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1.
Mol Cell Biol ; 44(2): 57-71, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38483114

RESUMO

Myocyte enhancer factor 2 (MEF2) proteins are involved in multiple developmental, physiological, and pathological processes in vertebrates. Protein-protein interactions underlie the plethora of biological processes impacted by MEF2A, necessitating a detailed characterization of the MEF2A interactome. A nanobody based affinity-purification/mass spectrometry strategy was employed to achieve this goal. Specifically, the MEF2A protein complexes were captured from myogenic lysates using a GFP-tagged MEF2A protein immobilized with a GBP-nanobody followed by LC-MS/MS proteomic analysis to identify MEF2A interactors. After bioinformatic analysis, we further characterized the interaction of MEF2A with a transcriptional repressor, FOXP1. FOXP1 coprecipitated with MEF2A in proliferating myogenic cells which diminished upon differentiation (myotube formation). Ectopic expression of FOXP1 inhibited MEF2A driven myogenic reporter genes (derived from the creatine kinase muscle and myogenin genes) and delayed induction of endogenous myogenin during differentiation. Conversely, FOXP1 depletion enhanced MEF2A transactivation properties and myogenin expression. The FoxP1:MEF2A interaction is also preserved in cardiomyocytes and FoxP1 depletion enhanced cardiomyocyte hypertrophy. FOXP1 prevented MEF2A phosphorylation and activation by the p38MAPK pathway. Overall, these data implicate FOXP1 in restricting MEF2A function in order to avoid premature differentiation in myogenic progenitors and also to possibly prevent re-activation of embryonic gene expression in cardiomyocyte hypertrophy.


Assuntos
Proteômica , Espectrometria de Massas em Tandem , Animais , Fatores de Transcrição MEF2/genética , Miogenina , Cromatografia Líquida , Músculo Esquelético/fisiologia , Hipertrofia
2.
Front Biosci (Landmark Ed) ; 29(2): 49, 2024 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-38420814

RESUMO

BACKGROUND: Myogenin is well known as a crucial transcription factor in skeletal muscle development, yet its other biological functions remain unexplored. Previous research showed that myogenin suppresses apoptosis induced by angiotensin II in human induced pluripotent stem cell-derived cardiomyocytes, and offered a new perspective on myogenin's role in cardioprotection. However, the detailed mechanism of this cardioprotection, especially under oxidative stress, is still unclear. METHODS: In this study, hydrogen peroxide (H2O2) was used to generate reactive oxygen species in myogenin-overexpressing cardiomyocytes. The apoptosis was examined by flow cytometry. Transcriptome sequencing (RNA-seq) was performed to identify genes regulated by myogenin. Western blotting was used to detect the protein level of DUSP13 and the phosphorylation level of p38 mitogen-activated protein kinase (MAPK). The dual-luciferase reporter assay and ChIP assay were used to confirm the binding of myogenin to the promoter region of DUSP13. DUSP13 overexpression and knockdown assays were performed to study its anti-apoptotic role. RESULTS: Flow cytometry analysis of apoptosis showed that overexpressing myogenin for 24 and 48 hours decreased the apoptotic ratio by 47.9% and 63.5%, respectively, compared with untreated controls. Transcriptome sequencing performed on cardiomyocytes that expressed myogenin for different amounts of time (6, 12, 24, and 48 hours) identified DUSP13 as being up-regulated by myogenin. Western blotting showed that overexpression of myogenin increased the expression of DUSP13 and decreased the phosphorylation level of p38 MAPK. A dual-luciferase reporter assay proved that myogenin bound directly to the promoter region of DUSP13 and led to strong relative luciferase activity. Direct expression of DUSP13A and DUSP13B significantly reduced the rates of apoptosis and necrosis in cells treated with H2O2. Knockdown of DUSP13B significantly increased the rate of apoptosis in cells treated with H2O2. CONCLUSIONS: The present findings suggest that myogenin might attenuate apoptosis induced by reactive oxygen species by up-regulating DUSP13 and inactivating the p38 MAPK pathway.


Assuntos
Peróxido de Hidrogênio , Células-Tronco Pluripotentes Induzidas , Humanos , Espécies Reativas de Oxigênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Miogenina/genética , Miogenina/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Apoptose , Estresse Oxidativo , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Luciferases/metabolismo
3.
Life Sci Alliance ; 7(5)2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38373797

RESUMO

Skeletal muscle development is a highly ordered process orchestrated transcriptionally by the myogenic regulatory factors. However, the downstream molecular mechanisms of myogenic regulatory factor functions in myogenesis are not fully understood. Here, we identified the RNA-binding protein Musashi2 (Msi2) as a myogenin target gene and a post-transcriptional regulator of myoblast differentiation. Msi2 knockdown in murine myoblasts blocked differentiation without affecting the expression of MyoD or myogenin. Msi2 overexpression was also sufficient to promote myoblast differentiation and myocyte fusion. Msi2 loss attenuated autophagosome formation via down-regulation of the autophagic protein MAPL1LC3/ATG8 (LC3) at the early phase of myoblast differentiation. Moreover, forced activation of autophagy effectively suppressed the differentiation defects incurred by Msi2 loss. Consistent with its functions in myoblasts in vitro, mice deficient for Msi2 exhibited smaller limb skeletal muscles, poorer exercise performance, and muscle fiber-type switching in vivo. Collectively, our study demonstrates that Msi2 is a novel regulator of mammalian myogenesis and establishes a new functional link between muscular development and autophagy regulation.


