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1.
Ageing Res Rev ; 67: 101309, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33626408

RESUMO

Mitochondria are highly dynamic organelles capable of adapting their network, morphology, and function, playing a role in oxidative phosphorylation and many cellular processes in most cell types. Skeletal muscle is a very plastic tissue, subjected to many morphological changes following diverse stimuli, such as during myogenic differentiation and regenerative myogenesis. For some time now, mitochondria have been reported to be involved in myogenesis by promoting a bioenergetic remodeling and assisting myoblasts in surviving the process. However, not much is known about the interplay between mitochondrial quality control and myogenic differentiation. Sestrin2 (SESN2) is a well described regulator of autophagy and antioxidant responses and has been gaining attention due to its role in aging-associated pathologies and redox signaling promoted by reactive oxygen species (ROS) in many tissues. Current evidence involving SESN2-associated pathways suggest that it can act as a potential regulator of mitochondrial quality control following induction by ROS under stress conditions, such as during myogenesis. Yet, there are no studies directly assessing SESN2 involvement in myogenic differentiation. This review provides novel insights pertaining the involvement of SESN2 in myogenic differentiation by analyzing the interactions between ROS and mitochondrial remodeling.


Assuntos
Mitocôndrias , Desenvolvimento Muscular , Diferenciação Celular , Mioblastos/metabolismo , Espécies Reativas de Oxigênio/metabolismo
2.
Nat Commun ; 12(1): 749, 2021 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-33531476

RESUMO

Fusion of nascent myoblasts to pre-existing myofibres is critical for skeletal muscle growth and repair. The vast majority of molecules known to regulate myoblast fusion are necessary in this process. Here, we uncover, through high-throughput in vitro assays and in vivo studies in the chicken embryo, that TGFß (SMAD2/3-dependent) signalling acts specifically and uniquely as a molecular brake on muscle fusion. While constitutive activation of the pathway arrests fusion, its inhibition leads to a striking over-fusion phenotype. This dynamic control of TGFß signalling in the embryonic muscle relies on a receptor complementation mechanism, prompted by the merging of myoblasts with myofibres, each carrying one component of the heterodimer receptor complex. The competence of myofibres to fuse is likely restored through endocytic degradation of activated receptors. Altogether, this study shows that muscle fusion relies on TGFß signalling to regulate its pace.


Assuntos
Fibras Musculares Esqueléticas/efeitos dos fármacos , Mioblastos/citologia , Mioblastos/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Comunicação Celular/fisiologia , Diferenciação Celular/fisiologia , Fusão Celular , Galinhas , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Fibras Musculares Esqueléticas/metabolismo , Miofibrilas/metabolismo , Transdução de Sinais/fisiologia
3.
Nature ; 591(7849): 281-287, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33568815

RESUMO

Skeletal muscle regenerates through the activation of resident stem cells. Termed satellite cells, these normally quiescent cells are induced to proliferate by wound-derived signals1. Identifying the source and nature of these cues has been hampered by an inability to visualize the complex cell interactions that occur within the wound. Here we use muscle injury models in zebrafish to systematically capture the interactions between satellite cells and the innate immune system after injury, in real time, throughout the repair process. This analysis revealed that a specific subset of macrophages 'dwell' within the injury, establishing a transient but obligate niche for stem cell proliferation. Single-cell profiling identified proliferative signals that are secreted by dwelling macrophages, which include the cytokine nicotinamide phosphoribosyltransferase (Nampt, which is also known as visfatin or PBEF in humans). Nampt secretion from the macrophage niche is required for muscle regeneration, acting through the C-C motif chemokine receptor type 5 (Ccr5), which is expressed on muscle stem cells. This analysis shows that in addition to their ability to modulate the immune response, specific macrophage populations also provide a transient stem-cell-activating niche, directly supplying proliferation-inducing cues that govern the repair process that is mediated by muscle stem cells. This study demonstrates that macrophage-derived niche signals for muscle stem cells, such as NAMPT, can be applied as new therapeutic modalities for skeletal muscle injury and disease.


