-
Frontiers in Bioscience (Landmark... Feb 2024Myogenin is well known as a crucial transcription factor in skeletal muscle development, yet its other biological functions remain unexplored. Previous research showed...
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 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 . 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 as being up-regulated by myogenin. Western blotting showed that overexpression of myogenin increased the expression of and decreased the phosphorylation level of p38 MAPK. A dual-luciferase reporter assay proved that myogenin bound directly to the promoter region of and led to strong relative luciferase activity. Direct expression of and significantly reduced the rates of apoptosis and necrosis in cells treated with H2O2. Knockdown of 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 and inactivating the p38 MAPK pathway.
Topics: Humans; Reactive Oxygen Species; Hydrogen Peroxide; Myogenin; Induced Pluripotent Stem Cells; Apoptosis; Oxidative Stress; p38 Mitogen-Activated Protein Kinases; Luciferases
PubMed: 38420814
DOI: 10.31083/j.fbl2902049 -
Journal of Translational Medicine Feb 2017Sarcopenia, osteoporosis and osteoarthritis are the most frequent musculoskeletal disorders affecting older people. The main aim of this study was to test the hypothesis...
BACKGROUND
Sarcopenia, osteoporosis and osteoarthritis are the most frequent musculoskeletal disorders affecting older people. The main aim of this study was to test the hypothesis that the balance between BMPs and myostatin pathways regulates the age-related muscle degeneration in OP and OA patients. To this end, we investigated the relationship among the expression of BMP-2/4-7, myostatin and phosphorylated Smads1-5-8 and the muscle quality, evaluated in term of fibers atrophy and satellite cells activity.
METHODS
In this retrospective study, we collected 123 biopsies of vastus lateralis: 48 biopsies from patients who underwent hip arthroplasty for subcapital fractures of the femur (OP), 55 biopsies from patients who underwent hip arthroplasty for osteoarthritis (OA) and 20 biopsies from patients who underwent hip arthroplasty for high-energy hip fractures (CTRL). Muscle biopsies were fixed in 4% paraformaldehyde and paraffin embedded. Serial sections were used for morphometrical and immunohistochemical analysis (BMP/2/4-7, myostatin, Smads1-5-8, Pax7 and myogenin). In addition, 1 mm of muscle tissue of each patient was embedded in epon for ultrastructural study.
RESULTS
Morphometric data indicated an increase of the number of atrophic fibers in OP patients compare to OA. In line with these data, we found an high regenerative potential in muscle tissues of OA patients due to the significant amount of both Pax7 and myogenin positive satellite cells detected in OA group. In addition, our data showed the decrease of BMP2/4 and -7 expression in OP patients compared to both OA group and CTRL. Conversely, OP patients were characterized by high levels of myostatin expression. A different expression profile was also found for phosphorylated Smad1-5-8 between OP and OA patients. In particular, OP patients showed a low number of positive phosphorylated Smad1-5-8 nuclei.
CONCLUSION
The identification of molecular pathways involved in the pathogenesis of sarcopenia open new prospective for the development of drugs able to prevent/treat the muscle impairment that occur in elderly. Results here reported, highlighting the role of BMPs and myostatin pathways in physio-pathogenesis of human sarcopenia, allow us to propose human recombinant BMP-2/7 and anti-myostatin antibodies as a possible therapeutic option for the sarcopenia.
Topics: Bone Morphogenetic Proteins; Case-Control Studies; Female; Humans; Immunohistochemistry; Male; Muscle Fibers, Skeletal; Myostatin; Phosphorylation; Sarcopenia; Satellite Cells, Skeletal Muscle; Smad Proteins
PubMed: 28202082
DOI: 10.1186/s12967-017-1143-6 -
Intractable & Rare Diseases Research Aug 2023Interferon-induced transmembrane proteins (IFITMs 1, 2, and 3) play a critical role in preventing pathogen infection in vertebrates. They are also involved in the...
