autophagy. Additionally, the activator of the PI3K/Akt/mTOR pathway, the expression of VMP1, and ULK1 were higher than the negative control, and the expression of mTOR was depressed. The expression of VMP1, ULK1, and mTOR was the opposite when the inhibitor was added to the myoblasts. These results show that the PI3K/Akt/mTOR pathway promoted the expression of VMP1 and ULK1. By using adenovirus-mediated apoptosis and proliferation assays, we found that that miR-124a inhibits myoblast proliferation and autophagy, and promotes their apoptosis by targeting VMP1. In conclusion, our results indicated that VMP1 was highly expressed in the LD muscle tissues of nine-month-old goats, and that it was regulated by miR-124a to inhibit myoblast cells' apoptosis through the PI3K/Akt/mTOR pathway, and to promote proliferation and autophagy. These findings contribute to the understanding of the molecular mechanisms involved in myoblast proliferation, autophagy, and apoptosis.


Assuntos
MicroRNAs , Fosfatidilinositol 3-Quinases , Animais , Apoptose/genética , Autofagia/genética , Proliferação de Células/genética , Cabras/metabolismo , MicroRNAs/metabolismo , Mioblastos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo
6.
Int J Mol Sci ; 23(14)2022 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-35886840

RESUMO

Human skeletal muscle contains three different types of fibers, each with a different metabolism. Exercise differently contributes to differentiation and metabolism in human myoblast cells. The aims of the present study were to investigate the effects of different types of chronic training on the human LHCN-M2 myoblast cell bioenergetic profile during differentiation in real time and on the ROS overproduction consequent to H2O2 injury. We demonstrated that exercise differently affects the myoblast bioenergetics: aerobic exercise induced the most efficient glycolytic and oxidative capacity and proton leak reduction compared to untrained or anaerobic trained sera-treated cells. Similarly, ROS overproduction after H2O2 stress was lower in cells treated with differently trained sera compared to untrained sera, indicating a cytoprotective effect of training on the reduction of oxidative stress, and thus the promotion of longevity. In conclusion, for the first time, this study has provided knowledge regarding the modifications induced by different types of chronic training on human myoblast cell bioenergetics during the differentiation process in real time, and on ROS overproduction due to stress, with positive implications in terms of longevity.


Assuntos
Peróxido de Hidrogênio , Mioblastos , Metabolismo Energético , Humanos , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo
7.
Int J Mol Sci ; 23(13)2022 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-35806481

RESUMO

Lactoferrin (Ltf), a naturally active glycoprotein, possesses anti-inflammatory, anti-microbial, anti-tumor, and immunomodulatory activities. Many published studies have indicated that Ltf modulates the proliferation of stem cells. However, the role of Ltf in the proliferation of satellite cells, an important cell type in muscle regeneration, has not yet been reported. Here, by using Ltf systemic knockout mice, we illustrate the role of Ltf in skeletal muscle. Results shows that Ltf deficiency impaired proliferation of satellite cells (SCs) and the regenerative capability of skeletal muscle. Mechanistic studies showed that ERK1/2 phosphorylation was significantly downregulated after Ltf deletion in SCs. Simultaneously, the cell cycle-related proteins cyclin D and CDK4 were significantly downregulated. Intervention with exogenous recombinant lactoferrin (R-Ltf) at a concentration of 1000 µg/mL promoted proliferation of SCs. In addition, intraperitoneal injection of Ltf effectively ameliorated the skeletal muscle of mice injured by 1.2% BaCl2 solution. Our results suggest a protective effect of Ltf in the repair of skeletal muscle damage. Ltf holds promise as a novel therapeutic agent for skeletal muscle injuries.