Assuntos
Macrófagos/metabolismo , Músculo Esquelético/citologia , Músculo Esquelético/lesões , Mioblastos/citologia , Nicotinamida Fosforribosiltransferase/metabolismo , Nicho de Células-Tronco , Peixe-Zebra/metabolismo , Animais , Proliferação de Células , Modelos Animais de Doenças , Humanos , Macrófagos/citologia , Masculino , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Mioblastos/metabolismo , Nicotinamida Fosforribosiltransferase/genética , Fator de Transcrição PAX7/metabolismo , RNA-Seq , Receptores CCR5/genética , Receptores CCR5/metabolismo , Regeneração/fisiologia , Análise de Célula Única , Peixe-Zebra/imunologia
4.
Nat Commun ; 12(1): 495, 2021 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-33479215

RESUMO

Myomerger is a muscle-specific membrane protein involved in formation of multinucleated muscle cells by mediating the transition from the early hemifusion stage to complete fusion. Here, we considered the physical mechanism of the Myomerger action based on the hypothesis that Myomerger shifts the spontaneous curvature of the outer membrane leaflets to more positive values. We predicted, theoretically, that Myomerger generates the outer leaflet elastic stresses, which propagate into the hemifusion diaphragm and accelerate the fusion pore formation. We showed that Myomerger ectodomain indeed generates positive spontaneous curvature of lipid monolayers. We substantiated the mechanism by experiments on myoblast fusion and influenza hemagglutinin-mediated cell fusion. In both processes, the effects of Myomerger ectodomain were strikingly similar to those of lysophosphatidylcholine known to generate a positive spontaneous curvature of lipid monolayers. The control of post-hemifusion stages by shifting the spontaneous curvature of proximal membrane monolayers may be utilized in diverse fusion processes.


Assuntos
Membrana Celular/metabolismo , Fusão de Membrana , Proteínas de Membrana/metabolismo , Mioblastos/metabolismo , Algoritmos , Animais , Fusão Celular , Linhagem Celular , Bicamadas Lipídicas/metabolismo , Lipídeos de Membrana/metabolismo , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Modelos Teóricos , Mioblastos/citologia , Células NIH 3T3
5.
Int J Mol Sci ; 22(2)2021 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-33467209

RESUMO

Skeletal muscle is the most abundant tissue and constitutes about 40% of total body mass. Herein, we report that crude water extract (CWE) of G. uralensis enhanced myoblast proliferation and differentiation. Pretreatment of mice with the CWE of G. uralensis prior to cardiotoxin-induced muscle injury was found to enhance muscle regeneration by inducing myogenic gene expression and downregulating myostatin expression. Furthermore, this extract reduced nitrotyrosine protein levels and atrophy-related gene expression. Of the five different fractions of the CWE of G. uralensis obtained, the ethyl acetate (EtOAc) fraction more significantly enhanced myoblast proliferation and differentiation than the other fractions. Ten bioactive compounds were isolated from the EtOAc fraction and characterized by GC-MS and NMR. Of these compounds (4-hydroxybenzoic acid, liquiritigenin, (R)-(-)-vestitol, isoliquiritigenin, medicarpin, tetrahydroxymethoxychalcone, licochalcone B, liquiritin, liquiritinapioside, and ononin), liquiritigenin, tetrahydroxymethoxychalcone, and licochalcone B were found to enhance myoblast proliferation and differentiation, and myofiber diameters in injured muscles were wider with the liquiritigenin than the non-treated one. Computational analysis showed these compounds are non-toxic and possess good drug-likeness properties. These findings suggest that G. uralensis-extracted components might be useful therapeutic agents for the management of muscle-associated diseases.