Interferon-induced transmembrane proteins (IFITMs 1, 2, and 3) play a critical role in preventing pathogen infection in vertebrates. They are also involved in the occurrence and prognosis of cancer. Myogenesis is a complex process regulated by several factors. This study disclosed that Ifitm1-3 were upregulated in the process of myogenic differentiation of C2C12 myoblasts on days 3, 5, and 7. This positively correlated with the expression of differentiation factors MyoD, myogenin, Mrf5, and desmin. Furthermore, knockdown of Ifitm1-3 by their individual siRNAs inhibited myogenesis of C2C12 myoblasts, with relative downregulation of MyoD, myogenin, Mrf5, and desmin. Subsequently, myotube formation and fusion percentage decreased. Co-immunoprecipitation combined with LC-MS/MS analysis uncovered the interaction proteins of IFITM1 and IFITM3 in C2C12 myoblasts. A total of 84 overlapped interaction proteins of IFITM1 and IFITM3 were identified, and one of the clusters was engaged in cytoskeletal and sarcomere proteins, including desmin, myosin, actin, vimentin, nestin, ankycorbin, and nucleolin. Hence, we hypothesize that these interacting proteins may function as scaffolds for IFITM1-3, possibly through the interaction protein desmin to initiate further interaction with other proteins to participate in myogenesis; however, the molecular mechanisms remain unclear. Our study may contribute to the development of novel therapeutics for myopathic diseases.
PubMed: 37662621
DOI: 10.5582/irdr.2023.01050 -
Cells Dec 2022During aging, muscle cell apoptosis increases and myogenesis gradually declines. The impaired myogenic and survival potential of the aged skeletal muscle can be...
BACKGROUND
During aging, muscle cell apoptosis increases and myogenesis gradually declines. The impaired myogenic and survival potential of the aged skeletal muscle can be ameliorated by its mechanical loading. However, the molecular responses of aged muscle cells to mechanical loading remain unclear. This study examined the effect of mechanical loading of aged, proliferating, and differentiated myoblasts on the gene expression and signaling responses associated with their myogenic lineage progression and survival.
METHODS
Control and aged C2C12 cells were cultured on elastic membranes and underwent passive stretching for 12 h at a low frequency (0.25 Hz) and different elongations, varying the strain on days 0 and 10 of myoblast differentiation. Activation of ERK1/2 and Akt, and the expression of focal adhesion kinase (FAK) and key myogenic regulatory factors (MRFs), MyoD and Myogenin, were determined by immunoblotting of the cell lysates derived from stretched and non-stretched myoblasts. Changes in the expression levels of the MRFs, muscle growth, atrophy, and pro-apoptotic factors in response to mechanical loading of the aged and control cells were quantified by real-time qRT-PCR.
RESULTS
Mechanical stretching applied on myoblasts resulted in the upregulation of FAK both in proliferating (day 0) and differentiated (day 10) cells, as well as in increased phosphorylation of ERK1/2 in both control and aged cells. Moreover, Akt activation and the expression of early differentiation factor MyoD increased significantly after stretching only in the control myoblasts, while the late differentiation factor Myogenin was upregulated in both the control and aged myoblasts. At the transcriptional level, mechanical loading of the proliferating myoblasts led to an increased expression of IGF-1 isoforms and MRFs, and to downregulation of muscle atrophy factors mainly in control cells, as well as in the upregulation of pro-apoptotic factors both in control and aged cells. In differentiated cells, mechanical loading resulted in an increased expression of the IGF-1Ea isoform and Myogenin, and in the downregulation of atrophy and pro-apoptotic factors in both the control and aged cells.
CONCLUSIONS
This study revealed a diminished beneficial effect of mechanical loading on the myogenic and survival ability of the senescent muscle cells compared with the controls, with a low strain (2%) loading being most effective in upregulating myogenic/anabolic factors and downregulating atrophy and pro-apoptotic genes mainly in the aged myotubes.
Topics: Myogenin; Proto-Oncogene Proteins c-akt; Myogenic Regulatory Factors; Myoblasts; Muscle Development
PubMed: 36552743
DOI: 10.3390/cells11243979 -
Journal of Cachexia, Sarcopenia and... Aug 2020Cachexia, a multifactorial syndrome affecting more than 50% of patients with advanced cancer and responsible for ~20% of cancer-associated deaths, is still a poorly...
