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1.
Curr Biol ; 34(9): R343-R345, 2024 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-38714160

RESUMO

Repeated rounds of fusion between apposing myoblasts allow muscles to become multinucleated. New research finds that myoblasts undergoing fusion in the Drosophila embryo respond to hormone signaling from a nearby tissue, resulting in the activation of a myoblast-specific gene necessary for the fusion process.


Assuntos
Fusão Celular , Mioblastos , Animais , Mioblastos/metabolismo , Mioblastos/fisiologia , Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Transdução de Sinais , Comunicação Celular
2.
Int J Mol Sci ; 25(9)2024 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-38732031

RESUMO

Skeletal muscle myogenesis hinges on gene regulation, meticulously orchestrated by molecular mechanisms. While the roles of transcription factors and non-coding RNAs in myogenesis are widely known, the contribution of RNA-binding proteins (RBPs) has remained unclear until now. Therefore, to investigate the functions of post-transcriptional regulators in myogenesis and uncover new functional RBPs regulating myogenesis, we employed CRISPR high-throughput RBP-KO (RBP-wide knockout) library screening. Through this approach, we successfully identified Eef1a1 as a novel regulatory factor in myogenesis. Using CRISPR knockout (CRISPRko) and CRISPR interference (CRISPRi) technologies, we successfully established cellular models for both CRISPRko and CRISPRi. Our findings demonstrated that Eef1a1 plays a crucial role in promoting proliferation in C2C12 myoblasts. Through siRNA inhibition and overexpression methods, we further elucidated the involvement of Eef1a1 in promoting proliferation and suppressing differentiation processes. RIP (RNA immunoprecipitation), miRNA pull-down, and Dual-luciferase reporter assays confirmed that miR-133a-3p targets Eef1a1. Co-transfection experiments indicated that miR-133a-3p can rescue the effect of Eef1a1 on C2C12 myoblasts. In summary, our study utilized CRISPR library high-throughput screening to unveil a novel RBP, Eef1a1, involved in regulating myogenesis. Eef1a1 promotes the proliferation of myoblasts while inhibiting the differentiation process. Additionally, it acts as an antagonist to miR-133a-3p, thus modulating the process of myogenesis.


Assuntos
Diferenciação Celular , Proliferação de Células , Desenvolvimento Muscular , Mioblastos , Fator 1 de Elongação de Peptídeos , Desenvolvimento Muscular/genética , Fator 1 de Elongação de Peptídeos/genética , Fator 1 de Elongação de Peptídeos/metabolismo , Animais , Camundongos , Proliferação de Células/genética , Diferenciação Celular/genética , Mioblastos/metabolismo , Mioblastos/citologia , Sistemas CRISPR-Cas , Linhagem Celular , MicroRNAs/genética , MicroRNAs/metabolismo , Humanos , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética
3.
Commun Biol ; 7(1): 518, 2024 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-38698103

RESUMO

Myoblast proliferation and differentiation are essential for skeletal muscle development. In this study, we generated the expression profiles of mRNAs, long noncoding RNAs (lncRNAs), and microRNAs (miRNAs) in different developmental stages of chicken primary myoblasts (CPMs) using RNA sequencing (RNA-seq) technology. The dual luciferase reporter system was performed using chicken embryonic fibroblast cells (DF-1), and functional studies quantitative real-time polymerase chain reaction (qPCR), cell counting kit-8 (CCK-8), 5-Ethynyl-2'-deoxyuridine (EdU), flow cytometry cycle, RNA fluorescence in situ hybridization (RNA-FISH), immunofluorescence, and western blotting assay. Our research demonstrated that miR-301a-5p had a targeted binding ability to lncMDP1 and ChaC glutathione-specific gamma-glutamylcyclotransferase 1 (CHAC1). The results revealed that lncMDP1 regulated the proliferation and differentiation of myoblasts via regulating the miR-301a-5p/CHAC1 axis, and CHAC1 promotes muscle regeneration. This study fulfilled the molecular regulatory network of skeletal muscle development and providing an important theoretical reference for the future improvement of chicken meat performance and meat quality.


Assuntos
Galinhas , Perfilação da Expressão Gênica , MicroRNAs , Desenvolvimento Muscular , RNA Longo não Codificante , Animais , MicroRNAs/genética , MicroRNAs/metabolismo , Desenvolvimento Muscular/genética , Galinhas/genética , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Diferenciação Celular/genética , Proliferação de Células , Mioblastos/metabolismo , Mioblastos/citologia , Embrião de Galinha
4.
PLoS One ; 19(5): e0301690, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38701072