Assuntos
Glycyrrhiza uralensis/química , Atrofia Muscular/tratamento farmacológico , Extratos Vegetais/química , Animais , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Chalconas/química , Chalconas/farmacologia , Chalconas/uso terapêutico , Flavanonas/química , Flavanonas/farmacologia , Flavanonas/uso terapêutico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mioblastos/citologia , Mioblastos/efeitos dos fármacos , Mioblastos/metabolismo , Miostatina/genética , Miostatina/metabolismo , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Tirosina/análogos & derivados , Tirosina/metabolismo
6.
Nat Commun ; 12(1): 470, 2021 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-33473109

RESUMO

Healthy aging can be promoted by enhanced metabolic fitness and physical capacity. Mitochondria are chief metabolic organelles with strong implications in aging that also coordinate broad physiological functions, in part, using peptides that are encoded within their independent genome. However, mitochondrial-encoded factors that actively regulate aging are unknown. Here, we report that mitochondrial-encoded MOTS-c can significantly enhance physical performance in young (2 mo.), middle-age (12 mo.), and old (22 mo.) mice. MOTS-c can regulate (i) nuclear genes, including those related to metabolism and proteostasis, (ii) skeletal muscle metabolism, and (iii) myoblast adaptation to metabolic stress. We provide evidence that late-life (23.5 mo.) initiated intermittent MOTS-c treatment (3x/week) can increase physical capacity and healthspan in mice. In humans, exercise induces endogenous MOTS-c expression in skeletal muscle and in circulation. Our data indicate that aging is regulated by genes encoded in both of our co-evolved mitochondrial and nuclear genomes.


Assuntos
Envelhecimento/genética , Homeostase/fisiologia , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Músculo Esquelético/metabolismo , Adulto , Animais , Linhagem Celular , Núcleo Celular , Regulação da Expressão Gênica , Humanos , Masculino , Metabolômica , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias Musculares/metabolismo , Mioblastos/metabolismo , Estresse Fisiológico , Adulto Jovem
7.
Nat Chem Biol ; 17(3): 307-316, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33510451

RESUMO

Glucocorticoids display remarkable anti-inflammatory activity, but their use is limited by on-target adverse effects including insulin resistance and skeletal muscle atrophy. We used a chemical systems biology approach, ligand class analysis, to examine ligands designed to modulate glucocorticoid receptor activity through distinct structural mechanisms. These ligands displayed diverse activity profiles, providing the variance required to identify target genes and coregulator interactions that were highly predictive of their effects on myocyte glucose disposal and protein balance. Their anti-inflammatory effects were linked to glucose disposal but not muscle atrophy. This approach also predicted selective modulation in vivo, identifying compounds that were muscle-sparing or anabolic for protein balance and mitochondrial potential. Ligand class analysis defined the mechanistic links between the ligand-receptor interface and ligand-driven physiological outcomes, a general approach that can be applied to any ligand-regulated allosteric signaling system.


Assuntos
Anti-Inflamatórios/farmacologia , Transportador de Glucose Tipo 4/genética , Atrofia Muscular/tratamento farmacológico , Receptores de Glucocorticoides/química , Transdução de Sinais/efeitos dos fármacos , Células A549 , Regulação Alostérica , Animais , Anti-Inflamatórios/síntese química , Linhagem Celular Transformada , Regulação da Expressão Gênica , Glucose/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Humanos , Lipopolissacarídeos/administração & dosagem , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Atrofia Muscular/induzido quimicamente , Atrofia Muscular/genética , Atrofia Muscular/metabolismo , Mioblastos/efeitos dos fármacos , Mioblastos/metabolismo , Ratos , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Relação Estrutura-Atividade
8.
J Agric Food Chem ; 69(1): 592-601, 2021 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-33346638

RESUMO

Beef is considered to be a good quality meat product because it contains linoleic acid and specific proteins, which can bring significant benefits to health. Circular RNAs (circRNAs) have been reported to regulate skeletal myogenesis. RNA-seq was used to investigate the circRNA molecular regulatory mechanisms with respect to differences in muscle quality between buffalo and cattle. A total of 10,449 circRNA candidates were detected, and 1128 of these were found to be differentially expressed between cattle and buffalo muscle tissue libraries. Differentially expressed 23 circRNAs were verified by qPCR. CircEch1, one of the most up-regulated circRNAs during muscle development, was subsequently characterized. CCK-8 (65.05 ± 2.33%, P < 0.0001), EdU (72.99 ± 0.04%, P < 0.001), and Western blotting assays showed that overexpression of circEch1 inhibited the proliferation of bovine myoblasts but promoted differentiation. In vivo studies suggested that circEch1 stimulates skeletal muscle regeneration. These results demonstrate that the novel regulator circEch1 induces myoblast differentiation and skeletal muscle regeneration. They also provide new insights into the mechanisms of circRNA regulation of beef quality.


