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Redox Biology Jul 2024Cysteine, the rate-controlling amino acid in cellular glutathione synthesis is imported as cystine, by the cystine/glutamate antiporter, xCT, and subsequently reduced to...
Cysteine, the rate-controlling amino acid in cellular glutathione synthesis is imported as cystine, by the cystine/glutamate antiporter, xCT, and subsequently reduced to cysteine. As glutathione redox is important in muscle regeneration in aging, we hypothesized that xCT exerts upstream control over skeletal muscle glutathione redox, metabolism and regeneration. Bioinformatic analyses of publicly available datasets revealed that expression levels of xCT and GSH-related genes are inversely correlated with myogenic differentiation genes. Muscle satellite cells (MuSCs) isolated from Slc7a11 mice, which harbour a mutation in the Slc7a11 gene encoding xCT, required media supplementation with 2-mercaptoethanol to support cell proliferation but not myotube differentiation, despite persistently lower GSH. Slc7a11 primary myotubes were larger compared to WT myotubes, and also exhibited higher glucose uptake and cellular oxidative capacities. Immunostaining of myogenic markers (Pax7, MyoD, and myogenin) in cardiotoxin-damaged tibialis anterior muscle fibres revealed greater MuSC activation and commitment to differentiation in Slc7a11 muscle compared to WT mice, culminating in larger myofiber cross-sectional areas at 21 days post-injury. Slc7a11 mice subjected to a 5-week exercise training protocol demonstrated enhanced insulin tolerance compared to WT mice, but blunted muscle mitochondrial biogenesis and respiration in response to exercise training. Our results demonstrate that the absence of xCT inhibits cell proliferation but promotes myotube differentiation by regulating cellular metabolism and glutathione redox. Altogether, these results support the notion that myogenesis is a redox-regulated process and may help inform novel therapeutic approaches for muscle wasting and dysfunction in aging and disease.
Topics: Animals; Mice; Oxidation-Reduction; Cell Differentiation; Glutathione; Muscle, Skeletal; Amino Acid Transport System y+; Energy Metabolism; Muscle Development; Satellite Cells, Skeletal Muscle; Muscle Fibers, Skeletal; Cystine
PubMed: 38815331
DOI: 10.1016/j.redox.2024.103213 -
PLoS Neglected Tropical Diseases May 2024Photobiomodulation has exhibited promise in mitigating the local effects induced by Bothrops snakebite envenoming; however, the mechanisms underlying this protection are...
BACKGROUND
Photobiomodulation has exhibited promise in mitigating the local effects induced by Bothrops snakebite envenoming; however, the mechanisms underlying this protection are not yet fully understood. Herein, the effectiveness of photobiomodulation effects on regenerative response of C2C12 myoblast cells following exposure to Bothrops jararacussu venom (BjsuV), as well as the mechanisms involved was investigated.
METHODOLOGY/PRINCIPAL FINDINGS
C2C12 myoblast cells were exposed to BjsuV (12.5 μg/mL) and irradiated once for 10 seconds with laser light of 660 nm (14.08 mW; 0.04 cm2; 352 mW/cm2) or 780 nm (17.6 mW; 0.04 cm2; 440 mW/ cm2) to provide energy densities of 3.52 and 4.4 J/cm2, and total energies of 0.1408 and 0.176 J, respectively. Cell migration was assessed through a wound-healing assay. The expression of MAPK p38-α, NF-Кβ, Myf5, Pax-7, MyoD, and myogenin proteins were assessed by western blotting analysis. In addition, interleukin IL1-β, IL-6, TNF-alfa and IL-10 levels were measured in the supernatant by ELISA. The PBM applied to C2C12 cells exposed to BjsuV promoted cell migration, increase the expression of myogenic factors (Pax7, MyF5, MyoD and myogenin), reduced the levels of proinflammatory cytokines, IL1-β, IL-6, TNF-alfa, and increased the levels of anti-inflammatory cytokine IL-10. In addition, PBM downregulates the expression of NF-kB, and had no effect on p38 MAKP.
CONCLUSION/SIGNIFICANCE
These data demonstrated that protection of the muscle cell by PBM seems to be related to the increase of myogenic factors as well as the modulation of inflammatory mediators. PBM therapy may offer a new therapeutic strategy to address the local effects of snakebite envenoming by promoting muscle regeneration and reducing the inflammatory process.
Topics: Animals; Bothrops; Myoblasts; Mice; Low-Level Light Therapy; Cytokines; Cell Line; Crotalid Venoms; Myogenin; PAX7 Transcription Factor; NF-kappa B; MyoD Protein; Cell Movement; Myogenic Regulatory Factor 5; p38 Mitogen-Activated Protein Kinases; Snake Bites; Venomous Snakes
PubMed: 38814992
DOI: 10.1371/journal.pntd.0012227 -
Journal of Biomedical Research May 2024Macrophages mediated inflammatory response is crucial for the recovery of skeletal muscle following ischemia. Thus, it's necessary to exploit macrophages based...