Assuntos
Desenvolvimento Muscular , Músculo Esquelético , Animais , Camundongos , Miogenina/genética , Miogenina/metabolismo , Músculo Esquelético/metabolismo , Desenvolvimento Muscular/genética , Autofagia/genética , Proteínas de Ligação a RNA/genética , Mamíferos/metabolismo
4.
Exp Physiol ; 108(12): 1531-1547, 2023 12.
Artigo em Inglês | MEDLINE | ID: mdl-37864311

RESUMO

NEW FINDINGS: What is the central question of this study? Does the hormone Klotho affect the myogenic response of muscle cells to mechanical loading or exercise? What is the main finding and its importance? Klotho prevents direct, mechanical activation of genes that regulate muscle differentiation, including genes that encode the myogenic regulatory factor myogenin and proteins in the canonical Wnt signalling pathway. Similarly, elevated levels of klotho expression in vivo prevent the exercise-induced increase in myogenin-expressing cells and reduce exercise-induced activation of the Wnt pathway. These findings demonstrate a new mechanism through which the responses of muscle to the mechanical environment are regulated. ABSTRACT: Muscle growth is influenced by changes in the mechanical environment that affect the expression of genes that regulate myogenesis. We tested whether the hormone Klotho could influence the response of muscle to mechanical loading. Applying mechanical loads to myoblasts in vitro increased RNA encoding transcription factors that are expressed in activated myoblasts (Myod) and in myogenic cells that have initiated terminal differentiation (Myog). However, application of Klotho to myoblasts prevented the loading-induced activation of Myog without affecting loading-induced activation of Myod. This indicates that elevated Klotho inhibits mechanically-induced differentiation of myogenic cells. Elevated Klotho also reduced the transcription of genes encoding proteins involved in the canonical Wnt pathway or their target genes (Wnt9a, Wnt10a, Ccnd1). Because the canonical Wnt pathway promotes differentiation of myogenic cells, these findings indicate that Klotho inhibits the differentiation of myogenic cells experiencing mechanical loading. We then tested whether these effects of Klotho occurred in muscles of mice experiencing high-intensity interval training (HIIT) by comparing wild-type mice and klotho transgenic mice. The expression of a klotho transgene combined with HIIT synergized to tremendously elevate numbers of Pax7+ satellite cells and activated MyoD+ cells. However, transgene expression prevented the increase in myogenin+ cells caused by HIIT in wild-type mice. Furthermore, transgene expression diminished the HIIT-induced activation of the canonical Wnt pathway in Pax7+ satellite cells. Collectively, these findings show that Klotho inhibits loading- or exercise-induced activation of muscle differentiation and indicate a new mechanism through which the responses of muscle to the mechanical environment are regulated.


Assuntos
Músculos , Células Satélites de Músculo Esquelético , Animais , Camundongos , Diferenciação Celular , Hormônios/metabolismo , Desenvolvimento Muscular/genética , Músculo Esquelético/metabolismo , Músculos/metabolismo , Proteína MyoD/metabolismo , Miogenina/metabolismo , Células Satélites de Músculo Esquelético/metabolismo
5.
Int J Mol Sci ; 24(19)2023 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-37834190

RESUMO

Mice are commonly used to study mandibular dynamics due to their similarity in chewing cycle patterns with humans. Adult mice treated unilaterally with botulinum toxin type A (BoNTA) in the masseter exhibit atrophy of this muscle characterized by an increase in the gene expression of atrophy-related molecular markers, and a reduction in both muscle fiber diameter and muscle mass at 14d. However, the impact of this muscle imbalance on the non-treated masticatory muscles remains unexplored. Here, we hypothesize that the unilateral masseter hypofunction leads to molecular and 3D morphometric signs of atrophy of the masseter and its agonist masticatory muscles in adult mice. Twenty-three 8-week-old male BALB/c mice received a single injection of BoNTA in the right masseter, whereas the left masseter received the same volume of saline solution (control side). Animals were euthanized at 2d, 7d, and 14d, and the masticatory muscles were analyzed for mRNA expression. Five heads were harvested at 14d, fixed, stained with a contrast-enhanced agent, and scanned using X-ray microtomography. The three-dimensional morphometric parameters (the volume and thickness) from muscles in situ were obtained. Atrogin-1/MAFbx, MuRF-1, and Myogenin mRNA gene expression were significantly increased at 2 and 7d for both the masseter and temporalis from the BoNTA side. For medial pterygoid, increased mRNA gene expression was found at 7d for Atrogin-1/MAFbx and at 2d-7d for Myogenin. Both the volume and thickness of the masseter, temporalis, and medial pterygoid muscles from the BoNTA side were significantly reduced at 14d. In contrast, the lateral pterygoid from the BoNTA side showed a significant increase in volume at 14d. Therefore, the unilateral hypofunction of the masseter leads to molecular and morphological signs of atrophy in both the BoNTA-injected muscle and its agonistic non-injected masticatory muscles. The generalized effect on the mouse masticatory apparatus when one of its components is intervened suggests the need for more clinical studies to determine the safety of BoNTA usage in clinical dentistry.