Assuntos
Bovinos/genética , Desenvolvimento Muscular , Músculo Esquelético/citologia , RNA Circular/metabolismo , Animais , Búfalos , Bovinos/crescimento & desenvolvimento , Bovinos/metabolismo , Proliferação de Células , Músculo Esquelético/crescimento & desenvolvimento , Músculo Esquelético/metabolismo , Mioblastos/citologia , Mioblastos/metabolismo , RNA Circular/genética
9.
PLoS One ; 15(12): e0244791, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33382817

RESUMO

Ageing and chronic diseases lead to muscle loss and impair the regeneration of skeletal muscle. Thus, it's crucial to seek for effective intervention to improve the muscle regeneration. Tid1, a mitochondrial co-chaperone, is important to maintain mitochondrial membrane potential and ATP synthesis. Previously, we demonstrated that mice with skeletal muscular specific Tid1 deficiency displayed muscular dystrophy and postnatal lethality. Tid1 can interact with STAT3 protein, which also plays an important role during myogenesis. In this study, we used GMI, immunomodulatory protein of Ganoderma microsporum, as an inducer in C2C12 myoblast differentiation. We observed that GMI pretreatment promoted the myogenic differentiation of C2C12 myoblasts. We also showed that the upregulation of mitochondria protein Tid1 with the GMI pre-treatment promoted myogenic differentiation ability of C2C12 cells. Strikingly, we observed the concomitant elevation of STAT3 acetylation (Ac-STAT3) during C2C12 myogenesis. Our study suggests that GMI promotes the myogenic differentiation through the activation of Tid1 and Ac-STAT3.


Assuntos
Proteínas Fúngicas/metabolismo , Ganoderma/metabolismo , Proteínas de Choque Térmico HSP40/metabolismo , Desenvolvimento Muscular/fisiologia , Mioblastos/citologia , Fator de Transcrição STAT3/metabolismo , Acetilação , Animais , Diferenciação Celular/fisiologia , Linhagem Celular , Proliferação de Células/fisiologia , Proteínas Fúngicas/genética , Proteínas de Choque Térmico HSP40/genética , Camundongos , Camundongos Knockout , Mioblastos/metabolismo , Regulação para Cima
10.
Cell Prolif ; 53(11): e12914, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32990380

RESUMO

OBJECTIVES: Mouse embryonic stem cell (mESC) culture contains various heterogeneous populations, which serve as excellent models to study gene regulation in early embryo development. The heterogeneity is typically defined by transcriptional activities, for example, the expression of Nanog or Rex1 mRNA. Our objectives were to identify mESC heterogeneity that are caused by mechanisms other than transcriptional control. MATERIALS AND METHODS: Klf3 mRNA and protein were analysed by RT-qPCR, Western blotting or immunofluorescence in mESCs, C2C12 cells, early mouse embryos and various mouse tissues. An ESC reporter line expressing KLF3-GFP fusion protein was made to study heterogeneity of KLF3 protein expression in ESCs. GFP-positive mESCs were sorted for further analysis including RT-qPCR and RNA-seq. RESULTS: In the majority of mESCs, KLF3 protein is actively degraded due to its proline-rich sequence and highly disordered structure. Interestingly, KLF3 protein is stabilized in a small subset of mESCs. Transcriptome analysis indicates that KLF3-positive mESCs upregulate genes that are initially activated in 8-cell embryos. Consistently, KLF3 protein but not mRNA is dramatically increased in 8-cell embryos. Forced expression of KLF3 protein in mESCs promotes the expression of 8-cell-embryo activated genes. CONCLUSIONS: Our study identifies previously unrecognized heterogeneity due to KLF3 protein expression in mESCs.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Fatores de Transcrição Kruppel-Like/genética , Células-Tronco Embrionárias Murinas/citologia , Animais , Diferenciação Celular , Linhagem Celular , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Mioblastos/citologia , Mioblastos/metabolismo , RNA Mensageiro/genética , Ativação Transcricional , Transcriptoma
11.
PLoS One ; 15(9): e0239047, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32941492