BACKGROUND
Cachexia, a multifactorial syndrome affecting more than 50% of patients with advanced cancer and responsible for ~20% of cancer-associated deaths, is still a poorly understood process without a standard cure available. Skeletal muscle atrophy caused by systemic inflammation is a major clinical feature of cachexia, leading to weight loss, dampening patients' quality of life, and reducing patients' response to anticancer therapy. RAGE (receptor for advanced glycation end-products) is a multiligand receptor of the immunoglobulin superfamily and a mediator of muscle regeneration, inflammation, and cancer.
METHODS
By using murine models consisting in the injection of colon 26 murine adenocarcinoma (C26-ADK) or Lewis lung carcinoma (LLC) cells in BALB/c and C57BL/6 or Ager (RAGE-null) mice, respectively, we investigated the involvement of RAGE signalling in the main features of cancer cachexia, including the inflammatory state. In vitro experiments were performed using myotubes derived from C2C12 myoblasts or primary myoblasts isolated from C57BL/6 wild type and Ager mice treated with the RAGE ligand, S100B (S100 calcium-binding protein B), TNF (tumor necrosis factor)α±IFN (interferon) γ, and tumour cell- or masses-conditioned media to analyse hallmarks of muscle atrophy. Finally, muscles of wild type and Ager mice were injected with TNFα/IFNγ or S100B in a tumour-free environment.
RESULTS
We demonstrate that RAGE is determinant to activate signalling pathways leading to muscle protein degradation in the presence of proinflammatory cytokines and/or tumour-derived cachexia-inducing factors. We identify the RAGE ligand, S100B, as a novel factor able to induce muscle atrophy per se via a p38 MAPK (p38 mitogen-activated protein kinase)/myogenin axis and STAT3 (signal transducer and activator of transcription 3)-dependent MyoD (myoblast determination protein 1) degradation. Lastly, we found that in cancer conditions, an increase in serum levels of tumour-derived S100B and HMGB1 (high mobility group box 1) occurs leading to chronic activation/overexpression of RAGE, which induces hallmarks of cancer cachexia (i.e. muscle wasting, systemic inflammation, and release of tumour-derived pro-cachectic factors). Absence of RAGE in mice translates into reduced serum levels of cachexia-inducing factors, delayed loss of muscle mass and strength, reduced tumour progression, and increased survival.
CONCLUSIONS
RAGE is a molecular determinant in inducing the hallmarks of cancer cachexia, and molecular targeting of RAGE might represent a therapeutic strategy to prevent or counteract the cachectic syndrome.
Topics: Animals; Cachexia; Humans; Mice; Neoplasms; Receptor for Advanced Glycation End Products
PubMed: 32159297
DOI: 10.1002/jcsm.12561 -
Biochemical and Biophysical Research... May 2020Although several studies have implied that a hypoxic environment may be a factor that influences muscle hypertrophy, scant attention has been paid to the effect of...
Although several studies have implied that a hypoxic environment may be a factor that influences muscle hypertrophy, scant attention has been paid to the effect of oxygen molecules on the morphological characteristics of muscle. The purpose of the present study was to examine the effect of semisevere (i.e., 5%) to moderate (i.e., 10% or 15%) hypoxic environments on the morphological characteristics of skeletal muscle and the associated mechanisms. C2C12 skeletal muscle cells were divided into various groups, namely, the normoxia group (20.9% O) and hypoxia groups (5% O, 10% O, and 15% O), and cell growth and the expression of associated proteins in the hypoxia groups were compared with those in the normoxia group. The myotube diameter and cell differentiation index were determined on day 6 by immunocytochemical analyses. The expression of proteins associated with muscle cell differentiation (MyoD and myogenin) and muscle hypertrophy (mTOR and p70s6K) were analyzed by Western blotting. We found that compared with normoxia, a 5% oxygen environment inhibited differentiation and caused muscle atrophy. However, compared with normoxia, a 10% oxygen environment promoted muscle differentiation, and 10% oxygen and 15% oxygen environments induced muscle hypertrophy. Compared with normoxia, a 10% oxygen environment promoted myogenin and the expression of mTOR, p70s6K, and the metabolic signal AMPK. We concluded that a hypoxic environment, if not too severe, may promote muscle differentiation and hypertrophy by increasing the expression of proteins associated with muscle cell differentiation and hypertrophy.