RESUMO

Myogenesis is regulated mainly by transcription factors known as Myogenic Regulatory Factors (MRFs), and the transcription is affected by epigenetic modifications. However, the epigenetic regulation of myogenesis is poorly understood. Here, we focused on the epigenomic modification enzyme, PHF2, which demethylates histone 3 lysine 9 dimethyl (H3K9me2) during myogenesis. Phf2 mRNA was expressed during myogenesis, and PHF2 was localized in the nuclei of myoblasts and myotubes. We generated Phf2 knockout C2C12 myoblasts using the CRISPR/Cas9 system and analyzed global transcriptional changes via RNA-sequencing. Phf2 knockout (KO) cells 2 d post differentiation were subjected to RNA sequencing. Gene ontology (GO) analysis revealed that Phf2 KO impaired the expression of the genes related to skeletal muscle fiber formation and muscle cell development. The expression levels of sarcomeric genes such as Myhs and Mybpc2 were severely reduced in Phf2 KO cells at 7 d post differentiation, and H3K9me2 modification of Mybpc2, Mef2c and Myh7 was increased in Phf2 KO cells at 4 d post differentiation. These findings suggest that PHF2 regulates sarcomeric gene expression via epigenetic modification.


Assuntos
Desenvolvimento Muscular , Sarcômeros , Animais , Camundongos , Diferenciação Celular/genética , Linhagem Celular , Epigênese Genética , Técnicas de Inativação de Genes , Histona Desmetilases/metabolismo , Histona Desmetilases/genética , Histonas/metabolismo , Fatores de Transcrição MEF2/genética , Fatores de Transcrição MEF2/metabolismo , Desenvolvimento Muscular/genética , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/citologia , Mioblastos/metabolismo , Mioblastos/citologia , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Sarcômeros/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Transcrição Gênica
5.
Sci Adv ; 10(18): eadl1922, 2024 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-38691604

RESUMO

The most common form of facioscapulohumeral dystrophy (FSHD1) is caused by a partial loss of the D4Z4 macrosatellite repeat array in the subtelomeric region of chromosome 4. Patients with FSHD1 typically carry 1 to 10 D4Z4 repeats, whereas nonaffected individuals have 11 to 150 repeats. The ~150-kilobyte subtelomeric region of the chromosome 10q exhibits a ~99% sequence identity to the 4q, including the D4Z4 array. Nevertheless, contractions of the chr10 array do not cause FSHD or any known disease, as in most people D4Z4 array on chr10 is flanked by the nonfunctional polyadenylation signal, not permitting the DUX4 expression. Here, we attempted to correct the FSHD genotype by a CRISPR-Cas9-induced exchange of the chr4 and chr10 subtelomeric regions. We demonstrated that the induced t(4;10) translocation can generate recombinant genotypes translated into improved FSHD phenotype. FSHD myoblasts with the t(4;10) exhibited reduced expression of the DUX4 targets, restored PAX7 target expression, reduced sensitivity to oxidative stress, and improved differentiation capacity.


Assuntos
Cromossomos Humanos Par 10 , Cromossomos Humanos Par 4 , Genótipo , Proteínas de Homeodomínio , Distrofia Muscular Facioescapuloumeral , Fenótipo , Telômero , Humanos , Cromossomos Humanos Par 10/genética , Cromossomos Humanos Par 4/genética , Sistemas CRISPR-Cas , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Distrofia Muscular Facioescapuloumeral/genética , Mioblastos/metabolismo , Fator de Transcrição PAX7/genética , Fator de Transcrição PAX7/metabolismo , Telômero/genética , Telômero/metabolismo , Translocação Genética
6.
Sci Adv ; 10(18): eadj8042, 2024 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-38691608

RESUMO

Overactivation of the transforming growth factor-ß (TGFß) signaling in Duchenne muscular dystrophy (DMD) is a major hallmark of disease progression, leading to fibrosis and muscle dysfunction. Here, we investigated the role of SETDB1 (SET domain, bifurcated 1), a histone lysine methyltransferase involved in muscle differentiation. Our data show that, following TGFß induction, SETDB1 accumulates in the nuclei of healthy myotubes while being already present in the nuclei of DMD myotubes where TGFß signaling is constitutively activated. Transcriptomics revealed that depletion of SETDB1 in DMD myotubes leads to down-regulation of TGFß target genes coding for secreted factors involved in extracellular matrix remodeling and inflammation. Consequently, SETDB1 silencing in DMD myotubes abrogates the deleterious effect of their secretome on myoblast differentiation by impairing myoblast pro-fibrotic response. Our findings indicate that SETDB1 potentiates the TGFß-driven fibrotic response in DMD muscles, providing an additional axis for therapeutic intervention.