RESUMO

Muscle aging is accompanied by blunted muscle regeneration in response to injury and disuse. Oxidative stress likely underlies this diminished response, but muscle redox sensors that act in regeneration have not yet been characterized. Calmodulin contains multiple redox sensitive methionines whose oxidation alters the regulation of numerous cellular targets. We have used the CRISPR-Cas9 system to introduce a single amino acid substitution M109Q that mimics oxidation of methionine to methionine sulfoxide in one or both alleles of the CALM1 gene, one of three genes encoding the muscle regulatory protein calmodulin, in C2C12 mouse myoblasts. When signaled to undergo myogenesis, mutated myoblasts failed to differentiate into myotubes. Although early myogenic regulatory factors were present, cells with the CALM1 M109Q mutation in one or both alleles were unable to withdraw from the cell cycle and failed to express late myogenic factors. We have shown that a single oxidative modification to a redox-sensitive muscle regulatory protein can halt myogenesis, suggesting a molecular target for mitigating the impact of oxidative stress in age-related muscle degeneration.


Assuntos
Calmodulina/metabolismo , Desenvolvimento Muscular/fisiologia , Animais , Calmodulina/genética , Calmodulina/fisiologia , Diferenciação Celular/fisiologia , Camundongos , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Atrofia Muscular/metabolismo , Mioblastos/metabolismo , Oxirredução , Estresse Oxidativo/fisiologia
12.
PLoS Comput Biol ; 16(9): e1008205, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32903255

RESUMO

Single-cell RNA sequencing (scRNA-seq) can map cell types, states and transitions during dynamic biological processes such as tissue development and regeneration. Many trajectory inference methods have been developed to order cells by their progression through a dynamic process. However, when time series data is available, most of these methods do not consider the available time information when ordering cells and are instead designed to work only on a single scRNA-seq data snapshot. We present Tempora, a novel cell trajectory inference method that orders cells using time information from time-series scRNA-seq data. In performance comparison tests, Tempora inferred known developmental lineages from three diverse tissue development time series data sets, beating state of the art methods in accuracy and speed. Tempora works at the level of cell clusters (types) and uses biological pathway information to help identify cell type relationships. This approach increases gene expression signal from single cells, processing speed, and interpretability of the inferred trajectory. Our results demonstrate the utility of a combination of time and pathway information to supervise trajectory inference for scRNA-seq based analysis.


Assuntos
Biologia Computacional/métodos , Perfilação da Expressão Gênica/métodos , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Software , Algoritmos , Animais , Células Cultivadas , Humanos , Camundongos , Mioblastos/metabolismo , RNA/genética , RNA/metabolismo , Reprodutibilidade dos Testes
13.
PLoS Negl Trop Dis ; 14(8): e0008282, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32817655

RESUMO

Muscle cells are potential targets of many arboviruses, such as Ross River, Dengue, Sindbis, and chikungunya viruses, that may be involved in the physiopathological course of the infection. During the recent outbreak of Zika virus (ZIKV), myalgia was one of the most frequently reported symptoms. We investigated the susceptibility of human muscle cells to ZIKV infection. Using an in vitro model of human primary myoblasts that can be differentiated into myotubes, we found that myoblasts can be productively infected by ZIKV. In contrast, myotubes were shown to be resistant to ZIKV infection, suggesting a differentiation-dependent susceptibility. Infection was accompanied by a caspase-independent cytopathic effect, associated with paraptosis-like cytoplasmic vacuolization. Proteomic profiling was performed 24h and 48h post-infection in cells infected with two different isolates. Proteome changes indicate that ZIKV infection induces an upregulation of proteins involved in the activation of the Interferon type I pathway, and a downregulation of protein synthesis. This work constitutes the first observation of primary human muscle cells susceptibility to ZIKV infection, and differentiation-dependent restriction of infection from myoblasts to myotubes. Since myoblasts constitute the reservoir of stem cells involved in reparation/regeneration in muscle tissue, the infection of muscle cells and the viral-induced alterations observed here could have consequences in ZIKV infection pathogenesis.


