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International Journal of Molecular... Apr 2024During 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...
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 , 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 , , and but strongly repressed that of , suggesting the maintenance of myogenic stemness amid repressed expression. The proliferation effect of GST-bFGF was conserved in C2C12 over-expressed with (C2C12-tTA-MyoD), implying its independence of the down-regulation of . In addition, the repressive effect of GST-bFGF on myogenic differentiation was almost totally rescued by the over-expression of . 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 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.
Topics: Muscle Development; Animals; Mice; MyoD Protein; Cell Proliferation; Fibroblast Growth Factor 2; Myoblasts; Cell Line; PAX7 Transcription Factor; PAX3 Transcription Factor; Myogenic Regulatory Factor 5; Cyclin D1; Satellite Cells, Skeletal Muscle; Cell Differentiation; Proto-Oncogene Proteins c-akt; Muscle, Skeletal
PubMed: 38673893
DOI: 10.3390/ijms25084308 -
Biomolecules Mar 2024Sarcopenia has a complex pathophysiology that encompasses metabolic dysregulation and muscle ultrastructural changes. Among the drivers of intracellular and... (Review)
Review
Sarcopenia has a complex pathophysiology that encompasses metabolic dysregulation and muscle ultrastructural changes. Among the drivers of intracellular and ultrastructural changes of muscle fibers in sarcopenia, mitochondria and their quality control pathways play relevant roles. Mononucleated muscle stem cells/satellite cells (MSCs) have been attributed a critical role in muscle repair after an injury. The involvement of mitochondria in supporting MSC-directed muscle repair is unclear. There is evidence that a reduction in mitochondrial biogenesis blunts muscle repair, thus indicating that the delivery of functional mitochondria to injured muscles can be harnessed to limit muscle fibrosis and enhance restoration of muscle function. Injection of autologous respiration-competent mitochondria from uninjured sites to damaged tissue has been shown to reduce infarct size and enhance cell survival in preclinical models of ischemia-reperfusion. Furthermore, the incorporation of donor mitochondria into MSCs enhances lung and cardiac tissue repair. This strategy has also been tested for regeneration purposes in traumatic muscle injuries. Indeed, the systemic delivery of mitochondria promotes muscle regeneration and restores muscle mass and function while reducing fibrosis during recovery after an injury. In this review, we discuss the contribution of altered MSC function to sarcopenia and illustrate the prospect of harnessing mitochondrial delivery and restoration of MSCs as a therapeutic strategy against age-related sarcopenia.
Topics: Sarcopenia; Humans; Satellite Cells, Skeletal Muscle; Animals; Signal Transduction; Mitochondria; Aging; Regeneration; Mitochondria, Muscle; Muscle, Skeletal
PubMed: 38672432
DOI: 10.3390/biom14040415 -
Skeletal Muscle Apr 2024Duchenne muscular dystrophy (DMD) is associated with impaired muscle regeneration, progressive muscle weakness, damage, and wasting. While the cause of DMD is an...
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.
Topics: Animals; Muscular Dystrophy, Duchenne; Connexins; Mice, Inbred mdx; Muscle, Skeletal; Male; Nerve Tissue Proteins; Humans; Mice; Myoblasts; Cell Line; Muscle Strength; Disease Models, Animal; Mice, Inbred C57BL; Mice, Knockout
PubMed: 38671506
DOI: 10.1186/s13395-024-00340-8 -
BMC Biotechnology Apr 2024Volumetric 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...
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.
Topics: Animals; Mice; Muscle Development; Muscle, Skeletal; Keratins; Cell Line; Hydrogels; Neovascularization, Physiologic; Tissue Engineering; Disease Models, Animal; Collagen; Myoblasts; Male; Tissue Scaffolds; Angiogenesis
PubMed: 38671404
DOI: 10.1186/s12896-024-00847-4 -
Scientific Reports Apr 2024Massive rotator cuff (RC) tendon tears are associated with progressive fibro-adipogenesis and muscle atrophy that altogether cause shoulder muscle wasting. Platelet...