Assuntos
Toxinas Botulínicas Tipo A , Músculos da Mastigação , Adulto , Humanos , Camundongos , Masculino , Animais , Miogenina , Músculo Masseter/patologia , Músculo Masseter/fisiologia , Atrofia Muscular/patologia , RNA Mensageiro
6.
Cells ; 12(18)2023 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-37759547

RESUMO

Olive flounder (Paralichthys olivaceus) muscle satellite cells (OFMCs) were obtained by enzymatic primary cell isolation and the explant method. Enzymatic isolation yielded cells that reached 80% confluence within 8 days, compared to 15 days for the explant method. Optimal OFMC growth was observed in 20% fetal bovine serum at 28 °C with 0.8 mM CaCl2 and the basic fibroblast growth factor (BFGF) to enhance cell growth. OFMCs have become permanent cell lines through the spontaneous immortalization crisis at the 20th passage. Olive flounder skeletal muscle myoblasts were induced into a mitogen-poor medium containing 2% horse serum for differentiation; they fused to form multinucleate myotubes. The results indicated complete differentiation of myoblasts into myotubes; we also detected the expression of the myogenic regulatory factors myoD, myogenin, and desmin. Upregulation (Myogenin, desmin) and downregulation (MyoD) of muscle regulation factors confirmed the differentiation in OFMCs.


Assuntos
Linguado , Células Satélites de Músculo Esquelético , Animais , Miogenina , Desmina , Fibras Musculares Esqueléticas , Músculo Esquelético
7.
J Cell Physiol ; 238(11): 2638-2650, 2023 11.
Artigo em Inglês | MEDLINE | ID: mdl-37683043

RESUMO

Skeletal muscle regeneration is a crucial physiological process that occurs in response to injury or disease. As an important transcriptome surveillance system that regulates tissue development, the role of nonsense-mediated mRNA decay (NMD) in muscle regeneration remains unclear. Here, we found that NMD inhibits myoblast differentiation by targeting the phosphoinositide-3-kinase regulatory subunit 5 gene, which leads to the suppression of the transcriptional activity of myogenic differentiation (MyoD), a key regulator of myoblast differentiation. This disruption of MyoD transcriptional activity subsequently affects the expression levels of myogenin and myosin heavy chain, crucial markers of myoblast differentiation. Additionally, through up-frameshift protein 1 knockdown experiments, we observed that inhibiting NMD can accelerate muscle regeneration in vivo. These findings highlight the potential of NMD as a novel therapeutic target for the treatment of muscle-related injuries and diseases.


Assuntos
Mioblastos , Degradação do RNAm Mediada por Códon sem Sentido , Animais , Masculino , Camundongos , Diferenciação Celular/genética , Linhagem Celular , Camundongos Endogâmicos C57BL , Desenvolvimento Muscular/genética , Músculo Esquelético/metabolismo , Músculos , Mioblastos/metabolismo , Proteína MyoD/genética , Proteína MyoD/metabolismo , Miogenina/genética , Miogenina/metabolismo , Degradação do RNAm Mediada por Códon sem Sentido/genética
8.
Cells ; 12(17)2023 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-37681900

RESUMO

Sarcopenia has a high prevalence among the aging population. Sarcopenia is of tremendous socioeconomic importance because it can lead to falls and hospitalization, subsequently increasing healthcare costs while limiting quality of life. In sarcopenic muscle fibers, the E3 ubiquitin ligase F-Box Protein 32 (Fbxo32) is expressed at substantially higher levels, driving ubiquitin-proteasomal muscle protein degradation. As one of the key regulators of muscular equilibrium, the transcription factor Forkhead Box O3 (FOXO3) can increase the expression of Fbxo32, making it a possible target for the regulation of this detrimental pathway. To test this hypothesis, murine C2C12 myoblasts were transduced with AAVs carrying a plasmid for four specific siRNAs against Foxo3. Successfully transduced myoblasts were selected via FACS cell sorting to establish single clone cell lines. Sorted myoblasts were further differentiated into myotubes and stained for myosin heavy chain (MHC) by immunofluorescence. The resulting area was calculated. Myotube contractions were induced by electrical stimulation and quantified. We found an increased Foxo3 expression in satellite cells in human skeletal muscle and an age-related increase in Foxo3 expression in older mice in silico. We established an in vitro AAV-mediated FOXO3 knockdown on protein level. Surprisingly, the myotubes with FOXO3 knockdown displayed a smaller myotube size and a lower number of nuclei per myotube compared to the control myotubes (AAV-transduced with a functionless control plasmid). During differentiation, a lower level of FOXO3 reduced the expression Fbxo32 within the first three days. Moreover, the expression of Myod1 and Myog via ATM and Tp53 was reduced. Functionally, the Foxo3 knockdown myotubes showed a higher contraction duration and time to peak. Early Foxo3 knockdown seems to terminate the initiation of differentiation due to lack of Myod1 expression, and mediates the inhibition of Myog. Subsequently, the myotube size is reduced and the excitability to electrical stimulation is altered.