Assuntos
Diferenciação Celular , Células Musculares/metabolismo , Células Musculares/virologia , Proteômica , Infecção por Zika virus , Morte Celular , Linhagem Celular , Efeito Citopatogênico Viral , Suscetibilidade a Doenças , Interações Hospedeiro-Patógeno , Humanos , Interferon Tipo I/metabolismo , Células Musculares/patologia , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/virologia , Mioblastos/metabolismo , Mioblastos/virologia , Proteínas/metabolismo , Células-Tronco , Replicação Viral , Zika virus/patogenicidade , Infecção por Zika virus/patologia , Infecção por Zika virus/virologia
14.
Life Sci ; 258: 118243, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32791154

RESUMO

AIMS: Although autophagy impairment is a well-established cause of muscle atrophy and P300 has recently been identified as an important regulator of autophagy, the effects of P300 on autophagy and muscle atrophy in type 2 diabetes (T2D) remain unexplored. We aimed at characterizing the role of P300 in diabetic muscle and its underlying mechanism. MAIN METHODS: Protein levels of phosphorylated P300, total P300, acetylated histone H3, LC3, p62 and myosin heavy chain, and mRNA levels of Atrogin-1 and MuRF1 were analyzed in palmitic acid (PA)-treated myotubes and db/db mice. Autophagic flux was assessed using transmission electron microscopy, immunofluorescence and mRFP-GFP-LC3 lentivirus transfection in cells. Muscle weight, blood glucose and grip strength were measured in mice. Hematoxylin and eosin (H&E) staining was performed to determine changes in muscle fiber size. To investigate the effects of P300 on autophagy and myofiber remodeling, a P300 specific inhibitor, c646, was utilized. 3-Methyladenine (3-MA) was utilized to inhibit autophagosomes formation, and chloroquine (CQ) was used to block autophagic flux. KEY FINDINGS: Phosphorylation of P300 in response to PA enhanced its activity and subsequently suppressed autophagic flux, leading to atrophy-related morphological and molecular changes in myotubes. Inhibition of P300 reestablished autophagic flux and ameliorated PA-induced myotubes atrophy. However, this effect was largely abolished by co-treatment with the autophagy inhibitor CQ. In vivo results demonstrated that inhibition of P300 partially rescued muscle wasting in db/db mice, accompanied with autophagy reactivation. SIGNIFICANCE: The findings revealed that T2D-induced overactivation of P300 contributes to muscle atrophy by blocking autophagic flux.


Assuntos
Autofagia/fisiologia , Diabetes Mellitus Tipo 2/metabolismo , Proteína p300 Associada a E1A/metabolismo , Atrofia Muscular/metabolismo , Animais , Linhagem Celular , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patologia , Proteína p300 Associada a E1A/genética , Força da Mão/fisiologia , Masculino , Camundongos , Camundongos Transgênicos , Atrofia Muscular/genética , Atrofia Muscular/patologia , Mioblastos/metabolismo , Mioblastos/patologia
15.
Proc Natl Acad Sci U S A ; 117(32): 19254-19265, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32719146

RESUMO

The appropriate arrangement of myonuclei within skeletal muscle myofibers is of critical importance for normal muscle function, and improper myonuclear localization has been linked to a variety of skeletal muscle diseases, such as centronuclear myopathy and muscular dystrophies. However, the molecules that govern myonuclear positioning remain elusive. Here, we report that skeletal muscle-specific CIP (sk-CIP) is a regulator of nuclear positioning. Genetic deletion of sk-CIP in mice results in misalignment of myonuclei along the myofibers and at specialized structures such as neuromuscular junctions (NMJs) and myotendinous junctions (MTJs) in vivo, impairing myonuclear positioning after muscle regeneration, leading to severe muscle dystrophy in mdx mice, a mouse model of Duchenne muscular dystrophy. sk-CIP is localized to the centrosome in myoblasts and relocates to the outer nuclear envelope in myotubes upon differentiation. Mechanistically, we found that sk-CIP interacts with the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex and the centriole Microtubule Organizing Center (MTOC) proteins to coordinately modulate myonuclear positioning and alignment. These findings indicate that sk-CIP may function as a muscle-specific anchoring protein to regulate nuclear position in multinucleated muscle cells.