Assuntos
Histona-Lisina N-Metiltransferase , Fibras Musculares Esqueléticas , Distrofia Muscular de Duchenne , Transdução de Sinais , Fator de Crescimento Transformador beta , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patologia , Histona-Lisina N-Metiltransferase/metabolismo , Histona-Lisina N-Metiltransferase/genética , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Fator de Crescimento Transformador beta/metabolismo , Humanos , Animais , Diferenciação Celular , Camundongos , Mioblastos/metabolismo , Fibrose , Regulação da Expressão Gênica
7.
Proc Natl Acad Sci U S A ; 121(19): e2321438121, 2024 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-38687782

RESUMO

Successful CRISPR/Cas9-based gene editing in skeletal muscle is dependent on efficient propagation of Cas9 to all myonuclei in the myofiber. However, nuclear-targeted gene therapy cargos are strongly restricted to their myonuclear domain of origin. By screening nuclear localization signals and nuclear export signals, we identify "Myospreader," a combination of short peptide sequences that promotes myonuclear propagation. Appending Myospreader to Cas9 enhances protein stability and myonuclear propagation in myoblasts and myofibers. AAV-delivered Myospreader dCas9 better inhibits transcription of toxic RNA in a myotonic dystrophy mouse model. Furthermore, Myospreader Cas9 achieves higher rates of gene editing in CRISPR reporter and Duchenne muscular dystrophy mouse models. Myospreader reveals design principles relevant to all nuclear-targeted gene therapies and highlights the importance of the spatial dimension in therapeutic development.


Assuntos
Sistemas CRISPR-Cas , Núcleo Celular , Edição de Genes , Terapia Genética , Músculo Esquelético , Distrofia Muscular de Duchenne , Edição de Genes/métodos , Animais , Camundongos , Músculo Esquelético/metabolismo , Núcleo Celular/metabolismo , Terapia Genética/métodos , Distrofia Muscular de Duchenne/terapia , Distrofia Muscular de Duchenne/genética , Humanos , Sinais de Localização Nuclear/genética , Proteína 9 Associada à CRISPR/metabolismo , Proteína 9 Associada à CRISPR/genética , Modelos Animais de Doenças , Mioblastos/metabolismo
8.
G3 (Bethesda) ; 14(5)2024 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-38577978

RESUMO

Genetic variability significantly contributes to individual differences in skeletal muscle mass; however, the specific genes involved in that process remain elusive. In this study, we examined the role of positional candidates, Rps6ka6 and Pou3f4, of a chromosome X locus, implicated in muscle mass variability in CFW laboratory mice. Histology of hindlimb muscles was studied in CFW male mice carrying the muscle "increasing" allele C (n = 15) or "decreasing" allele T (n = 15) at the peak marker of the locus, rs31308852, and in the Pou3f4y/- and their wild-type male littermates. To study the role of the Rps6ka6 gene, we deleted exon 7 (Rps6ka6-ΔE7) using clustered regularly interspaced palindromic repeats-Cas9 based method in H2Kb myogenic cells creating a severely truncated RSK4 protein. We then tested whether that mutation affected myoblast proliferation, migration, and/or differentiation. The extensor digitorum longus muscle was 7% larger (P < 0.0001) due to 10% more muscle fibers (P = 0.0176) in the carriers of the "increasing" compared with the "decreasing" CFW allele. The number of fibers was reduced by 15% (P = 0.0268) in the slow-twitch soleus but not in the fast-twitch extensor digitorum longus (P = 0.2947) of Pou3f4y/- mice. The proliferation and migration did not differ between the Rps6ka6-ΔE7 and wild-type H2Kb myoblasts. However, indices of differentiation (myosin expression, P < 0.0001; size of myosin-expressing cells, P < 0.0001; and fusion index, P = 0.0013) were significantly reduced in Rps6ka6-ΔE7 cells. This study suggests that the effect of the X chromosome locus on muscle fiber numbers in the fast-twitch extensor digitorum longus is mediated by the Rps6ka6 gene, whereas the Pou3f4 gene affects fiber number in slow-twitch soleus.


Assuntos
Músculo Esquelético , Animais , Camundongos , Músculo Esquelético/metabolismo , Masculino , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Diferenciação Celular/genética , Alelos , Proliferação de Células , Movimento Celular/genética , Mioblastos/metabolismo , Loci Gênicos
9.
J Cell Physiol ; 239(5): e31226, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38591363

RESUMO

Understanding how skeletal muscle fiber proportions are regulated is essential for understanding muscle function and improving the quality of mutton. While circular RNA (circRNA) has a critical function in myofiber type transformation, the specific mechanisms are not yet fully understood. Prior evidence indicates that circular ubiquitin-specific peptidase 13 (circUSP13) can promote myoblast differentiation by acting as a ceRNA, but its potential role in myofiber switching is still unknown. Herein, we found that circUSP13 enhanced slow myosin heavy chain (MyHC-slow) and suppressed MyHC-fast expression in goat primary myoblasts (GPMs). Meanwhile, circUSP13 evidently enhanced the remodeling of the mitochondrial network while inhibiting the autophagy of GPMs. We obtained fast-dominated myofibers, via treatment with rotenone, and further demonstrated the positive role of circUSP13 in the fast-to-slow transition. Mechanistically, activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway significantly impaired the slow-to-fast shift in fully differentiated myotubes, which was restored by circUSP13 or IGF1 overexpression. In conclusion, circUSP13 promoted the fast-to-slow myofiber type transition through MAPK/ERK signaling in goat skeletal muscle. These findings provide novel insights into the role of circUSP13 in myofiber type transition and contribute to a better understanding of the genetic mechanisms underlying meat quality.