Assuntos
Proteína Forkhead Box O3 , Proteína MyoD , Miogenina , Qualidade de Vida , Sarcopenia , Idoso , Animais , Humanos , Camundongos , Proteína Forkhead Box O3/genética , Fibras Musculares Esqueléticas , Músculo Esquelético , Mioblastos , Miogenina/metabolismo , Proteína MyoD/metabolismo
9.
Int J Mol Sci ; 24(17)2023 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-37685838

RESUMO

Various pathological alterations, including lipid-deposition-induced comparative cardiac lipotoxicity, contribute to cardiac aging in the failing heart. A decline in endogenous myogenin proteins can lead to the reversal of muscle cell differentiation and the creation of mononucleated muscle cells. Myogenin may be a specific regulator of adaptive responses to avoid pathological hypertrophy in the heart. Hence, it is important to understand the regulation of myogenin expression and functions in response to exposure to varied stresses. In this study, we first examined and verified the cytotoxic effect of palmitic acid on H9c2 cells. The reduction in myogenin mRNA and protein expression by palmitic acid was independent of the effect of glucose. Meanwhile, the induction of cyclooxygenase 2 and activating transcription factor 3 mRNAs and proteins by palmitic acid was dependent on the presence of glucose. In addition, palmitic acid failed to disrupt cell cycle progression when H9c2 cells were treated with no glucose. Next, we examined the functional role of myogenin in palmitic-acid-treated H9c2 cells and found that myogenin may be involved in palmitic-acid-induced mitochondrial and cytosolic ROS generation, cellular senescence, and mitochondrial membrane potential. Finally, the GSE150059 dataset was deposited in the Gene Expression Omnibus website and the dataset was further analyzed via the molecular microscope diagnostic system (MMDx), demonstrating that many heart transplant biopsies currently diagnosed as no rejection have mild molecular-antibody-mediated rejection-related changes. Our data show that the expression levels of myogenin were lower than the average level in the studied population. Combining these results, we uncover part of the functional role of myogenin in lipid- and glucose-induced cardiac cell stresses. This finding provides valuable insight into the differential role of fatty-acid-associated gene expression in cardiovascular tissues. Additionally, the question of whether this gene expression is regulated by myogenin also highlights the usefulness of a platform such as MMDx-Heart and can help elucidate the functional role of myogenin in heart transplantation.


Assuntos
Transplante de Coração , Ácido Palmítico , Ácido Palmítico/farmacologia , Miogenina , Coração
10.
Cell Death Differ ; 30(8): 1900-1915, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37400716

RESUMO

Skeletal muscle regeneration relies on muscle stem (satellite) cells. We previously demonstrated that satellite cells efficiently and accurately repair radiation-induced DNA double-strand breaks (DSBs) via the DNA-dependent kinase DNA-PKcs. We show here that DNA-PKcs affects myogenesis independently of its role in DSB repair. Consequently, this process does not require the accumulation of DSBs and it is also independent of caspase-induced DNA damage. We report that in myogenic cells DNA-PKcs is essential for the expression of the differentiation factor Myogenin in an Akt2-dependent manner. DNA-PKcs interacts with the p300-containing complex that activates Myogenin transcription. We show also that SCID mice that are deficient in DNA-PKcs, and are used for transplantation and muscle regeneration studies, display altered myofiber composition and delayed myogenesis upon injury. These defects are exacerbated after repeated injury/regeneration events resulting in reduced muscle size. We thus identify a novel, caspase-independent, regulation of myogenic differentiation, and define a differentiation phase that does not involve the DNA damage/repair process.


Assuntos
Reparo do DNA , Proteínas Proto-Oncogênicas c-akt , Animais , Camundongos , Miogenina/genética , Miogenina/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Camundongos SCID , Dano ao DNA , Desenvolvimento Muscular , Caspases/metabolismo , DNA
11.
Mol Ther ; 31(9): 2612-2632, 2023 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-37452493

RESUMO

Rhabdomyosarcoma is the most common pediatric soft tissue tumor, comprising two major subtypes: the PAX3/7-FOXO1 fusion-negative embryonal and the PAX3/7-FOXO1 fusion-positive alveolar subtype. Here, we demonstrate that the expression levels of the transcriptional repressor TRPS1 are specifically enhanced in the embryonal subtype, resulting in impaired terminal myogenic differentiation and tumor growth. During normal myogenesis, expression levels of TRPS1 have to decrease to allow myogenic progression, as demonstrated by overexpression of TRPS1 in myoblasts impairing myotube formation. Consequentially, myogenic differentiation in embryonal rhabdomyosarcoma in vitro as well as in vivo can be achieved by reducing TRPS1 levels. Furthermore, we show that TRPS1 levels in RD cells, the bona fide model cell line for embryonal rhabdomyosarcoma, are regulated by miR-1 and that TRPS1 and MYOD1 share common genomic binding sites. The myogenin (MYOG) promoter is one of the critical targets of TRPS1 and MYOD1; we demonstrate that TRPS1 restricts MYOG expression and thereby inhibits terminal myogenic differentiation. Therefore, reduction of TRPS1 levels in embryonal rhabdomyosarcoma might be a therapeutic approach to drive embryonal rhabdomyosarcoma cells into myogenic differentiation, thereby generating postmitotic myotubes.