Assuntos
Lactoferrina , Sistema de Sinalização das MAP Quinases , Animais , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células/fisiologia , Regulação para Baixo , Lactoferrina/deficiência , Lactoferrina/metabolismo , Lactoferrina/farmacologia , Camundongos , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , Proteínas Recombinantes/farmacologia , Transdução de Sinais
8.
PLoS One ; 17(7): e0272231, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35901044

RESUMO

Arecoline is known to induce reactive oxygen species (ROS). Our previous studies showed that arecoline inhibited myogenic differentiation and acetylcholine receptor cluster formation of C2C12 myoblasts. N-acetyl-cysteine (NAC) is a known ROS scavenger. We hypothesize that NAC scavenges the excess ROS caused by arecoline. In this article we examined the effect of NAC on the inhibited myoblast differentiation by arecoline and related mechanisms. We found that NAC less than 2 mM is non-cytotoxic to C2C12 by viability analysis. We further demonstrated that NAC attenuated the decreased number of myotubes and nuclei in each myotube compared to arecoline treatment by H & E staining. We also showed that NAC prevented the decreased expression level of the myogenic markers, myogenin and MYH caused by arecoline, using immunocytochemistry and western blotting. Finally, we found that NAC restored the decreased expression level of p-ERK1/2 by arecoline. In conclusion, our results indicate that NAC attenuates the damage of the arecoline-inhibited C2C12 myoblast differentiation by the activation/phosphorylation of ERK. This is the first report to demonstrate that NAC has beneficial effects on skeletal muscle myogenesis through ERK1/2 upon arecoline treatment. Since defects of skeletal muscle associates with several diseases, NAC can be a potent drug candidate in diseases related to defects in skeletal muscle myogenesis.


Assuntos
Arecolina , Sistema de Sinalização das MAP Quinases , Acetilcisteína/metabolismo , Acetilcisteína/farmacologia , Arecolina/farmacologia , Diferenciação Celular , Desenvolvimento Muscular , Mioblastos/metabolismo , Fosforilação , Espécies Reativas de Oxigênio/metabolismo
9.
Sci Rep ; 12(1): 13020, 2022 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-35906363

RESUMO

Muscle fibres are broadly categorised into types I and II; the fibre-type ratio determines the contractile and metabolic properties of skeletal muscle tissue. The maintenance of type I fibres is essential for the prevention of obesity and the treatment of muscle atrophy caused by type 2 diabetes or unloading. Some reports suggest that myokines are related to muscle fibre type determination. We thus explored whether a myokine determines whether satellite cells differentiate to type I fibres. By examining the fibre types separately, we identified R-spondin 3 (Rspo3) as a myokine of interest, a secreted protein known as an activator of Wnt signalling pathways. To examine whether Rspo3 induces type I fibres, primary myoblasts prepared from mouse soleus muscles were exposed to a differentiation medium containing the mouse recombinant Rspo3 protein. Expression of myosin heavy chain (MyHC) I, a marker of type I fibre, significantly increased in the differentiated myotubes compared with a control. The Wnt/ß-catenin pathway was shown to be the dominant signalling pathway which induces Rspo3-induced MyHC I expression. These results revealed Rspo3 as a myokine that determines whether satellite cells differentiate to type I fibres.


Assuntos
Diabetes Mellitus Tipo 2 , Animais , Diabetes Mellitus Tipo 2/metabolismo , Camundongos , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Trombospondinas/metabolismo
10.
J Anim Sci ; 100(8)2022 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-35908792