Macrophages mediated inflammatory response is crucial for the recovery of skeletal muscle following ischemia. Thus, it's necessary to exploit macrophages based therapeutic targets for ischemic disease. Here, we found mRNA level of SR-A1 was elevated in patients with critical limb ischemia by analysis of gene expression omnibus (GEO) database. Then we investigated the role and the underlined mechanisms of macrophage SR-A1 in a mouse HLI model. Compared with the SR-A1 mice, the Lyz /SR-A1 (SR-A1 ) mice showed significantly lower laser doppler blood flow in the ischemic limb at day 7 after HLI. Consistently, histological analysis exhibited that ischemic limb of SR-A1 mice displayed more sever and sustained necrotic morphology, inflammation and fibrosis, decreased vessel density and regeneration rate, compared with which of control SR-A1 mice. Furthermore, restoration of wild-type myeloid cells to SR-A1 knock-out mice effectively relieved the doppler perfusion in the ischemic limb and restrained skeletal muscle damage 7 days post HLI. In line with findings, when co-cultivating macrophages with the mouse myoblast line C2C12, SR-A1 bone marrow macrophage significantly inhibited myoblast differentiation . Mechanically, SR-A1 enhanced skeletal muscle regeneration response to HLI by inhibiting the oncostatin M (OSM) production via suppressed NF-κB signaling activation. These results indicates that SR-A1 is a promising candidate molecule to improve tissue repair and regeneration in peripheral ischemic arterial disease.
PubMed: 38807379
DOI: 10.7555/JBR.38.20240117 -
Proceedings of the National Academy of... Jun 2024Myogenesis is a multistep process that requires a spatiotemporal regulation of cell events resulting finally in myoblast fusion into multinucleated myotubes. Most major...
Myogenesis is a multistep process that requires a spatiotemporal regulation of cell events resulting finally in myoblast fusion into multinucleated myotubes. Most major insights into the mechanisms underlying fusion seem to be conserved from insects to mammals and include the formation of podosome-like protrusions (PLPs) that exert a driving force toward the founder cell. However, the machinery that governs this process remains poorly understood. In this study, we demonstrate that MTM1 is the main enzyme responsible for the production of phosphatidylinositol 5-phosphate, which in turn fuels PI5P 4-kinase α to produce a minor and functional pool of phosphatidylinositol 4,5-bisphosphate that concentrates in PLPs containing the scaffolding protein Tks5, Dynamin-2, and the fusogenic protein Myomaker. Collectively, our data reveal a functional crosstalk between a PI-phosphatase and a PI-kinase in the regulation of PLP formation.
Topics: Animals; Phosphatidylinositol Phosphates; Mice; Cell Fusion; Myoblasts; Podosomes; Protein Tyrosine Phosphatases, Non-Receptor; Muscle Development
PubMed: 38805272
DOI: 10.1073/pnas.2217971121 -
Biotech (Basel (Switzerland)) Apr 2024A myogenetic oligodeoxynucleotide (myoDN), iSN04 (5'-AGA TTA GGG TGA GGG TGA-3'), is a single-stranded 18-base telomeric DNA that serves as an anti-nucleolin aptamer and...
A myogenetic oligodeoxynucleotide (myoDN), iSN04 (5'-AGA TTA GGG TGA GGG TGA-3'), is a single-stranded 18-base telomeric DNA that serves as an anti-nucleolin aptamer and induces myogenic differentiation, which is expected to be a nucleic acid drug for the prevention of disease-associated muscle wasting. To improve the drug efficacy and synthesis cost of myoDN, shortening the sequence while maintaining its structure-based function is a major challenge. Here, we report the novel 12-base non-telomeric myoDN, iMyo01 (5'-TTG GGT GGG GAA-3'), which has comparable myogenic activity to iSN04. iMyo01 as well as iSN04 promoted myotube formation of primary-cultured human myoblasts with upregulation of myogenic gene expression. Both iMyo01 and iSN04 interacted with nucleolin, but iMyo01 did not bind to berberine, the isoquinoline alkaloid that stabilizes iSN04. Nuclear magnetic resonance revealed that iMyo01 forms a G-quadruplex structure despite its short sequence. Native polyacrylamide gel electrophoresis and a computational molecular dynamics simulation indicated that iMyo01 forms a homodimer to generate a G-quadruplex. These results provide new insights into the aptamer truncation technology that preserves aptamer conformation and bioactivity for the development of efficient nucleic acid drugs.
PubMed: 38804293
DOI: 10.3390/biotech13020011 -
Scientific Reports May 2024Telocytes are a unique interstitial cell type that functions in adulthood and embryogenesis. They have characteristic immunohistochemical phenotypes while acquiring...