Assuntos
MicroRNAs , Rabdomiossarcoma Embrionário , Humanos , Criança , Rabdomiossarcoma Embrionário/genética , Rabdomiossarcoma Embrionário/metabolismo , Rabdomiossarcoma Embrionário/patologia , Miogenina/genética , Miogenina/metabolismo , Diferenciação Celular/genética , MicroRNAs/genética , Desenvolvimento Muscular/genética , Linhagem Celular Tumoral , Proteínas Repressoras
12.
Parasitol Int ; 96: 102773, 2023 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-37330041

RESUMO

Trichinella spiralis (T. spiralis)-induced myopathy is an inflammatory myopathy that is difficult to treat unless the parasite is combated in its early intestinal phase before it reaches the muscles. This study aimed to evaluate the effect of local mesenchymal stem cell (MSC) therapy on T. spiralis-induced inflammatory myopathy in rats. Rats were divided into four groups: Group 1 (non-infected non-treated group); Group 2 (infected non-treated group); Group 3 (infected albendazole (ABZ)-treated group); and Group 4 (infected MSC-treated group). Their muscle status was assessed physiologically with the righting reflex and electromyography (EMG), parasitologically with the total muscle larval count, histopathologically with hematoxylin and eosin and Mallory's trichrome stains, as well as immunohistochemically for myogenin as a marker of muscle regeneration. Additionally, serum muscle enzymes creatine kinase (CK) and lactate dehydrogenase (LDH), as well as muscle matrix metalloproteinases MMP1 and MMP9, were assayed. Finally, the immunological response was assessed by measuring the levels of the muscle inflammatory cytokines tumor necrosis factor-alpha (TNF-α), interferon-gamma (INF-γ), and interleukin-4 (IL-4). Our findings revealed that MSC therapy markedly improved muscle EMG and righting reflex, as well as the histopathological appearance of the muscles, reduced inflammatory cellular infiltrates, and increased myogenin immunostaining. It also reduced serum CK and LDH levels, as well as muscle INF-γ, TNF-α, IL-4, MMP1, and MMP9 levels. However, it had no effect on the total muscle larval count. Accordingly, due to its anti-inflammatory properties and muscle-regenerative effect, MSC therapy could be a promising new remedy for T. spiralis-induced myopathy.


Assuntos
Doenças Musculares , Miosite , Trichinella spiralis , Triquinelose , Ratos , Animais , Triquinelose/parasitologia , Interleucina-4 , Metaloproteinase 9 da Matriz , Metaloproteinase 1 da Matriz , Miogenina , Fator de Necrose Tumoral alfa , Miosite/terapia , Interferon gama , Células-Tronco , Terapia Biológica
13.
Cells ; 12(9)2023 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-37174683

RESUMO

Myoblast fusion is essential for skeletal muscle development, growth, and regeneration. However, the molecular mechanisms underlying myoblast fusion and differentiation are not fully understood. Previously, we reported that interleukin-4 (IL-4) promotes myoblast fusion; therefore, we hypothesized that IL-4 signaling might regulate the expression of the molecules involved in myoblast fusion. In this study, we showed that in addition to fusion, IL-4 promoted the differentiation of C2C12 myoblast cells by inducing myoblast determination protein 1 (MyoD) and myogenin, both of which regulate the expression of myomerger and myomaker, the membrane proteins essential for myoblast fusion. Unexpectedly, IL-4 treatment increased the expression of myomerger, but not myomaker, in C2C12 cells. Knockdown of IL-4 receptor alpha (IL-4Rα) in C2C12 cells by small interfering RNA impaired myoblast fusion and differentiation. We also demonstrated a reduction in the expression of MyoD, myogenin, and myomerger by knockdown of IL-4Rα in C2C12 cells, while the expression level of myomaker remained unchanged. Finally, cell mixing assays and the restoration of myomerger expression partially rescued the impaired fusion in the IL-4Rα-knockdown C2C12 cells. Collectively, these results suggest that the IL-4/IL-4Rα axis promotes myoblast fusion and differentiation via the induction of myogenic regulatory factors, MyoD and myogenin, and myomerger.


Assuntos
Interleucina-4 , Fatores de Regulação Miogênica , Diferenciação Celular/genética , Interleucina-4/farmacologia , Interleucina-4/metabolismo , Mioblastos/metabolismo , Fatores de Regulação Miogênica/metabolismo , Miogenina/genética , Miogenina/metabolismo , Animais , Camundongos
14.
Histochem Cell Biol ; 160(2): 135-146, 2023 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-37179509

RESUMO

The aim of the current study was to investigate the effect of glutamine supplementation on the expression of HSP70 and the calcium-binding proteins from the S100 superfamily in the recovering extensor digitorum longus (EDL) muscle after injury. Two-month-old Wistar rats were subjected to cryolesion of the EDL muscle and then randomly divided into two groups (with or without glutamine supplementation). Starting immediately after the injury, the supplemented group received daily doses of glutamine (1 g/kg/day, via gavage) for 3 and 10 days orally. Then, muscles were subjected to histological, molecular, and functional analysis. Glutamine supplementation induced an increase in myofiber size of regenerating EDL muscles and prevented the decline in maximum tetanic strength of these muscles evaluated 10 days after injury. An accelerated upregulation of myogenin mRNA levels was detected in glutamine-supplemented injured muscles on day 3 post-cryolesion. The HSP70 expression increased only in the injured group supplemented with glutamine for 3 days. The increase in mRNA levels of NF-κB, the pro-inflammatory cytokines IL-1ß and TNF-α, and the calcium-binding proteins S100A8 and S100A9 on day 3 post-cryolesion in EDL muscles was attenuated by glutamine supplementation. In contrast, the decrease in S100A1 mRNA levels in the 3-day-injured EDL muscles was minimized by glutamine supplementation. Overall, our results suggest that glutamine supplementation accelerates the recovery of myofiber size and contractile function after injury by modulating the expression of myogenin, HSP70, NF-κB, pro-inflammatory cytokines, and S100 calcium-binding proteins.