Assuntos
Proteínas de Transporte/metabolismo , Núcleo Celular/metabolismo , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , Miopatias Congênitas Estruturais/fisiopatologia , Proteínas Nucleares/metabolismo , Animais , Proteínas de Transporte/genética , Núcleo Celular/genética , Proteínas Correpressoras , Humanos , Camundongos , Camundongos Endogâmicos mdx , Camundongos Knockout , Músculo Esquelético/fisiopatologia , Miopatias Congênitas Estruturais/genética , Miopatias Congênitas Estruturais/metabolismo , Proteínas Nucleares/genética , Especificidade de Órgãos
16.
Am J Physiol Cell Physiol ; 319(2): C441-C454, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32639872

RESUMO

Skeletal muscle atrophy is caused by a decrease in muscle size and strength and results from a range of physiological conditions, including denervation, immobilization, corticosteroid exposure and aging. Newly named dual-specificity phosphatase 29 (Dusp29) has been identified as a novel neurogenic atrophy-induced gene in skeletal muscle. Quantitative PCR analysis revealed that Dusp29 expression is significantly higher in differentiated myotubes compared with proliferating myoblasts. To determine how Dusp29 is transcriptionally regulated in skeletal muscle, fragments of the promoter region of Dusp29 were cloned, fused to a reporter gene, and found to be highly inducible in response to ectopic expression of the myogenic regulatory factors (MRF), MyoD and myogenin. Furthermore, site-directed mutagenesis of conserved E-box elements within the proximal promoter of Dusp29 rendered a Dusp29 reporter gene unresponsive to MRF overexpression. Dusp29, an atypical Dusp also known as Dupd1/Dusp27, was found to attenuate the ERK1/2 branch of the MAP kinase signaling pathway in muscle cells and inhibit muscle cell differentiation when ectopically expressed in proliferating myoblasts. Interestingly, Dusp29 was also found to destabilize AMPK protein while simultaneously enriching the phosphorylated pool of AMPK in muscle cells. Additionally, Dusp29 overexpression resulted in a significant increase in the glucocorticoid receptor (GR) protein and elevation in GR phosphorylation. Finally, Dusp29 was found to significantly impair the ability of the glucocorticoid receptor to function as a transcriptional activator in muscle cells treated with dexamethasone. Identifying and characterizing the function of Dusp29 in muscle provides novel insights into the molecular and cellular mechanisms for skeletal muscle atrophy.


Assuntos
Fosfatases de Especificidade Dupla/genética , Atrofia Muscular/genética , Proteína MyoD/genética , Miogenina/genética , Animais , Diferenciação Celular/genética , Linhagem Celular , Proliferação de Células/genética , Regulação da Expressão Gênica/genética , Humanos , Sistema de Sinalização das MAP Quinases/genética , Células Musculares/metabolismo , Células Musculares/patologia , Atrofia Muscular/patologia , Mioblastos/metabolismo , Fosforilação/genética , Receptores de Glucocorticoides/genética , Transdução de Sinais , Ativação Transcricional/genética
17.
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
18.
Am J Physiol Cell Physiol ; 319(2): C432-C440, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32608991

RESUMO

microRNAs (miRNAs) are important regulators of cellular homeostasis and exert their effect by directly controlling protein expression. We have previously reported an age-dependent negative association between microRNA-99b (miR-99b-5p) expression and muscle protein synthesis in human muscle in vivo. Here we investigated the role of miR-99b-5p as a potential negative regulator of protein synthesis via inhibition of mammalian target for rapamycin (MTOR) signaling in human primary myocytes. Overexpressing miR-99b-5p in human primary myotubes from young and old subjects significantly decreased protein synthesis with no effect of donor age. A binding interaction between miR-99b-5p and its putative binding site within the MTOR 3'-untranslated region (UTR) was confirmed in C2C12 myoblasts. The observed decline in protein synthesis was, however, not associated with a suppression of the MTOR protein but of its regulatory associated protein of mTOR complex 1 (RPTOR). These results demonstrate that modulating the expression levels of a miRNA can regulate protein synthesis in human muscle cells and provide a potential mechanism for muscle wasting in vivo.