Massive rotator cuff (RC) tendon tears are associated with progressive fibro-adipogenesis and muscle atrophy that altogether cause shoulder muscle wasting. Platelet derived growth factor β (PDGFRβ) lineage cells, that co-express PDGFRα have previously been shown to directly contribute to scar formation and fat accumulation in a mouse model of irreversible tendon and nerve transection (TTDN). Conversely, PDGFRβ lineage cells have also been shown to be myogenic in cultures and in other models of skeletal muscle injury. We therefore hypothesized that PDGFRβ demarcates two distinct RC residing subpopulations, fibro-adipogenic and myogenic, and aimed to elucidate the identity of the PDGFRβ myogenic precursors and evaluate their contribution, if any, to RC myo-regeneration. Lineage tracing revealed increasing contribution of PDGFRβ myo-progenitors to the formation of GFP myofibers, which were the most abundant myofiber type in regenerated muscle at 2 weeks post-TTDN. Muscle regeneration preceded muscle atrophy and both advanced from the lateral site of tendon transection to the farthest medial region. GFP/PDGFRβSca-1linCXCR4Integrin-β1 marked a novel subset of satellite cells with confirmed myogenic properties. Further studies are warranted to identify the existence of PDGFRβ satellite cells in human and other mouse muscles and to define their myo-regenerative potential following acute and chronic muscle injury.
Topics: Animals; Receptor, Platelet-Derived Growth Factor beta; Satellite Cells, Skeletal Muscle; Regeneration; Mice; Cell Lineage; Rotator Cuff Injuries; Muscle Development; Disease Models, Animal; Rotator Cuff; Male
PubMed: 38671006
DOI: 10.1038/s41598-024-58926-7 -
Poultry Science Jul 2024The Wooden Breast myopathy results in the necrosis and fibrosis of breast muscle fibers in fast-growing heavy weight meat-type broiler chickens. Myogenic satellite cells...
The Wooden Breast myopathy results in the necrosis and fibrosis of breast muscle fibers in fast-growing heavy weight meat-type broiler chickens. Myogenic satellite cells are required to repair and regenerate the damaged muscle fibers. Using Genome Wide Association, candidate genes affected with Wooden Breast have been previously reported. The effect of these genes on satellite cell proliferation, differentiation, and the synthesis of lipids by satellite cells is unknown. Satellite cells isolated from the pectoralis major muscle from commercial Ross 708 broilers and a Randombred chicken (RBch) line were used. Expression of calponin 1 (CNN1) and PHD and ring fingers domains 1 (PHRF1) were knocked down by silent interfering RNA to determine their effect on satellite cell-mediated proliferation, differentiation, and lipid accumulation. CNN1 and PHRF1 affected satellite cell activity and lipid accumulation in both lines. Proliferation was reduced in the Ross 708 and RBch lines by knocking down the expression of both genes, and differentiation was affected with a line and treatment interaction when gene expression was reduced at the beginning of proliferation. During differentiation lipid accumulation was decreased with knocking down the expression of CNN1 and PHRF1. Both CNN1 and PHRF1 have not been reported previously in skeletal muscle and further research is required to determine their effect on satellite cell-mediated growth and regeneration of the pectoralis major (breast) muscle.
Topics: Animals; Satellite Cells, Skeletal Muscle; Chickens; Avian Proteins; Pectoralis Muscles; Calcium-Binding Proteins; Microfilament Proteins; Calponins; Cell Proliferation; Cell Differentiation; Poultry Diseases; Gene Knockdown Techniques
PubMed: 38669818
DOI: 10.1016/j.psj.2024.103781 -
Cells Apr 2024Meat 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....
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.
Topics: Animals; Muscle Development; LIM Domain Proteins; Swine; Cell Proliferation; Cell Differentiation; Protein Isoforms; Muscle, Skeletal; Polymorphism, Single Nucleotide; Myoblasts; Promoter Regions, Genetic
PubMed: 38667334
DOI: 10.3390/cells13080720 -
Cells Apr 2024Muscle satellite cells (MuSCs) are crucial for muscle development and regeneration. The primary pig MuSCs (pMuSCs) is an ideal in vitro cell model for studying the pig's...
Muscle satellite cells (MuSCs) are crucial for muscle development and regeneration. The primary pig MuSCs (pMuSCs) is an ideal in vitro cell model for studying the pig's muscle development and differentiation. However, the long-term in vitro culture of pMuSCs results in the gradual loss of their stemness, thereby limiting their application. To address this conundrum and maintain the normal function of pMuSCs during in vitro passaging, we generated an immortalized pMuSCs (SV40 T-pMuSCs) by stably expressing SV40 T-antigen (SV40 T) using a lentiviral-based vector system. The SV40 T-pMuSCs can be stably sub-cultured for over 40 generations in vitro. An evaluation of SV40 T-pMuSCs was conducted through immunofluorescence staining, quantitative real-time PCR, EdU assay, and SA-β-gal activity. Their proliferation capacity was similar to that of primary pMuSCs at passage 1, and while their differentiation potential was slightly decreased. SiRNA-mediated interference of SV40 T-antigen expression restored the differentiation capability of SV40 T-pMuSCs. Taken together, our results provide a valuable tool for studying pig skeletal muscle development and differentiation.