Assuntos
Glutamina , NF-kappa B , Ratos , Animais , Glutamina/farmacologia , Glutamina/metabolismo , Miogenina/metabolismo , Miogenina/farmacologia , NF-kappa B/metabolismo , Ratos Wistar , Músculo Esquelético/metabolismo , Contração Muscular/fisiologia , Citocinas/metabolismo , RNA Mensageiro/metabolismo , Suplementos Nutricionais , Proteínas de Ligação ao Cálcio
15.
Int J Mol Sci ; 24(10)2023 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-37240382

RESUMO

All-trans retinoic acid (ATRA) promotes myoblast differentiation into myotubes. Leucine-rich repeat-containing G-protein-coupled receptor 6 (LGR6) is a candidate ATRA-responsive gene; however, its role in skeletal muscles remains unclear. Here, we demonstrated that during the differentiation of murine C2C12 myoblasts into myotubes, Lgr6 mRNA expression transiently increased before the increase in the expression of the mRNAs encoding myogenic regulatory factors, such as myogenin, myomaker, and myomerger. The loss of LGR6 decreased the differentiation and fusion indices. The exogenous expression of LGR6 up to 3 and 24 h after the induction of differentiation increased and decreased the mRNA levels of myogenin, myomaker, and myomerger, respectively. Lgr6 mRNA was transiently expressed after myogenic differentiation in the presence of a retinoic acid receptor α (RARα) agonist and an RARγ agonist in addition to ATRA, but not in the absence of ATRA. Furthermore, a proteasome inhibitor or Znfr3 knockdown increased exogenous LGR6 expression. The loss of LGR6 attenuated the Wnt/ß-catenin signaling activity induced by Wnt3a alone or in combination with Wnt3a and R-spondin 2. These results indicate that LGR6 promotes myogenic differentiation and that ATRA is required for the transient expression of LGR6 during differentiation. Furthermore, LGR6 expression appeared to be downregulated by the ubiquitin-proteasome system involving ZNRF3.


Assuntos
Tretinoína , Via de Sinalização Wnt , Camundongos , Animais , Miogenina/genética , Miogenina/metabolismo , Tretinoína/farmacologia , Tretinoína/metabolismo , Mioblastos/metabolismo , RNA Mensageiro/genética , Diferenciação Celular/genética , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo
16.
J Cachexia Sarcopenia Muscle ; 14(4): 1721-1736, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37209006

RESUMO

BACKGROUND: Sepsis-induced intensive care unit-acquired weakness (ICUAW) features profound muscle atrophy and attenuated muscle regeneration related to malfunctioning satellite cells. Transforming growth factor beta (TGF-ß) is involved in both processes. We uncovered an increased expression of the TGF-ß receptor II (TßRII)-inhibitor SPRY domain-containing and SOCS-box protein 1 (SPSB1) in skeletal muscle of septic mice. We hypothesized that SPSB1-mediated inhibition of TßRII signalling impairs myogenic differentiation in response to inflammation. METHODS: We performed gene expression analyses in skeletal muscle of cecal ligation and puncture- (CLP) and sham-operated mice, as well as vastus lateralis of critically ill and control patients. Pro-inflammatory cytokines and specific pathway inhibitors were used to quantitate Spsb1 expression in myocytes. Retroviral expression plasmids were used to investigate the effects of SPSB1 on TGF-ß/TßRII signalling and myogenesis in primary and immortalized myoblasts and differentiated myotubes. For mechanistical analyses we used coimmunoprecipitation, ubiquitination, protein half-life, and protein synthesis assays. Differentiation and fusion indices were determined by immunocytochemistry, and differentiation factors were quantified by qRT-PCR and Western blot analyses. RESULTS: SPSB1 expression was increased in skeletal muscle of ICUAW patients and septic mice. Tumour necrosis factor (TNF), interleukin-1ß (IL-1ß), and IL-6 increased the Spsb1 expression in C2C12 myotubes. TNF- and IL-1ß-induced Spsb1 expression was mediated by NF-κB, whereas IL-6 increased the Spsb1 expression via the glycoprotein 130/JAK2/STAT3 pathway. All cytokines reduced myogenic differentiation. SPSB1 avidly interacted with TßRII, resulting in TßRII ubiquitination and destabilization. SPSB1 impaired TßRII-Akt-Myogenin signalling and diminished protein synthesis in myocytes. Overexpression of SPSB1 decreased the expression of early (Myog, Mymk, Mymx) and late (Myh1, 3, 7) differentiation-markers. As a result, myoblast fusion and myogenic differentiation were impaired. These effects were mediated by the SPRY- and SOCS-box domains of SPSB1. Co-expression of SPSB1 with Akt or Myogenin reversed the inhibitory effects of SPSB1 on protein synthesis and myogenic differentiation. Downregulation of Spsb1 by AAV9-mediated shRNA attenuated muscle weight loss and atrophy gene expression in skeletal muscle of septic mice. CONCLUSIONS: Inflammatory cytokines via their respective signalling pathways cause an increase in SPSB1 expression in myocytes and attenuate myogenic differentiation. SPSB1-mediated inhibition of TßRII-Akt-Myogenin signalling and protein synthesis contributes to a disturbed myocyte homeostasis and myogenic differentiation that occurs during inflammation.