Assuntos
Cabras , Sistema de Sinalização das MAP Quinases , Cadeias Pesadas de Miosina , Animais , Sistema de Sinalização das MAP Quinases/fisiologia , Cadeias Pesadas de Miosina/metabolismo , Cadeias Pesadas de Miosina/genética , Diferenciação Celular , Fibras Musculares de Contração Lenta/metabolismo , Fibras Musculares de Contração Rápida/metabolismo , Músculo Esquelético/metabolismo , Autofagia/fisiologia , Mioblastos/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/genética , Células Cultivadas , Fibras Musculares Esqueléticas/metabolismo , Desenvolvimento Muscular/genética
10.
Exp Gerontol ; 190: 112428, 2024 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-38604253

RESUMO

BACKGROUND: Mitochondrial dysregulation in skeletal myocytes is considered a major factor in aged sarcopenia. In this study, we aimed to study the effects of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) on Sestrin2-mediated mechanistic target of rapamycin complex 1 (mTORC1) in aged skeletal muscles. METHODS: C2C12 myoblasts were stimulated by 50 µM 7ß-hydroxycholesterol (7ß-OHC) to observe the changes of DNA damage, mitochondrial membrane potential (Δψm), mitochondrial ROS and PGC-1α protein. The PGC-1α silence in the C2C12 cells was established by siRNA transfection. The levels of DNA damage, Δψm, mitochondrial ROS, Sestrin2 and p-S6K1/S6K1 proteins were observed after the PGC-1α silence in the C2C12 cells. Recombinant Sestrin2 treatment was used to observe the changes of DNA damage, Δψm, mitochondrial ROS and p-S6K1/S6K1 protein in the 7ß-OHC-treated or PGC-1α siRNA-transfected C2C12 cells. Wild-type (WT) mice and muscle-specific PGC-1α conditional knockout (MKO) mice, including young and old, were used to analyse the effects of PGC-1α on muscle function and the levels of Sestrin2 and p-S6K1 in the white gastrocnemius muscles. Recombinant Sestrin2 was administrated to analyse its effects on muscle function in the old WT mice and old MKO mice. RESULTS: 7ß-OHC treatment induced DNA damage, mitochondrial dysfunction and decrease of PGC-1α protein in the C2C12 cells. PGC-1α silence also induced DNA damage and mitochondrial dysfunction in the C2C12 cells. Additionally, PGC-1α silence or 7ß-OHC treatment decreased the levels of Sestrin2 and p-S6K1/S6K1 protein in the C2C12 cells. Recombinant Sestrin2 treatment significantly improved the DNA damage and mitochondrial dysfunction in the 7ß-OHC-treated or PGC-1α siRNA-transfected C2C12 cells. At the same age, muscle-specific PGC-1α deficiency aggravated aged sarcopenia and decreased the levels of Sestrin2 and p-S6K1 in the white gastrocnemius muscles when compared to the WT mice. Recombinant Sestrin2 treatment improved muscle function and increased p-S6K1 levels in the old two genotypes. CONCLUSION: This research demonstrates that PGC-1α participates in regulating mitochondrial function in aged sarcopenia through effects on the Sestrin2-mediated mTORC1 pathway.


Assuntos
Dano ao DNA , Alvo Mecanístico do Complexo 1 de Rapamicina , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Proteínas Quinases S6 Ribossômicas 90-kDa , Sarcopenia , Sestrinas , Animais , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Camundongos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Sarcopenia/metabolismo , Camundongos Knockout , Potencial da Membrana Mitocondrial , Espécies Reativas de Oxigênio/metabolismo , Envelhecimento/fisiologia , Envelhecimento/metabolismo , Transdução de Sinais , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Masculino , Músculo Esquelético/metabolismo , Linhagem Celular , Mitocôndrias/metabolismo , Peroxidases/metabolismo , Camundongos Endogâmicos C57BL , Mioblastos/metabolismo
11.
Proc Natl Acad Sci U S A ; 121(17): e2312330121, 2024 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-38625936

RESUMO

The apolipoprotein B messenger RNA editing enzyme, catalytic polypeptide (APOBEC) family is composed of nucleic acid editors with roles ranging from antibody diversification to RNA editing. APOBEC2, a member of this family with an evolutionarily conserved nucleic acid-binding cytidine deaminase domain, has neither an established substrate nor function. Using a cellular model of muscle differentiation where APOBEC2 is inducibly expressed, we confirmed that APOBEC2 does not have the attributed molecular functions of the APOBEC family, such as RNA editing, DNA demethylation, and DNA mutation. Instead, we found that during muscle differentiation APOBEC2 occupied a specific motif within promoter regions; its removal from those regions resulted in transcriptional changes. Mechanistically, these changes reflect the direct interaction of APOBEC2 with histone deacetylase (HDAC) transcriptional corepressor complexes. We also found that APOBEC2 could bind DNA directly, in a sequence-specific fashion, suggesting that it functions as a recruiter of HDAC to specific genes whose promoters it occupies. These genes are normally suppressed during muscle cell differentiation, and their suppression may contribute to the safeguarding of muscle cell fate. Altogether, our results reveal a unique role for APOBEC2 within the APOBEC family.