RESUMO

Intrauterine growth restriction (IUGR) is linked to lifelong reductions in muscle mass due to intrinsic functional deficits in myoblasts, but the mechanisms underlying these deficits are not known. Our objective was to determine if the deficits were associated with changes in inflammatory and adrenergic regulation of IUGR myoblasts, as was previously observed in IUGR muscle. Primary myoblasts were isolated from IUGR fetal sheep produced by hyperthermia-induced placental insufficiency (PI-IUGR; n = 9) and their controls (n = 9) and from IUGR fetal sheep produced by maternofetal inflammation (MI-IUGR; n = 6) and their controls (n = 7). Proliferation rates were less (P < 0.05) for PI-IUGR myoblasts than their controls and were not affected by incubation with IL-6, TNF-α, norepinephrine, or insulin. IκB kinase inhibition reduced (P < 0.05) proliferation of control myoblasts modestly in basal media but substantially in TNF-α-added media and reduced (P < 0.05) PI-IUGR myoblast proliferation substantially in basal and TNF-α-added media. Proliferation was greater (P < 0.05) for MI-IUGR myoblasts than their controls and was not affected by incubation with TNF-α. Insulin increased (P < 0.05) proliferation in both MI-IUGR and control myoblasts. After 72-h differentiation, fewer (P < 0.05) PI-IUGR myoblasts were myogenin+ than controls in basal and IL-6 added media but not TNF-α-added media. Fewer (P < 0.05) PI-IUGR myoblasts were desmin+ than controls in basal media only. Incubation with norepinephrine did not affect myogenin+ or desmin+ percentages, but insulin increased (P < 0.05) both markers in control and PI-IUGR myoblasts. After 96-h differentiation, fewer (P < 0.05) MI-IUGR myoblasts were myogenin+ and desmin+ than controls regardless of media, although TNF-α reduced (P < 0.05) desmin+ myoblasts for both groups. Differentiated PI-IUGR myoblasts had greater (P < 0.05) TNFR1, ULK2, and TNF-α-stimulated TLR4 gene expression, and PI-IUGR semitendinosus muscle had greater (P < 0.05) TNFR1 and IL6 gene expression, greater (P < 0.05) c-Fos protein, and less (P < 0.05) IκBα protein. Differentiated MI-IUGR myoblasts had greater (P < 0.05) TNFR1 and IL6R gene expression, tended to have greater (P = 0.07) ULK2 gene expression, and had greater (P < 0.05) ß-catenin protein and TNF-α-stimulated phosphorylation of NFκB. We conclude that these enriched components of TNF-α/TNFR1/NFκB and other inflammatory pathways in IUGR myoblasts contribute to their dysfunction and help explain impaired muscle growth in the IUGR fetus.


Myoblasts are stems cells whose functional capacity can limit muscle growth. However, stressful intrauterine conditions cause these cells to be intrinsically dysfunctional. This restricts muscle growth capacity, leading to intrauterine growth restriction (IUGR) of the fetus, low birth weight, and less muscle mass after birth. Consequently, meat yield is reduced in IUGR-born food animals and glucose homeostasis is impaired in IUGR-born humans, which contributes to metabolic dysfunction. Intrinsic dysfunction of IUGR myoblasts has been previously observed, but the fetal programming changes (i.e., permanent changes in the development of cellular mechanisms that explains different functional outcomes) have not been identified. This study shows that one mechanism is the enhancement of signaling pathways for TNF-α and other inflammatory cytokines. These cytokines have roles in stress responses and regulation of muscle growth. Programmed enhancement of these pathways means that IUGR myoblasts are more responsive to even normal amounts of circulating cytokines. Unfortunately, the primary response of myoblasts to cytokines is slower differentiation (i.e., cellular transformation necessary for muscle growth). Programmed enhancement of this response directly impedes myoblast-dependent muscle growth, and the deficit is lifelong. However, identifying this mechanism is a fundamental step for developing strategies to improve muscle growth in low birth weight offspring.


Assuntos
Retardo do Crescimento Fetal , Doenças dos Ovinos , Animais , Proliferação de Células , Desmina/metabolismo , Feminino , Retardo do Crescimento Fetal/veterinária , Feto/metabolismo , Insulina/metabolismo , Insulina/farmacologia , Interleucina-6/metabolismo , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , Miogenina/metabolismo , Norepinefrina , Placenta/metabolismo , Gravidez , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Ovinos , Transdução de Sinais
11.
Cell Commun Signal ; 20(1): 107, 2022 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-35850889

RESUMO

BACKGROUND: Type 2 diabetes mellitus is a global health problem. It often leads to a decline in the differentiation capacity of myoblasts and progressive loss of muscle mass, which in turn results in deterioration of skeletal muscle function. However, effective therapies against skeletal muscle diseases are unavailable. METHODS: Skeletal muscle mass and differentiation ability were determined in db/+ and db/db mice. Transcriptomics and metabolomics approaches were used to explore the genetic mechanism regulating myoblast differentiation in C2C12 myoblasts. RESULTS: In this study, the relatively uncharacterized solute carrier family gene Slc2a6 was found significantly up-regulated during myogenic differentiation and down-regulated during diabetes-induced muscle atrophy. Moreover, RNAi of Slc2a6 impaired the differentiation and myotube formation of C2C12 myoblasts. Both metabolomics and RNA-seq analyses showed that the significantly differentially expressed genes (e.g., LDHB) and metabolites (e.g., Lactate) during the myogenic differentiation of C2C12 myoblasts post-Slc2a6-RNAi were enriched in the glycolysis pathway. Furthermore, we show that Slc2a6 regulates the myogenic differentiation of C2C12 myoblasts partly through the glycolysis pathway by targeting LDHB, which affects lactic acid accumulation. CONCLUSION: Our study broadens the understanding of myogenic differentiation and offers the Slc2a6-LDHB axis as a potential therapeutic target for the treatment of diabetes-associated muscle atrophy. Video abstract.