Assuntos
Interleucina-6 , Proteínas Proto-Oncogênicas c-akt , Animais , Camundongos , Citocinas , Inflamação , Desenvolvimento Muscular , Músculo Esquelético/metabolismo , Miogenina/genética , Fator de Crescimento Transformador beta/metabolismo , Fator de Necrose Tumoral alfa
17.
Physiol Rep ; 11(8): e15657, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-37078370

RESUMO

Pemphigus Vulgaris (PV) is a blistering autoimmune disease caused by autoantibodies against desmoglein 1 and 3. Treatment options are limited to corticosteroids and immunosuppressants. The myotoxic effect of glucocorticoids is a fact that has been elucidated. So, the development of efficacious treatment approaches to combat muscle wasting is of great importance. Considering the adverse effect of glucocorticoid therapy in pemphigus patients and altered muscle metabolism, this study aimed to investigate the effect of l-carnitine supplementation which can be useful in combating muscle-wasting impact of glucocorticoid therapy. In this randomized double-blind placebo-controlled trial 44 pemphigus patients aged from 30 to 65 years, receiving glucocorticoid therapy were selected to evaluate the suitability of l-carnitine (LC) as an anti-wasting substance. Patients were randomly divided into two groups to receive 2 g/d l-carnitine or placebo for 8 weeks; serum markers of muscle metabolism (IGF-1, creatine kinase, myogenin, myostatin) was evaluated before and after the l-carnitine supplementation. Paired T-test was used to analyze the differences between variables before and after the intervention. Therefore, the student's t-test was performed to find any differences in baseline characteristics and dietary intakes between the trial groups. LC intake led to a significant rise in serum IGF-1 and a reduction in CK and myostatin levels compared to baseline (p < 0.05) but there were no significant inter-group differences in IGF-1 and CK levels; There was also a significant reduction in myostatin level in LC group (p < 0/05). Myogenin levels decreased in both LC and placebo groups but the decrease in the placebo group was significant (p = 0/008); it means LC prevent the myogenin decreasing trend in the LC group compared to placebo. In conclusion, LC supplementation beneficially changes the level of IGF-1 and myostatin and improves muscle metabolism and regeneration in PV patients.


Assuntos
Carnitina , Pênfigo , Humanos , Adulto , Pessoa de Meia-Idade , Idoso , Carnitina/uso terapêutico , Glucocorticoides/efeitos adversos , Pênfigo/tratamento farmacológico , Fator de Crescimento Insulin-Like I , Miogenina , Miostatina , Atrofia Muscular/tratamento farmacológico , Músculos , Método Duplo-Cego , Suplementos Nutricionais
18.
Int J Mol Sci ; 24(7)2023 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-37047747

RESUMO

Myogenic differentiation is a complex biological process that is regulated by multiple factors, among which long noncoding RNAs (lncRNAs) play an essential role. However, in-depth studies on the regulatory mechanisms of long noncoding RNAs (lncRNAs) in myogenic differentiation are limited. In this study, we characterized the role of the novel lncRNA TCONS_00323213, which is upregulated during porcine skeletal muscle satellite cell (PSC) differentiation in myogenesis. We found that TCONS_00323213 affected the proliferation and differentiation of PSC in vitro. We performed quantitative polymerase chain reaction (qPCR), 5-ethynyl-20-deoxyuridine (EdU), western blotting, immunofluorescence staining, pull-down assays, and cleavage under targets and tagmentation (CUT and Tag) assays to clarify the effects and action mechanisms of TCONS_00323213. LncRNA TCONS_00323213 inhibited myoblast proliferation based on analyses of cell survival rates during PSC proliferation. Functional analyses revealed that TCONS_00323213 promotes cell differentiation and enhances myogenin (MyoG), myosin heavy chain (MyHC), and myocyte enhancer factor 2 (MEF2C) during myoblast differentiation. As determined by pull-down and RNA immunoprecipitation (RIP) assays, the lncRNA TCONS_00323213 interacted with PBX/Knotted Homeobox 2 (PKNOX2). CUT and Tag assays showed that PKNOX2 was significantly enriched on the MyoG promoter after lncRNA TCONS_00323213 knockdown. Our findings demonstrate that the interaction between lncRNA TCONS_00323213 and PKNOX2 relieves the inhibitory effect of PKNOX2 on the MyoG promoter, increases its expression, and promotes PSC differentiation. This novel role of lncRNA TCONS_00323213 sheds light on the molecular mechanisms by which lncRNAs regulate porcine myogenesis.