Telocytes are a unique interstitial cell type that functions in adulthood and embryogenesis. They have characteristic immunohistochemical phenotypes while acquiring different immunohistochemical properties related to the organ microenvironment. The present study aims to investigate the immunohistochemical features of embryonic telocytes during myogenesis and describe their morphology using light microscopy and TEM. Telocytes represent a major cellular constituent in the interstitial elements. They had distinguished telopodes and podoms and formed a 3D interstitial network in the developing muscles. They formed heterocellular contact with myoblasts and nascent myotubes. Telocytes also had distinctive secretory activity. Telocytes identified by CD34. They also express CD68 and MMP-9 to facilitate the development of new tissues. Expression of CD21 by telocytes may reveal their function in immune defense. They also express VEGF, which regulates angiogenesis. In conclusion, the distribution and immunological properties of telocytes in the myogenic tissue indicate that telocytes provide biological and structural support in the development of the myogenic tissue architecture and organization.
Topics: Telocytes; Animals; Muscle Development; Immunohistochemistry; Mice; Antigens, CD; Antigens, CD34; Cellular Microenvironment; Matrix Metalloproteinase 9; Antigens, Differentiation, Myelomonocytic; Vascular Endothelial Growth Factor A; Myoblasts
PubMed: 38802438
DOI: 10.1038/s41598-024-62103-1 -
Journal of Diabetes Research 2024Islet transplantation (ITx) is an established and safe alternative to pancreas transplantation for type 1 diabetes mellitus (T1DM) patients. However, most ITx recipients...
Islet transplantation (ITx) is an established and safe alternative to pancreas transplantation for type 1 diabetes mellitus (T1DM) patients. However, most ITx recipients lose insulin independence by 3 years after ITx due to early graft loss, such that multiple donors are required to achieve insulin independence. In the present study, we investigated whether skeletal myoblast cells could be beneficial for promoting angiogenesis and maintaining the differentiated phenotypes of islets. In vitro experiments showed that the myoblast cells secreted angiogenesis-related cytokines (vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and stromal-derived factor-1 (SDF-1)), contributed to maintenance of differentiated islet phenotypes, and enhanced islet cell insulin secretion capacity. To verify these findings in vivo, we transplanted islets alone or with myoblast cells under the kidney capsule of streptozotocin-induced diabetic mice. Compared with islets alone, the group bearing islets with myoblast cells had a significantly lower average blood glucose level. Histological examination revealed that transplants with islets plus myoblast cells were associated with a significantly larger insulin-positive area and significantly higher number of CD31-positive microvessels compared to islets alone. Furthermore, islets cotransplanted with myoblast cells showed JAK-STAT signaling activation. Our results suggest two possible mechanisms underlying enhancement of islet graft function with myoblast cells cotransplantation: "indirect effects" mediated by angiogenesis and "direct effects" of myoblast cells on islets via the JAK-STAT cascade. Overall, these findings suggest that skeletal myoblast cells enhance the function of transplanted islets, implying clinical potential for a novel ITx procedure involving myoblast cells for patients with diabetes.
Topics: Animals; Islets of Langerhans Transplantation; Diabetes Mellitus, Experimental; Myoblasts, Skeletal; Mice; Male; Insulin; Neovascularization, Physiologic; Hepatocyte Growth Factor; Mice, Inbred C57BL; Vascular Endothelial Growth Factor A; Islets of Langerhans; Chemokine CXCL12; Blood Glucose; Diabetes Mellitus, Type 1; Signal Transduction; Insulin Secretion; Cell Differentiation
PubMed: 38800586
DOI: 10.1155/2024/5574968 -
European Journal of Cell Biology Jun 2024The RAS-MAPK-pathway is aberrantly regulated in cancer and developmental diseases called RASopathies. While typically the impact of Ras on the proliferation of various...
The RAS-MAPK-pathway is aberrantly regulated in cancer and developmental diseases called RASopathies. While typically the impact of Ras on the proliferation of various cancer cell lines is assessed, it is poorly established how Ras affects cellular differentiation. Here we implement the C2C12 myoblast cell line to systematically study the effect of Ras mutants and Ras-pathway drugs on differentiation. We first provide evidence that a minor pool of Pax7+ progenitors replenishes a major pool of transit amplifying cells that are ready to differentiate. Our data indicate that Ras isoforms have distinct roles in the differentiating culture, where K-Ras depletion increases and H-Ras depletion decreases terminal differentiation. This assay could therefore provide significant new insights into Ras biology and Ras-driven diseases. In line with this, we found that all oncogenic Ras mutants block terminal differentiation of transit amplifying cells. By contrast, RASopathy associated K-Ras variants were less able to block differentiation. Profiling of eight targeted Ras-pathway drugs on seven oncogenic Ras mutants revealed their allele-specific activities and distinct abilities to restore normal differentiation as compared to triggering cell death. In particular, the MEK-inhibitor trametinib could broadly restore differentiation, while the mTOR-inhibitor rapamycin broadly suppressed differentiation. We expect that this quantitative assessment of the impact of Ras-pathway mutants and drugs on cellular differentiation has great potential to complement cancer cell proliferation data.