Assuntos
Diabetes Mellitus Tipo 2 , Animais , Diferenciação Celular , Diabetes Mellitus Tipo 2/metabolismo , Camundongos , Desenvolvimento Muscular/genética , Fibras Musculares Esqueléticas/metabolismo , Atrofia Muscular/metabolismo , Mioblastos/metabolismo
12.
FASEB J ; 36(7): e22385, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35734962

RESUMO

Skeletal muscles can regenerate over the lifetime from resident muscle stem cells (MuSCs). Interactions between MuSCs and extracellular matrix (ECM) proteins are essential for muscle regeneration. The best-known receptors for ECM proteins are integrins, a family composed of twenty-some heterodimeric combinations of an α- and a ß-subunit. ß1-integrin (encoded by Itgb1) is required for quiescence, proliferation, migration, and fusion of Pax7+ MuSCs in the mouse model. ß3-integrin (encoded by Itgb3) has been reported to be critical for the myogenic differentiation of C2C12 myoblasts, and Itgb3 germline mutant mice were shown to regenerate few if any myofibers after injury. To investigate the autonomous role of Itgb3 in the myogenic lineage in vivo, we conditionally inactivated a floxed Itgb3 allele (Itgb3F ) by constitutive Pax7-Cre and tamoxifen-inducible Pax7-CreERT2 drivers. Unexpectedly, we found no defects in muscle regeneration in both conditional knockout models. In vitro studies using Itgb3 mutant myoblasts or RNAi knockdown of Itgb3 in myoblasts also did not reveal a role for myogenic differentiation. As ß1- and ß3-integrins share ECM ligands and downstream signaling effectors, we further examined Itgb3's role in a Itgb1 haploid background. Still, we found no evidence for an autonomous role of Itgb3 in muscle regeneration in vivo. Thus, while Itgb3 is critical for the differentiation of C2C12 cells, the regenerative defects reported for the Itgb3 germline mutant are not due to its role in the MuSC. We conclude that if ß3-integrin does have a role in Pax7+ MuSCs, it is compensated by ß1- and/or another ß-integrin(s).


Assuntos
Desenvolvimento Muscular , Mioblastos , Animais , Diferenciação Celular , Camundongos , Desenvolvimento Muscular/fisiologia , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , Transdução de Sinais
13.
Int J Mol Sci ; 23(11)2022 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-35682680

RESUMO

Myogenesis is a central step in prenatal myofiber formation, postnatal myofiber hypertrophy, and muscle damage repair in adulthood. RNA-Seq technology has greatly helped reveal the molecular mechanism of myogenesis, but batch effects in different experiments inevitably lead to misinterpretation of differentially expressed genes (DEGs). We previously applied the robust rank aggregation (RRA) method to effectively circumvent batch effects across multiple RNA-Seq datasets from 3T3-L1 cells. Here, we also used the RRA method to integrate nine RNA-Seq datasets from C2C12 cells and obtained 3140 robust DEGs between myoblasts and myotubes, which were then validated with array expression profiles and H3K27ac signals. The upregulated robust DEGs were highly enriched in gene ontology (GO) terms related to muscle cell differentiation and development. Considering that the cooperative binding of transcription factors (TFs) to enhancers to regulate downstream gene expression is a classical epigenetic mechanism, differentially expressed TFs (DETFs) were screened, and potential novel myogenic factors (MAF, BCL6, and ESR1) with high connection degree in protein-protein interaction (PPI) network were presented. Moreover, KLF5 cooperatively binds with the three key myogenic factors (MYOD, MYOG, and MEF2D) in C2C12 cells. Motif analysis speculates that the binding of MYOD and MYOG is KLF5-independent, while MEF2D is KLF5-dependent. It was revealed that KLF5-binding sites could be exploited to filter redundant MYOD-, MYOG-, and MEF2D-binding sites to focus on key enhancers for myogenesis. Further functional annotation of KLF5-binding sites suggested that KLF5 may regulate myogenesis through the PI3K-AKt signaling pathway, Rap1 signaling pathway, and the Hippo signaling pathway. In general, our study provides a wealth of untapped candidate targets for myogenesis and contributes new insights into the core regulatory mechanisms of myogenesis relying on KLF5-binding signal.