Assuntos
Desenvolvimento Muscular , RNA Longo não Codificante , Células Satélites de Músculo Esquelético , RNA Longo não Codificante/genética , RNA Longo não Codificante/fisiologia , Desenvolvimento Muscular/genética , Diferenciação Celular/genética , Células Satélites de Músculo Esquelético/citologia , Células Satélites de Músculo Esquelético/fisiologia , Animais , Suínos , Miogenina/genética , Miogenina/metabolismo , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Fatores de Transcrição MEF2/genética , Fatores de Transcrição MEF2/metabolismo , Técnicas de Silenciamento de Genes
19.
Mol Metab ; 71: 101704, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-36907509

RESUMO

OBJECTIVE: Skeletal muscle regeneration is markedly impaired during aging. How adult muscle stem cells contribute to this decrease in regenerative capacity is incompletely understood. We investigated mechanisms of age-related changes in myogenic progenitor cells using the tissue-specific microRNA 501. METHODS: Young and old C57Bl/6 mice were used (3 months or 24 months of age, respectively) with or without global or tissue-specific genetic deletion of miR-501. Muscle regeneration was induced using intramuscular cardiotoxin injection or treadmill exercise and analysed using single cell and bulk RNA sequencing, qRT-PCR and immunofluorescence. Muscle fiber damage was assessed with Evan`s blue dye (EBD). In vitro analysis was performed in primary muscle cells obtained from mice and humans. RESULTS: Single cell sequencing revealed myogenic progenitor cells in miR-501 knockout mice at day 6 after muscle injury that are characterized by high levels of myogenin and CD74. In control mice these cells were less in number and already downregulated after day 3 of muscle injury. Muscle from knockout mice had reduced myofiber size and reduced myofiber resilience to injury and exercise. miR-501 elicits this effect by regulating sarcomeric gene expression through its target gene estrogen-related receptor gamma (Esrrg). Importantly, in aged skeletal muscle where miR-501 was significantly downregulated and its target Esrrg significantly upregulated, the number of myog+/CD74+ cells during regeneration was upregulated to similar levels as observed in 501 knockout mice. Moreover, myog+/CD74+-aged skeletal muscle exhibited a similar decrease in the size of newly formed myofibers and increased number of necrotic myofibers after injury as observed in mice lacking miR-501. CONCLUSIONS: miR-501 and Esrrg are regulated in muscle with decreased regenerative capacity and loss of miR-501 is permissive to the appearance of CD74+ myogenic progenitors. Our data uncover a novel link between the metabolic transcription factor Esrrg and sarcomere formation and demonstrate that stem cell heterogeneity in skeletal muscle during aging is under miRNA control. Targeting Esrrg or myog+/CD74+ progenitor cells might improve fiber size and myofiber resilience to exercise in aged skeletal muscle.


Assuntos
MicroRNAs , Regeneração , Adulto , Idoso , Animais , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/genética , MicroRNAs/metabolismo , Músculo Esquelético/metabolismo , Miogenina/genética , Miogenina/metabolismo , Miogenina/farmacologia , Regeneração/genética , Células-Tronco/metabolismo
20.
Int J Legal Med ; 137(3): 875-886, 2023 May.
Artigo em Inglês | MEDLINE | ID: mdl-36797435

RESUMO

From the perspective of forensic wound age estimation, experiments related to skeletal muscle regeneration after injury have rarely been reported. Here, we examined the time-dependent expression patterns of multiple biomarkers associated with satellite cell fate, including the transcription factor paired box 7 (Pax7), myoblast determination protein (MyoD), myogenin, and insulin-like growth factor (IGF-1), using immunohistochemistry, western blotting, and quantitative real-time PCR in contused skeletal muscle. An animal model of skeletal muscle contusion was established in 30 Sprague-Dawley male rats, and another five rats were employed as non-contused controls. Morphometrically, the data obtained from the numbers of Pax7 + , MyoD + , and myogenin + cells were highly correlated with the wound age. Pax7, MyoD, myogenin, and IGF-1 expression patterns were upregulated after injury at both the mRNA and protein levels. Pax7, MyoD, and myogenin protein expression levels confirmed the results of the morphometrical analysis. Additionally, the relative quantity of IGF-1 protein > 0.92 suggested a wound age of 3 to 7 days. The relative quantity of Pax7 mRNA > 2.44 also suggested a wound age of 3 to 7 days. Relative quantities of Myod1, Myog, and Igf1 mRNA expression > 2.78, > 7.80, or > 3.13, respectively, indicated a wound age of approximately 3 days. In conclusion, the expression levels of Pax7, MyoD, myogenin, and IGF-1 were upregulated in a time-dependent manner during skeletal muscle wound healing, suggesting the potential for using them as candidate biomarkers for wound age estimation in skeletal muscle.


Assuntos
Contusões , Células Satélites de Músculo Esquelético , Ratos , Animais , Masculino , Miogenina/genética , Miogenina/metabolismo , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Ratos Sprague-Dawley , Músculo Esquelético/metabolismo , Contusões/metabolismo , Biomarcadores/metabolismo , RNA Mensageiro/metabolismo , Células Satélites de Músculo Esquelético/metabolismo , Proteína MyoD/genética , Proteína MyoD/metabolismo
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