Assuntos
Cromatina , Proteínas Musculares , Desaminases APOBEC/genética , Desaminase APOBEC-1/genética , Diferenciação Celular/genética , Cromatina/genética , Citidina Desaminase/metabolismo , DNA , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/metabolismo , Mioblastos/metabolismo , RNA Mensageiro/genética , Animais , Camundongos
12.
BMC Biotechnol ; 24(1): 23, 2024 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-38671404

RESUMO

Volumetric loss is one of the challenging issues in muscle tissue structure that causes functio laesa. Tissue engineering of muscle tissue using suitable hydrogels is an alternative to restoring the physiological properties of the injured area. Here, myogenic properties of type I collagen (0.5%) and keratin (0.5%) were investigated in a mouse model of biceps femoris injury. Using FTIR, gelation time, and rheological analysis, the physicochemical properties of the collagen (Col)/Keratin scaffold were analyzed. Mouse C2C12 myoblast-laden Col/Keratin hydrogels were injected into the injury site and histological examination plus western blotting were performed to measure myogenic potential after 15 days. FTIR indicated an appropriate interaction between keratin and collagen. The blend of Col/Keratin delayed gelation time when compared to the collagen alone group. Rheological analysis revealed decreased stiffening in blended Col/Keratin hydrogel which is favorable for the extrudability of the hydrogel. Transplantation of C2C12 myoblast-laden Col/Keratin hydrogel to injured muscle tissues led to the formation of newly generated myofibers compared to cell-free hydrogel and collagen groups (p < 0.05). In the C2C12 myoblast-laden Col/Keratin group, a low number of CD31+ cells with minimum inflammatory cells was evident. Western blotting indicated the promotion of MyoD in mice that received cell-laden Col/Keratin hydrogel compared to the other groups (p < 0.05). Despite the increase of the myosin cell-laden Col/Keratin hydrogel group, no significant differences were obtained related to other groups (p > 0.05). The blend of Col/Keratin loaded with myoblasts provides a suitable myogenic platform for the alleviation of injured muscle tissue.


Assuntos
Queratinas , Desenvolvimento Muscular , Músculo Esquelético , Animais , Camundongos , Músculo Esquelético/lesões , Músculo Esquelético/metabolismo , Queratinas/metabolismo , Linhagem Celular , Hidrogéis/química , Neovascularização Fisiológica/efeitos dos fármacos , Engenharia Tecidual/métodos , Modelos Animais de Doenças , Colágeno/metabolismo , Mioblastos/metabolismo , Mioblastos/citologia , Masculino , Alicerces Teciduais/química , Angiogênese
13.
Skelet Muscle ; 14(1): 8, 2024 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-38671506

RESUMO

BACKGROUND: Duchenne muscular dystrophy (DMD) is associated with impaired muscle regeneration, progressive muscle weakness, damage, and wasting. While the cause of DMD is an X-linked loss of function mutation in the gene encoding dystrophin, the exact mechanisms that perpetuate the disease progression are unknown. Our laboratory has demonstrated that pannexin 1 (Panx1 in rodents; PANX1 in humans) is critical for the development, strength, and regeneration of male skeletal muscle. In normal skeletal muscle, Panx1 is part of a multiprotein complex with dystrophin. We and others have previously shown that Panx1 levels and channel activity are dysregulated in various mouse models of DMD. METHODS: We utilized myoblast cell lines derived from DMD patients to assess PANX1 expression and function. To investigate how Panx1 dysregulation contributes to DMD, we generated a dystrophic (mdx) mouse model that lacks Panx1 (Panx1-/-/mdx). In depth characterization of this model included histological analysis, as well as locomotor, and physiological tests such as muscle force and grip strength assessments. RESULTS: Here, we demonstrate that PANX1 levels and channel function are reduced in patient-derived DMD myoblast cell lines. Panx1-/-/mdx mice have a significantly reduced lifespan, and decreased body weight due to lean mass loss. Their tibialis anterior were more affected than their soleus muscles and displayed reduced mass, myofiber loss, increased centrally nucleated myofibers, and a lower number of muscle stem cells compared to that of Panx1+/+/mdx mice. These detrimental effects were associated with muscle and locomotor functional impairments. In vitro, PANX1 overexpression in patient-derived DMD myoblasts improved their differentiation and fusion. CONCLUSIONS: Collectively, our findings suggest that PANX1/Panx1 dysregulation in DMD exacerbates several aspects of the disease. Moreover, our results suggest a potential therapeutic benefit to increasing PANX1 levels in dystrophic muscles.