Assuntos
Desenvolvimento Muscular , Fosfatidilinositol 3-Quinases , Diferenciação Celular/genética , Desenvolvimento Muscular/genética , Mioblastos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fatores de Transcrição/metabolismo
14.
J Agric Food Chem ; 70(26): 8145-8163, 2022 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-35749701

RESUMO

Circular RNA (circRNA) is a form of endogenous RNA that can regulate gene expression and participate in the regulation of myogenesis. However, the molecular mechanisms and potential roles of circRNAs in bovine muscle development remain largely unknown. Nevertheless, the RNA splicing factors regulating the biogenesis of bovine circRNA have not yet been characterized. In this study, we identified a novel circRNA, circMEF2D, formed by back-splicing of constitutive exons (exons 5-7) of the bovine MEF2D gene. Functional assays showed that circMEF2D inhibited the proliferation and differentiation of bovine myoblasts. Importantly, we showed that circMEF2D regulated the PI3K-AKT signaling pathway through direct and competitive binding to miR-486. Furthermore, to explore the formation mechanism of circMEF2D, we explored the MEF2D gene alternative splicing progress. Four alternative linear variants of MEF2D were found. Due to its role in alternative splicing, the RNA-binding protein HNRNPA1 was selected for further study and the modulation of HNRNPA1 levels showed that it negatively regulated both back-splicing and linear splicing of MEF2D gene. Overall, in addition to the characterization of bovine circRNAs, these findings revealed the crucial role of HNRNPA1 in MEF2D gene alternative splicing and demonstrated a regulatory circMEF2D-miR-486-PI3K-AKT axis.


Assuntos
MicroRNAs , RNA Circular , Animais , Bovinos , Proliferação de Células , MicroRNAs/genética , MicroRNAs/metabolismo , Mioblastos/metabolismo , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , RNA Circular/genética
15.
Cell Stem Cell ; 29(6): 933-947.e6, 2022 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-35597234

RESUMO

Many tissues harbor quiescent stem cells that are activated upon injury, subsequently proliferating and differentiating to repair tissue damage. Mechanisms by which stem cells sense injury and transition from quiescence to activation, however, remain largely unknown. Resident skeletal muscle stem cells (MuSCs) are essential orchestrators of muscle regeneration and repair. Here, with a combination of in vivo and ex vivo approaches, we show that quiescent MuSCs have elaborate, Rac GTPase-promoted cytoplasmic projections that respond to injury via the upregulation of Rho/ROCK signaling, facilitating projection retraction and driving downstream activation events. These early events involve rapid cytoskeletal rearrangements and occur independently of exogenous growth factors. This mechanism is conserved across a broad range of MuSC activation models, including injury, disease, and genetic loss of quiescence. Our results redefine MuSC activation and present a central mechanism by which quiescent stem cells initiate responses to injury.


Assuntos
Células Satélites de Músculo Esquelético , Proteínas rho de Ligação ao GTP , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético , Mioblastos/metabolismo , Células Satélites de Músculo Esquelético/metabolismo , Células-Tronco/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo
16.
Sci Rep ; 12(1): 8388, 2022 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-35589804