Topics: Cell Differentiation; Mutation; Animals; Mice; Protein Isoforms; ras Proteins; Cell Line; Humans
PubMed: 38795504
DOI: 10.1016/j.ejcb.2024.151425 -
Biomolecules May 2024Chimerism-based strategies represent a pioneering concept which has led to groundbreaking advancements in regenerative medicine and transplantation. This new approach... (Review)
Review
Chimerism-based strategies represent a pioneering concept which has led to groundbreaking advancements in regenerative medicine and transplantation. This new approach offers therapeutic potential for the treatment of various diseases, including inherited disorders. The ongoing studies on chimeric cells prompted the development of Dystrophin-Expressing Chimeric (DEC) cells which were introduced as a potential therapy for Duchenne Muscular Dystrophy (DMD). DMD is a genetic condition that leads to premature death in adolescent boys and remains incurable with current methods. DEC therapy, created via the fusion of human myoblasts derived from normal and DMD-affected donors, has proven to be safe and efficacious when tested in experimental models of DMD after systemic-intraosseous administration. These studies confirmed increased dystrophin expression, which correlated with functional and morphological improvements in DMD-affected muscles, including cardiac, respiratory, and skeletal muscles. Furthermore, the application of DEC therapy in a clinical study confirmed its long-term safety and efficacy in DMD patients. This review summarizes the development of chimeric cell technology tested in preclinical models and clinical studies, highlighting the potential of DEC therapy in muscle regeneration and repair, and introduces chimeric cell-based therapies as a promising, novel approach for muscle regeneration and the treatment of DMD and other neuromuscular disorders.
Topics: Muscular Dystrophy, Duchenne; Humans; Regeneration; Animals; Cell- and Tissue-Based Therapy; Muscle, Skeletal; Dystrophin; Myoblasts
PubMed: 38785982
DOI: 10.3390/biom14050575 -
Molecular Medicine Reports Jul 2024Promotion of myoblast differentiation by activating mitochondrial biogenesis and protein synthesis signaling pathways provides a potential alternative strategy to...
Promotion of myoblast differentiation by activating mitochondrial biogenesis and protein synthesis signaling pathways provides a potential alternative strategy to balance energy and overcome muscle loss and muscle disorders. (Lour.) Baill. extract (SCE) has been used extensively as a traditional herbal medicine and has several physiological activities, including anti‑asthmatic, anti‑oxidant, anti‑inflammatory, anti‑atopic, anticancer and hepatoprotective properties. However, the effects and mechanisms of action of SCE on muscle differentiation have not yet been clarified. In the present study, it was investigated whether SCE affects skeletal muscle cell differentiation through the regulation of mitochondrial biogenesis and protein synthesis in murine C2C12 myoblasts. The XTT colorimetric assay was used to determine cell viability, and myosin heavy chain (MyHC) levels were determined using immunocytochemistry. SCE was applied to C2C12 myotube at different concentrations (1, 5, or 10 ng/ml) and times (1,3, or 5 days). Reverse transcription‑quantitative PCR and western blotting were used to analyze the mRNA and protein expression change of factors related to differentiation, mitochondrial biogenesis and protein synthesis. Treatment of C2C12 cells with SCE at 1,5, and 10 ng/ml did not affect cell viability. SCE promoted C2C12 myotube formation and significantly increased MyHC expression in a concentration‑ and time‑dependent manner. SCE significantly increased the mRNA and protein expression of muscle differentiation‑specific markers, such as MyHC, myogenic differentiation 1, myogenin, Myogenic Factor 5, and β‑catenin, mitochondrial biosynthesis‑related factors, such as peroxisome proliferator‑activated receptor‑gamma coactivator‑1α, nuclear respirator factor‑1, AMP‑activated protein kinase phosphorylation, and histone deacetylase 5 and AKT/mTOR signaling factors related to protein synthesis. SCE may prevent skeletal muscle dysfunction by enhancing myoblast differentiation through the promotion of mitochondrial biogenesis and protein synthesis.
Topics: Animals; Mice; Cell Differentiation; Signal Transduction; TOR Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Plant Extracts; Organelle Biogenesis; Cell Line; Saururaceae; Cell Survival; Myoblasts; Mitochondria; Muscle Development; Muscle Fibers, Skeletal; Myosin Heavy Chains; Muscle, Skeletal
PubMed: 38785149
DOI: 10.3892/mmr.2024.13250