Assuntos
Conexinas , Camundongos Endogâmicos mdx , Músculo Esquelético , Distrofia Muscular de Duchenne , Proteínas do Tecido Nervoso , Animais , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patologia , Distrofia Muscular de Duchenne/fisiopatologia , Conexinas/genética , Conexinas/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Masculino , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Humanos , Camundongos , Mioblastos/metabolismo , Linhagem Celular , Força Muscular , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Camundongos Knockout
14.
Cells ; 13(8)2024 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-38667334

RESUMO

Meat yield, determined by muscle growth and development, is an important economic trait for the swine industry and a focus of research in animal genetics and breeding. PDZ and LIM domain 5 (PDLIM5) are cytoskeleton-related proteins that play key roles in various tissues and cells. These proteins have multiple isoforms, primarily categorized as short (PDLIM5-short) and long (PDLIM5-long) types, distinguished by the absence and presence of an LIM domain, respectively. However, the expression patterns of swine PDLIM5 isoforms and their regulation during porcine skeletal muscle development remain largely unexplored. We observed that PDLIM5-long was expressed at very low levels in pig muscles and that PDLIM5-short and total PDLIM5 were highly expressed in the muscles of slow-growing pigs, suggesting that PDLIM5-short, the dominant transcript in pigs, is associated with a slow rate of muscle growth. PDLIM5-short suppressed myoblast proliferation and myogenic differentiation in vitro. We also identified two single nucleotide polymorphisms (-258 A > T and -191 T > G) in the 5' flanking region of PDLIM5, which influenced the activity of the promoter and were associated with muscle growth rate in pigs. In summary, we demonstrated that PDLIM5-short negatively regulates myoblast proliferation and differentiation, providing a theoretical basis for improving pig breeding programs.


Assuntos
Proteínas com Domínio LIM , Desenvolvimento Muscular , Animais , Desenvolvimento Muscular/genética , Proteínas com Domínio LIM/genética , Proteínas com Domínio LIM/metabolismo , Suínos , Proliferação de Células/genética , Diferenciação Celular/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/crescimento & desenvolvimento , Polimorfismo de Nucleotídeo Único/genética , Mioblastos/metabolismo , Mioblastos/citologia , Regiões Promotoras Genéticas/genética
15.
Matrix Biol ; 129: 44-58, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38582404

RESUMO

Extracellular matrix (ECM) pathologic remodeling underlies many disorders, including muscular dystrophy. Tissue decellularization removes cellular components while leaving behind ECM components. We generated "on-slide" decellularized tissue slices from genetically distinct dystrophic mouse models. The ECM of dystrophin- and sarcoglycan-deficient muscles had marked thrombospondin 4 deposition, while dysferlin-deficient muscle had excess decorin. Annexins A2 and A6 were present on all dystrophic decellularized ECMs, but annexin matrix deposition was excessive in dysferlin-deficient muscular dystrophy. Muscle-directed viral expression of annexin A6 resulted in annexin A6 in the ECM. C2C12 myoblasts seeded onto decellularized matrices displayed differential myoblast mobility and fusion. Dystrophin-deficient decellularized matrices inhibited myoblast mobility, while dysferlin-deficient decellularized matrices enhanced myoblast movement and differentiation. Myoblasts treated with recombinant annexin A6 increased mobility and fusion like that seen on dysferlin-deficient decellularized matrix and demonstrated upregulation of ECM and muscle cell differentiation genes. These findings demonstrate specific fibrotic signatures elicit effects on myoblast activity.


Assuntos
Diferenciação Celular , Movimento Celular , Disferlina , Matriz Extracelular , Mioblastos , Sarcoglicanas , Animais , Mioblastos/metabolismo , Mioblastos/citologia , Matriz Extracelular/metabolismo , Camundongos , Sarcoglicanas/genética , Sarcoglicanas/metabolismo , Disferlina/genética , Disferlina/metabolismo , Distrofias Musculares/genética , Distrofias Musculares/metabolismo , Distrofias Musculares/patologia , Distrofina/genética , Distrofina/metabolismo , Anexina A2/genética , Anexina A2/metabolismo , Decorina/genética , Decorina/metabolismo , Linhagem Celular , Modelos Animais de Doenças , Músculo Esquelético/metabolismo
16.
Int J Mol Sci ; 25(8)2024 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-38673893