Topics: Animals; Satellite Cells, Skeletal Muscle; Swine; Cell Differentiation; Antigens, Polyomavirus Transforming; Cell Proliferation; Muscle Development; Antigens, Viral, Tumor; Simian virus 40
PubMed: 38667318
DOI: 10.3390/cells13080703 -
Cells Apr 2024Skeletal muscle degeneration is responsible for major mobility complications, and this muscle type has little regenerative capacity. Several biomaterials have been...
Skeletal muscle degeneration is responsible for major mobility complications, and this muscle type has little regenerative capacity. Several biomaterials have been proposed to induce muscle regeneration and function restoration. Decellularized scaffolds present biological properties that allow efficient cell culture, providing a suitable microenvironment for artificial construct development and being an alternative for in vitro muscle culture. For translational purposes, biomaterials derived from large animals are an interesting and unexplored source for muscle scaffold production. Therefore, this study aimed to produce and characterize bovine muscle scaffolds to be applied to muscle cell 3D cultures. Bovine muscle fragments were immersed in decellularizing solutions for 7 days. Decellularization efficiency, structure, composition, and three-dimensionality were evaluated. Bovine fetal myoblasts were cultured on the scaffolds for 10 days to attest cytocompatibility. Decellularization was confirmed by DAPI staining and DNA quantification. Histological and immunohistochemical analysis attested to the preservation of main ECM components. SEM analysis demonstrated that the 3D structure was maintained. In addition, after 10 days, fetal myoblasts were able to adhere and proliferate on the scaffolds, attesting to their cytocompatibility. These data, even preliminary, infer that generated bovine muscular scaffolds were well structured, with preserved composition and allowed cell culture. This study demonstrated that biomaterials derived from bovine muscle could be used in tissue engineering.
Topics: Animals; Cattle; Tissue Scaffolds; Muscle, Skeletal; Tissue Engineering; Myoblasts; Biocompatible Materials; Decellularized Extracellular Matrix; Cells, Cultured; Cell Proliferation; Extracellular Matrix
PubMed: 38667303
DOI: 10.3390/cells13080688 -
ELife Apr 2024Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular disorder characterized by progressive weakness of almost all skeletal muscles, whereas extraocular muscles...
Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular disorder characterized by progressive weakness of almost all skeletal muscles, whereas extraocular muscles (EOMs) are comparatively spared. While hindlimb and diaphragm muscles of end-stage SOD1G93A (G93A) mice (a familial ALS mouse model) exhibit severe denervation and depletion of Pax7satellite cells (SCs), we found that the pool of SCs and the integrity of neuromuscular junctions (NMJs) are maintained in EOMs. In cell sorting profiles, SCs derived from hindlimb and diaphragm muscles of G93A mice exhibit denervation-related activation, whereas SCs from EOMs of G93A mice display spontaneous (non-denervation-related) activation, similar to SCs from wild-type mice. Specifically, cultured EOM SCs contain more abundant transcripts of axon guidance molecules, including , along with more sustainable renewability than the diaphragm and hindlimb counterparts under differentiation pressure. In neuromuscular co-culture assays, AAV-delivery of to G93A-hindlimb SC-derived myotubes enhances motor neuron axon extension and innervation, recapitulating the innervation capacity of EOM SC-derived myotubes. G93A mice fed with sodium butyrate (NaBu) supplementation exhibited less NMJ loss in hindlimb and diaphragm muscles. Additionally, SCs derived from G93A hindlimb and diaphragm muscles displayed elevated expression of and improved renewability following NaBu treatment in vitro. Thus, the NaBu-induced transcriptomic changes resembling the patterns of EOM SCs may contribute to the beneficial effects observed in G93A mice. More broadly, the distinct transcriptomic profile of EOM SCs may offer novel therapeutic targets to slow progressive neuromuscular functional decay in ALS and provide possible 'response biomarkers' in pre-clinical and clinical studies.
Topics: Animals; Neuromuscular Junction; Amyotrophic Lateral Sclerosis; Mice; Satellite Cells, Skeletal Muscle; Disease Models, Animal; Transcriptome; Mice, Transgenic; Oculomotor Muscles
PubMed: 38661532
DOI: 10.7554/eLife.92644