Assuntos
MicroRNAs/genética , Fibras Musculares Esqueléticas/metabolismo , Biossíntese de Proteínas/genética , Serina-Treonina Quinases TOR/genética , Regiões 3' não Traduzidas/genética , Animais , Proliferação de Células/genética , Regulação da Expressão Gênica/genética , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Camundongos , Mioblastos/metabolismo , Transdução de Sinais/genética
19.
Proc Natl Acad Sci U S A ; 117(28): 16509-16515, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32601200

RESUMO

Facioscapulohumeral muscular dystrophy (FSHD), characterized by progressive muscle weakness and deterioration, is genetically linked to aberrant expression of DUX4 in muscle. DUX4, in its full-length form, is cytotoxic in nongermline tissues. Here, we designed locked nucleic acid (LNA) gapmer antisense oligonucleotides (AOs) to knock down DUX4 in immortalized FSHD myoblasts and the FLExDUX4 FSHD mouse model. Using a screening method capable of reliably evaluating the knockdown efficiency of LNA gapmers against endogenous DUX4 messenger RNA in vitro, we demonstrate that several designed LNA gapmers selectively and effectively reduced DUX4 expression with nearly complete knockdown. We also found potential functional benefits of AOs on muscle fusion and structure in vitro. Finally, we show that one of the LNA gapmers was taken up and induced effective silencing of DUX4 upon local treatment in vivo. The LNA gapmers developed here will help facilitate the development of FSHD therapies.


Assuntos
Terapia Genética , Proteínas de Homeodomínio/genética , Distrofia Muscular Facioescapuloumeral/terapia , Oligonucleotídeos Antissenso/administração & dosagem , Animais , Modelos Animais de Doenças , Técnicas de Silenciamento de Genes , Proteínas de Homeodomínio/metabolismo , Humanos , Camundongos , Distrofia Muscular Facioescapuloumeral/genética , Distrofia Muscular Facioescapuloumeral/metabolismo , Mioblastos/metabolismo , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/metabolismo
20.
Proc Natl Acad Sci U S A ; 117(28): 16383-16390, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32601238

RESUMO

Calcium uptake by the mitochondrial calcium uniporter coordinates cytosolic signaling events with mitochondrial bioenergetics. During the past decade all protein components of the mitochondrial calcium uniporter have been identified, including MCU, the pore-forming subunit. However, the specific lipid requirements, if any, for the function and formation of this channel complex are currently not known. Here we utilize yeast, which lacks the mitochondrial calcium uniporter, as a model system to address this problem. We use heterologous expression to functionally reconstitute human uniporter machinery both in wild-type yeast as well as in mutants defective in the biosynthesis of phosphatidylethanolamine, phosphatidylcholine, or cardiolipin (CL). We uncover a specific requirement of CL for in vivo reconstituted MCU stability and activity. The CL requirement of MCU is evolutionarily conserved with loss of CL triggering rapid turnover of MCU homologs and impaired calcium transport. Furthermore, we observe reduced abundance and activity of endogenous MCU in mammalian cellular models of Barth syndrome, which is characterized by a partial loss of CL. MCU abundance is also decreased in the cardiac tissue of Barth syndrome patients. Our work raises the hypothesis that impaired mitochondrial calcium transport contributes to the pathogenesis of Barth syndrome, and more generally, showcases the utility of yeast phospholipid mutants in dissecting the phospholipid requirements of ion channel complexes.


Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Mitocôndrias/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Animais , Síndrome de Barth/genética , Síndrome de Barth/metabolismo , Transporte Biológico , Canais de Cálcio/química , Canais de Cálcio/genética , Cardiolipinas/genética , Cardiolipinas/metabolismo , Humanos , Camundongos , Mitocôndrias/química , Mitocôndrias/genética , Mioblastos/metabolismo , Fosfolipídeos , Estabilidade Proteica , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
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