RESUMO

During embryogenesis, basic fibroblast growth factor (bFGF) is released from neural tube and myotome to promote myogenic fate in the somite, and is routinely used for the culture of adult skeletal muscle (SKM) stem cells (MuSC, called satellite cells). However, the mechanism employed by bFGF to promote SKM lineage and MuSC proliferation has not been analyzed in detail. Furthermore, the question of if the post-translational modification (PTM) of bFGF is important to its stemness-promoting effect has not been answered. In this study, GST-bFGF was expressed and purified from E.coli, which lacks the PTM system in eukaryotes. We found that both GST-bFGF and commercially available bFGF activated the Akt-Erk pathway and had strong cell proliferation effect on C2C12 myoblasts and MuSC. GST-bFGF reversibly compromised the myogenesis of C2C12 myoblasts and MuSC, and it increased the expression of Myf5, Pax3/7, and Cyclin D1 but strongly repressed that of MyoD, suggesting the maintenance of myogenic stemness amid repressed MyoD expression. The proliferation effect of GST-bFGF was conserved in C2C12 over-expressed with MyoD (C2C12-tTA-MyoD), implying its independence of the down-regulation of MyoD. In addition, the repressive effect of GST-bFGF on myogenic differentiation was almost totally rescued by the over-expression of MyoD. Together, these evidences suggest that (1) GST-bFGF and bFGF have similar effects on myogenic cell proliferation and differentiation, and (2) GST-bFGF can promote MuSC stemness and proliferation by differentially regulating MRFs and Pax3/7, (3) MyoD repression by GST-bFGF is reversible and independent of the proliferation effect, and (4) GST-bFGF can be a good substitute for bFGF in sustaining MuSC stemness and proliferation.


Assuntos
Proliferação de Células , Fator 2 de Crescimento de Fibroblastos , Desenvolvimento Muscular , Proteína MyoD , Mioblastos , Desenvolvimento Muscular/genética , Animais , Camundongos , Proteína MyoD/metabolismo , Proteína MyoD/genética , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fator 2 de Crescimento de Fibroblastos/farmacologia , Fator 2 de Crescimento de Fibroblastos/genética , Mioblastos/metabolismo , Mioblastos/citologia , Linhagem Celular , Fator de Transcrição PAX7/metabolismo , Fator de Transcrição PAX7/genética , Fator de Transcrição PAX3/metabolismo , Fator de Transcrição PAX3/genética , Fator Regulador Miogênico 5/metabolismo , Fator Regulador Miogênico 5/genética , Ciclina D1/metabolismo , Ciclina D1/genética , Células Satélites de Músculo Esquelético/metabolismo , Células Satélites de Músculo Esquelético/citologia , Diferenciação Celular , Proteínas Proto-Oncogênicas c-akt/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/citologia
17.
Food Funct ; 15(8): 4575-4585, 2024 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-38587267

RESUMO

Previous studies have shown that vitamin C (VC), an essential vitamin for the human body, can promote the differentiation of muscle satellite cells (MuSCs) in vitro and play an important role in skeletal muscle post-injury regeneration. However, the molecular mechanism of VC regulating MuSC proliferation has not been elucidated. In this study, the role of VC in promoting MuSC proliferation and its molecular mechanism were explored using cell molecular biology and animal experiments. The results showed that VC accelerates the progress of skeletal muscle post-injury regeneration by promoting MuSC proliferation in vivo. VC can also promote skeletal muscle regeneration in the case of atrophy. Using the C2C12 myoblast murine cell line, we observed that VC also stimulated cell proliferation. In addition, after an in vitro study establishing the occurrence of a physical interaction between VC and Pax7, we observed that VC also upregulated the total and nuclear Pax7 protein levels. This mechanism increased the expression of Myf5 (Myogenic Factor 5), a Pax7 target gene. This study establishes a theoretical foundation for understanding the regulatory mechanisms underlying VC-mediated MuSC proliferation and skeletal muscle regeneration. Moreover, it develops the application of VC in animal muscle nutritional supplements and treatment of skeletal muscle-related diseases.


Assuntos
Ácido Ascórbico , Proliferação de Células , Músculo Esquelético , Mioblastos , Fator de Transcrição PAX7 , Regeneração , Animais , Masculino , Camundongos , Ácido Ascórbico/farmacologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Mioblastos/efeitos dos fármacos , Mioblastos/metabolismo , Fator Regulador Miogênico 5/metabolismo , Fator Regulador Miogênico 5/genética , Fator de Transcrição PAX7/metabolismo , Fator de Transcrição PAX7/genética , Regeneração/efeitos dos fármacos , Células Satélites de Músculo Esquelético/metabolismo , Células Satélites de Músculo Esquelético/efeitos dos fármacos
18.
Commun Biol ; 7(1): 515, 2024 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-38688991

RESUMO

Adaptation to hypothermia is important for skeletal muscle cells under physiological stress and is used for therapeutic hypothermia (mild hypothermia at 32 °C). We show that hypothermic preconditioning at 32 °C for 72 hours improves the differentiation of skeletal muscle myoblasts using both C2C12 and primary myoblasts isolated from 3 month and 18-month-old mice. We analyzed the cold-shock proteome of myoblasts exposed to hypothermia (32 °C for 6 and 48 h) and identified significant changes in pathways related to RNA processing and central carbon, fatty acid, and redox metabolism. The analysis revealed that levels of the cold-shock protein RBM3, an RNA-binding protein, increases with both acute and chronic exposure to hypothermic stress, and is necessary for the enhanced differentiation and maintenance of mitochondrial metabolism. We also show that overexpression of RBM3 at 37 °C is sufficient to promote mitochondrial metabolism, cellular proliferation, and differentiation of C2C12 and primary myoblasts. Proteomic analysis of C2C12 myoblasts overexpressing RBM3 show significant enrichment of pathways involved in fatty acid metabolism, RNA metabolism and the electron transport chain. Overall, we show that the cold-shock protein RBM3 is a critical factor that can be used for controlling the metabolic network of myoblasts.