RESUMO

WNT signalling is essential for regulating a diverse range of cellular processes. In skeletal muscle, the WNT pathway plays crucial roles in maintenance of the stem cell pool and myogenic differentiation. Focus is usually directed at examining the function of central components of the WNT pathway, including ß-CATENIN and the GSK3ß complex and TCF/LEF transcription factors, in tissue homeostasis and cancer. Other core components of the WNT pathway though, are three dishevelled (DVL) proteins: membrane associated proteins that propagate WNT signalling from membrane to nucleus. Here we examined DVL function in human myogenesis and the muscle-related cancer alveolar rhabdomyosarcoma. We demonstrate that DVL1 and DVL3 are necessary for efficient proliferation in human myoblasts and are important for timely myogenic differentiation. DVL1 and DVL3 also contribute to regulation of proliferation in rhabdomyosarcoma. DVL1 or DVL3 must be present in the nucleus to regulate proliferation, but they operate through different protein domains: DVL3 requires the DIX and PDZ domains, while DVL1 does not. Importantly, DVL1 and DVL3 activity is independent of markedly increased translocation of ß-CATENIN to the nucleus, normally a hallmark of active canonical WNT signalling.


Assuntos
Neoplasias , beta Catenina , Proliferação de Células , Proteínas Desgrenhadas/metabolismo , Humanos , Mioblastos/metabolismo , Fosfoproteínas/metabolismo , Via de Sinalização Wnt , beta Catenina/metabolismo
17.
Int J Mol Sci ; 23(9)2022 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-35562902

RESUMO

Long noncoding RNA (lncRNA) plays a crucial part in all kinds of life activities, especially in myogenesis. SMARCD3 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily d, member 3) is a member of the SWI/SNF protein complex and was reported to be required for cell proliferation and myoblast differentiation. In this study, we identified a new lncRNA named SMARCD3-OT1 (SMARCD3overlappinglncRNA), which strongly regulated the development of myogenesis by improving the expression of SMARCD3X4 (SMARCD3transcripts4). We overexpressed and knockdown the expression of SMARCD3-OT1 and SMARCD3X4 to investigate their function on myoblast proliferation and differentiation. Cell experiments proved that SMARCD3-OT1 and SMARCD3X4 promoted myoblast proliferation through the CDKN1A pathway and improved differentiation of differentiated myoblasts through the MYOD pathway. Moreover, they upregulated the fast-twitch fiber-related genes and downregulated the slow-twitch fiber-related genes, which indicated that they facilitated the slow-twitch fiber to transform into the fast-twitch fiber. The animals' experiments supported the results above, demonstrating that SMARCD3-OT1 could induce muscle hypertrophy and fast-twitch fiber transformation. In conclusion, SMARCD3-OT1 can improve the expression of SMARCD3X4, thus inducing muscle hypertrophy. In addition, SMARCD3-OT1 can facilitate slow-twitch fibers to transform into fast-twitch fibers.


Assuntos
RNA Longo não Codificante , Animais , Diferenciação Celular/genética , Hipertrofia/genética , Hipertrofia/metabolismo , Desenvolvimento Muscular/genética , Músculos , Mioblastos/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo
18.
Cells ; 11(9)2022 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-35563742

RESUMO

For the purpose of skeletal muscle tissue engineering, different cell types have been investigated regarding their myogenic differentiation potential, including co-cultured myoblasts and adipogenic mesenchymal stromal cells (Mb/ADSC). As neural cells enhance synaptic junction formation, the aim of this study was to co-culture Schwann cells (SCs) with Mb/ADSC on biocompatible electrospun aligned poly-ε-polycaprolacton (PCL)-collagen I-nanofibers. It was hypothesized that SCs, as part of the peripheral nervous system, promote the myogenic differentiation of Mb/ADSC co-cultures. Mb/ADSC were compared to Mb/ADSC/SC regarding their capacity for myogenic differentiation via immunofluorescent staining and gene expression of myogenic markers. Mb/ADSC/SC showed more myotubes after 28 days of differentiation (p ≤ 0.05). After 28 days of differentiation on electrospun aligned PCL-collagen I-nanofibers, gene expression of myosin heavy chains (MYH2) and myogenin (MYOG) was upregulated in Mb/ADSC/SC compared to Mb/ADSC (p ≤ 0.01 and p ≤ 0.05, respectively). Immunofluorescent staining for MHC showed highly aligned multinucleated cells as possible myotube formation in Mb/ADSC/SC. In conclusion, SCs promote myogenic differentiation of Mb/ADSC. The co-culture of primary Mb/ADSC/SC on PCL-collagen I-nanofibers serves as a physiological model for skeletal muscle tissue engineering, applicable to future clinical applications.