Topics: Animals; Cell Differentiation; Cell Hypoxia; Cell Line; Cell Proliferation; Mice; Muscle, Skeletal; MyoD Protein; Myoblasts, Skeletal; Myogenin; Oxygen; Ribosomal Protein S6 Kinases, 70-kDa; TOR Serine-Threonine Kinases
PubMed: 32173524
DOI: 10.1016/j.bbrc.2020.02.152 -
PloS One 2017The development of ectothermic embryos is strongly affected by incubation temperature, and thermal imprinting of body growth and muscle phenotype has been reported in...
The development of ectothermic embryos is strongly affected by incubation temperature, and thermal imprinting of body growth and muscle phenotype has been reported in various teleost fishes. The complex epigenetic regulation of muscle development in vertebrates involves DNA methylation of the myogenin promoter. Body growth is a heritable and highly variable trait among fish populations that allows for local adaptations, but also for selective breeding. Here we studied the epigenetic effects of embryonic temperature and genetic background on body growth, muscle cellularity and myogenin expression in farmed Atlantic salmon (Salmo salar). Eggs from salmon families with either high or low estimated breeding values for body growth, referred to as Fast and Slow genotypes, were incubated at 8°C or 4°C until the embryonic 'eyed-stage' followed by rearing at the production temperature of 8°C. Rearing temperature strongly affected the growth rates, and the 8°C fish were about twice as heavy as the 4°C fish in the order Fast8>Slow8>Fast4>Slow4 prior to seawater transfer. Fast8 was the largest fish also at harvest despite strong growth compensation in the low temperature groups. Larval myogenin expression was approximately 4-6 fold higher in the Fast8 group than in the other groups and was associated with relative low DNA methylation levels, but was positively correlated with the expression levels of the DNA methyltransferase genes dnmt1, dnmt3a and dnmt3b. Juvenile Fast8 fish displayed thicker white muscle fibres than Fast4 fish, while Slow 8 and Slow 4 showed no difference in muscle cellularity. The impact of genetic background on the thermal imprinting of body growth and muscle development in Atlantic salmon suggests that epigenetic variation might play a significant role in the local adaptation to fluctuating temperatures over short evolutionary time.
Topics: Animals; DNA Methylation; Epigenesis, Genetic; Gene Expression Regulation, Developmental; Muscle Development; Myogenin; Promoter Regions, Genetic; Salmo salar
PubMed: 28662198
DOI: 10.1371/journal.pone.0179918 -
Current Issues in Molecular Biology Oct 2021Profound skeletal muscle loss can lead to severe disability and cosmetic deformities. Mesenchymal stem cell (MSC)-derived exosomes have shown potential as an effective...
Profound skeletal muscle loss can lead to severe disability and cosmetic deformities. Mesenchymal stem cell (MSC)-derived exosomes have shown potential as an effective therapeutic tool for tissue regeneration. This study aimed to determine the regenerative capacity of MSC-derived exosomes for skeletal muscle regeneration. Exosomes were isolated from human adipose tissue-derived MSCs (AD-MSCs). The effects of MSC-derived exosomes on satellite cells were investigated using cell viability, relevant genes, and protein analyses. Moreover, NOD-SCID mice were used and randomly assigned to the healthy control ( = 4), muscle defect ( = 6), and muscle defect + exosome ( = 6) groups. Muscle defects were created using a biopsy punch on the quadriceps of the hind limb. Four weeks after the surgery, the quadriceps muscles were harvested, weighed, and histologically analyzed. MSC-derived exosome treatment increased the proliferation and expression of myocyte-related genes, and immunofluorescence analysis for myogenin revealed a similar trend. Histologically, MSC-derived exosome-treated mice showed relatively preserved shapes and sizes of the muscle bundles. Immunohistochemical staining revealed greater expression of myogenin and myoblast determination protein 1 in the MSC-derived exosome-treated group. These results indicate that exosomes extracted from AD-MSCs have the therapeutic potential for skeletal muscle regeneration.