Assuntos
Diferenciação Celular , Mitocôndrias , Mioblastos , Proteoma , Proteínas de Ligação a RNA , Animais , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Camundongos , Mioblastos/metabolismo , Mitocôndrias/metabolismo , Proteoma/metabolismo , Resposta ao Choque Frio , Linhagem Celular
19.
Int J Biol Macromol ; 266(Pt 2): 131049, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38522687

RESUMO

Long non-coding RNAs (lncRNAs) play an essential role in vertebrate myogenesis and muscle diseases. However, the dynamic expression patterns, biological functions, and mechanisms of lncRNAs in skeletal muscle development and regeneration remain largely unknown. In this study, a novel lncRNA (named lncMGR) was differentially expressed during breast muscle development in fast- and slow-growing chickens. Functionally, lncMGR promoted myoblast differentiation, inhibited myoblast proliferation in vitro, and promoted myofiber hypertrophy and injury repair in vivo. Mechanistically, lncMGR increased the mRNA and protein expression of skeletal muscle myosin heavy chain 1 A (MYH1A) via both transcriptional and post-transcriptional regulation. Nuclear lncMGR recruited cyclin-dependent kinase 9 (CDK9) to the core transcriptional activation region of the MYH1A gene to activate MYH1A transcription. Cytoplasmic lncMGR served as a competitive endogenous RNA (ceRNA) to competitively absorb miR-2131-5p away from MYH1A and subsequently protected the MYH1A from miR-2131-5p-mediated degradation. Besides miR-2131-5p, cytoplasmic lncMGR could also sponge miR-143-3p to reconcile the antagonist between the miR-2131-5p/MYH1A-mediated inhibition effects and miR-143-3p-mediated promotion effects on myoblast proliferation, thereby inhibiting myoblast proliferation. Collectively, lncMGR could recruit CDK9 and sponge multiple miRNAs to regulate skeletal muscle development and regeneration, and could be a therapeutic target for muscle diseases.


Assuntos
Galinhas , MicroRNAs , Desenvolvimento Muscular , RNA Longo não Codificante , Animais , Diferenciação Celular/genética , Proliferação de Células/genética , Quinase 9 Dependente de Ciclina/metabolismo , Quinase 9 Dependente de Ciclina/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Desenvolvimento Muscular/genética , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiologia , Mioblastos/metabolismo , Mioblastos/citologia , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Regeneração/genética , RNA Longo não Codificante/genética
20.
Biomed Pharmacother ; 173: 116380, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38447450

RESUMO

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease affecting motor neurons. Considerable evidence indicates that early skeletal muscle atrophy plays a crucial role in the disease pathogenesis, leading to an altered muscle-motor neuron crosstalk that, in turn, may contribute to motor neuron degeneration. Currently, there is no effective treatment for ALS, highlighting the need to dig deeper into the pathological mechanisms for developing innovative therapeutic strategies. FM19G11 is a novel drug able to modulate the global cellular metabolism, but its effects on ALS skeletal muscle atrophy and mitochondrial metabolism have never been evaluated, yet. This study investigated whether FM19G11-loaded nanoparticles (NPs) may affect the bioenergetic status in myoblasts isolated from G93A-SOD1 mice at different disease stages. We found that FM19G1-loaded NP treatment was able to increase transcriptional levels of Akt1, Akt3, Mef2a, Mef2c and Ucp2, which are key genes associated with cell proliferation (Akt1, Akt3), muscle differentiation (Mef2c), and mitochondrial activity (Ucp2), in G93A-SOD1 myoblasts. These cells also showed a significant reduction of mitochondrial area and networks, in addition to decreased ROS production after treatment with FM19G11-loaded NPs, suggesting a ROS clearance upon the amelioration of mitochondrial dynamics. Our overall findings demonstrate a significant impact of FM19G11-loaded NPs on muscle cell function and bioenergetic status in G93A-SOD1 myoblasts, thus promising to open new avenues towards possible adoption of FM19G11-based nanotherapies to slow muscle degeneration in the frame of ALS and muscle disorders.


Assuntos
Esclerose Lateral Amiotrófica , Benzamidas , Nanopartículas , Doenças Neurodegenerativas , Camundongos , Animais , Superóxido Dismutase-1/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Esclerose Lateral Amiotrófica/tratamento farmacológico , Doenças Neurodegenerativas/patologia , Mioblastos/metabolismo , Atrofia/patologia , Camundongos Transgênicos , Modelos Animais de Doenças , Superóxido Dismutase/metabolismo
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