Assuntos
Células-Tronco Mesenquimais , Nanofibras , Caproatos , Colágeno/metabolismo , Colágeno Tipo I/metabolismo , Lactonas , Células-Tronco Mesenquimais/metabolismo , Mioblastos/metabolismo , Células de Schwann
19.
J Nutr Sci Vitaminol (Tokyo) ; 68(2): 79-86, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35491208

RESUMO

Skeletal muscle plays crucial roles in locomotion, protein reservoir, and maintenance of metabolic homeostasis. Loss of muscle, known as muscle atrophy, causes the metabolic diseases such as type 2 diabetes mellitus, hypertension, and so on. Therefore, great efforts have been devoted to prevent the muscle atrophy. Policosanols are a mixture of long chain fatty alcohols extracted from various natural sources. They have long been used as functional foods to lower the level of serum lipids, including triacylglycerol and cholesterol, and to protect against inflammatory stress. In this study, we examine the protective effect and molecular mechanism of Cuban policosanol on skeletal muscle cell death and mitochondrial dysfunction using lipopolysaccharide-treated C2C12 cells. Our results demonstrated that policosanol significantly rescued cell survival (40% vs. 88%; LPS vs. LPS+policosanol) via activation of the Akt pathway, resulting in inhibition of apoptosis (p<0.05). Moreover, policosanol restored the LPS-induced repression of collagen by two fold (0.33±0.04 vs. 0.67±0.03 compared to that of control; LPS vs. LPS+policosanol) via activation of ERK-mTOR-p70S6K pathways. In addition, policosanol increased the mitochondrial fusion by regulating the activities of DRP1 and Mfn2, leading to ameliorate the mitochondrial dysfunction induced by LPS. Improved mitochondria function increased the oxygen consumption rate with glucose as fuel source, indicating that policosanol could shift the glucose metabolism from lactate fermentation, induced by lipopolysaccharide, to oxidative phosphorylation. Thus, policosanol is a promising agent for preventing the inflammation-induced muscle cell death and mitochondrial dysfunction.


Assuntos
Diabetes Mellitus Tipo 2 , Lipopolissacarídeos , Animais , Apoptose , Linhagem Celular , Diabetes Mellitus Tipo 2/metabolismo , Álcoois Graxos/farmacologia , Sistema de Sinalização das MAP Quinases , Camundongos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Atrofia Muscular/metabolismo , Mioblastos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo
20.
Biochim Biophys Acta Mol Cell Res ; 1869(9): 119294, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35597451

RESUMO

Tinagl1 (tubulointerstitial nephritis antigen-like 1) is a matricellular protein involved in female infertility and breast cancer tumorigenesis. In this study, we analyzed the function of Tinagl1 in skeletal muscle using knockout mice and cell experiments. Although primary myoblasts isolated from Tinagl1-decifient (Tinagl1-/-) mice differentiated into normal myotubes, and treatment with recombinant Tinagl1 did not affect the proliferation or differentiation of C2C12 myoblasts, Tinagl1-/- mice exhibited reduced body mass and calf muscle weights compared to the control group (Tinagl1flox/flox). Furthermore, Tinagl1-/- mice showed myofibers with centrally located nuclei, which is a morphological marker of regenerating muscle or myopathy. In addition, the capillary density in the soleus muscle of Tinagl1-/- mice showed a decreasing trend compared to that of the control group. Importantly, si-RNA-mediated knockdown of TINAGL1 resulted in reduced tube formation in human umbilical vein endothelial cells (HUVECs), whereas treatment with Tinagl1 promoted tube formation. Immunoblot analysis revealed that Tinagl1 activates ERK signaling in both HUVECs and C2C12 myoblasts and myotubes, which are involved in the regulation of myogenic differentiation, proliferation, metabolism, and angiogenesis. Our results demonstrate that Tinagl1 may be required for normal muscle and capillary development through the activation of ERK signaling.


Assuntos
Células Endoteliais , Lipocalinas/metabolismo , Desenvolvimento Muscular , Proteínas de Neoplasias/metabolismo , Animais , Feminino , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Knockout , Desenvolvimento Muscular/genética , Músculo Esquelético , Mioblastos/metabolismo , Fatores de Transcrição/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...