Topics: Animals; Biomarkers; Exosomes; Fluorescent Antibody Technique; Humans; Immunohistochemistry; Mesenchymal Stem Cells; Mice; Muscle, Skeletal; Regeneration; Satellite Cells, Skeletal Muscle
PubMed: 34698065
DOI: 10.3390/cimb43030104 -
FASEB Journal : Official Publication of... Jan 2021Myogenesis includes sequential stages of progenitor cell proliferation, myogenic commitment and differentiation, myocyte fusion, and myotube maturation. Different stages...
Myogenesis includes sequential stages of progenitor cell proliferation, myogenic commitment and differentiation, myocyte fusion, and myotube maturation. Different stages of myogenesis are orchestrated and regulated by myogenic regulatory factors and various downstream cellular signaling. Here we identify phosphatase orphan 1 (Phospho1) as a new player in myogenesis. During activation, proliferation, and differentiation of quiescent satellite cells, the expression of Phospho1 gradually increases. Overexpression of Phospho1 inhibits myoblast proliferation but promotes their differentiation and fusion. Conversely, knockdown of Phospho1 accelerates myoblast proliferation but impairs myotube formation. Moreover, knockdown of Phospho1 decreases the OXPHO protein levels and mitochondria density, whereas overexpression of Phospho1 upregulates OXPHO protein levels and promotes mitochondrial oxygen consumption. Finally, we show that Phospho1 expression is controlled by myogenin, which binds to the promoter of Phospho1 to regulate its transcription. These results indicate a key role of Phospho1 in regulating myogenic differentiation and mitochondrial function.
Topics: Animals; Cell Differentiation; Cell Proliferation; Gene Expression Regulation, Enzymologic; Mice; Mitochondria, Muscle; Muscle Development; Myoblasts, Skeletal; Myogenin; Phosphoric Monoester Hydrolases
PubMed: 33140469
DOI: 10.1096/fj.202001672R -
Journal of Orthopaedic Research :... Feb 2020The recommended treatment varies depending on the severity of muscle injuries. The aim of this study was to evaluate the in vitro myoblast proliferation and the in vivo...
The recommended treatment varies depending on the severity of muscle injuries. The aim of this study was to evaluate the in vitro myoblast proliferation and the in vivo histologic and physiologic effects of hyperbaric oxygen treatment on muscle healing after contusion. Cells from the C2C12 myoblast cell line were exposed to 100% O for 25 min then to air for 5 min at 2.5 atmospheres absolute in a hyperbaric chamber for a total treatment duration of 90 min per 48 h at intervals of 2, 4, 6 and 8 days. Cell growth measurements and western blot analysis of myogenin and actin were performed. Then, 18 mice aged 8-10 weeks were used in the muscle contusion model. The histologic and physiologic effects and muscle regeneration after hyperbaric oxygen treatment were evaluated. The myoblast growth rate was significantly higher (p < 0.05) after hyperbaric oxygen treatment. Densitometric evaluation demonstrated a 39% (p < 0.05) and 25% (p < 0.05) increase in myogenin and actin protein levels, respectively, in the cells treated with 1 dose of hyperbaric oxygen. Similarly, the myogenin and actin protein levels increased for samples receiving multiple hyperbaric oxygen treatments when compared with the control. Physiologic evaluation of fast twitch and tetanus strength revealed a significant difference between the control group and the 14-day hyperbaric oxygen group. In conclusion, hyperbaric oxygen treatment increases the myoblast growth rate and myogenin and actin production. Better histologic and physiologic performance were found after hyperbaric oxygen treatment in animal contusion model. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:329-335, 2020.
Topics: Actins; Animals; Cell Line; Cell Proliferation; Contusions; Hyperbaric Oxygenation; Mice; Muscle Strength; Muscles; Myoblasts; Myogenin; Oxygen; Regeneration
PubMed: 31531986
DOI: 10.1002/jor.24478