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Experimental Gerontology Oct 2023Low back pain is a general phenomenon of aging, and surgery is an unavoidable choice to relieve severe back pain. The discarded surgical site during surgery is of high...
BACKGROUND
Low back pain is a general phenomenon of aging, and surgery is an unavoidable choice to relieve severe back pain. The discarded surgical site during surgery is of high value for muscle and muscle-related research. This study investigated the age-dependent properties of patients' paraspinal muscles at the cellular level.
METHODS
To define an association of paraspinal muscle degeneration with sarcopenia, we analyzed lumbar paraspinal muscle and myoblasts isolated from donors of various ages (25-77 years). Preoperative evaluations were performed by bioimpedance analysis using the InBody 720, magnetic resonance (MR) imaging of the lumbar spine, and lumbar extension strength using a lumbar extension dynamometer. In addition, the growth and differentiation capacity of myoblasts obtained from the donor was determined using proliferation assay and western blotting.
RESULTS
The cross-sectional area of the lumbar paraspinal muscle decreased with age and was also correlated with the appendicular skeletal muscle index (ASM/height). Human primary myoblasts isolated from paraspinal muscle preserved their proliferative capacity in vitro, which tended to decrease with donor age. The age-dependent decline in myoblast proliferation was correlated with levels of cell cycle inhibitory proteins (p16INK4a, p21CIP1, and p27KIP1) associated with cellular senescence. Primary myoblasts isolated from younger donors differentiated into multinucleate myotubes earlier and at a higher rate than those from older donors in vitro. Age-dependent decline in myogenic potential of the isolated primary myoblasts was likely correlated with the inactivation of myogenic transcription factors such as MyoD, myogenin, and MEF2c.
CONCLUSIONS
Myoblasts isolated from human paraspinal muscle preserve myogenic potential that correlates with donor age, providing an in vitro model of sarcopenia.
Topics: Humans; Sarcopenia; Paraspinal Muscles; Myoblasts; Muscle Fibers, Skeletal; Cell Cycle Proteins; Models, Theoretical
PubMed: 37591335
DOI: 10.1016/j.exger.2023.112273 -
Cellular and Molecular Life Sciences :... Nov 2023Adipose-derived stem cells (ADSC) therapy shows promise as an effective treatment for dystrophinopathy. Fibro-/adipogenic progenitors (FAPs) play an essential role in...
Adipose-derived stem cells (ADSC) therapy shows promise as an effective treatment for dystrophinopathy. Fibro-/adipogenic progenitors (FAPs) play an essential role in the myogenesis of muscle satellite cells and contribute to muscle fibrosis and adipocyte infiltration. The interleukin 4 (IL-4) pathway acts as a switch that regulates the functions of FAPs. The interaction between FAPs and engrafted cells remains unclear. In this study, we used a co-culture system to investigate possible crosstalk between the FAPs of dystrophic mice and ADSC overexpressing IL4 (IL4-ADSC) and control ADSC. Systemic transplantation of IL4-ADSC and control ADSC in dystrophic mice was conducted for 16 weeks, after which motor function and molecular improvements were evaluated. Overexpression of IL4 in ADSC significantly promoted myogenesis in vitro, increasing the expression of Pax7, Myogenin, and MyHC. Co-culture indicated that although myoblasts derived from control ADSC promoted adipogenic and fibrogenic differentiation of FAPs, FAPs did not significantly affect myogenesis of ADSC-derived myoblasts. However, overexpression of IL4 in ADSC inhibited their myotube-dependent promotion of FAPs differentiation on the one hand and promoted FAPs to enhance myogenesis on the other. Dystrophic mice administered with IL4-ADSC-derived myoblasts displayed significantly better motor ability, more engrafted cells showing dystrophin expression, and less muscle fibrosis, intramuscular adipocytes, and macrophage infiltration than mice administered control-ADSC-derived myoblasts. In conclusion, IL4 activation enhanced the therapeutic potential of ADSC transplantation in dystrophic mice, possibly by improving the myogenesis of IL4-ADSC and altering the crosstalk between engrafted stem cells and resident FAPs.
Topics: Mice; Animals; Interleukin-4; Adipogenesis; Cell Differentiation; Stem Cells; Satellite Cells, Skeletal Muscle; Fibrosis; Muscle, Skeletal
PubMed: 38010513
DOI: 10.1007/s00018-023-05020-2 -
Cells Aug 2023Sarcopenia has a high prevalence among the aging population. Sarcopenia is of tremendous socioeconomic importance because it can lead to falls and hospitalization,...
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.
Topics: Aged; Animals; Humans; Mice; Forkhead Box Protein O3; Muscle Fibers, Skeletal; Muscle, Skeletal; Myoblasts; Quality of Life; Sarcopenia; Myogenin; MyoD Protein
PubMed: 37681900
DOI: 10.3390/cells12172167 -
Cell Reports. Medicine Mar 2024Tissue regeneration following an injury requires dynamic cell-state transitions that allow for establishing the cell identities required for the restoration of tissue...
Tissue regeneration following an injury requires dynamic cell-state transitions that allow for establishing the cell identities required for the restoration of tissue homeostasis and function. Here, we present a biochemical intervention that induces an intermediate cell state mirroring a transition identified during normal differentiation of myoblasts and other multipotent and pluripotent cells to mature cells. When applied in somatic differentiated cells, the intervention, composed of one-carbon metabolites, reduces some dedifferentiation markers without losing the lineage identity, thus inducing limited reprogramming into a more flexible cell state. Moreover, the intervention enabled accelerated repair after muscle injury in young and aged mice. Overall, our study uncovers a conserved biochemical transitional phase that enhances cellular plasticity in vivo and hints at potential and scalable biochemical interventions of use in regenerative medicine and rejuvenation interventions that may be more tractable than genetic ones.
Topics: Mice; Animals; Cell Differentiation; Myoblasts; Muscles
PubMed: 38508141
DOI: 10.1016/j.xcrm.2024.101449 -
Nature Chemical Biology Feb 2024Targeting proximity-labeling enzymes to specific cellular locations is a viable strategy for profiling subcellular proteomes. Here, we generated transgenic mice (MAX-Tg)...
Targeting proximity-labeling enzymes to specific cellular locations is a viable strategy for profiling subcellular proteomes. Here, we generated transgenic mice (MAX-Tg) expressing a mitochondrial matrix-targeted ascorbate peroxidase. Comparative analysis of matrix proteomes from the muscle tissues showed differential enrichment of mitochondrial proteins. We found that reticulon 4-interacting protein 1 (RTN4IP1), also known as optic atrophy-10, is enriched in the mitochondrial matrix of muscle tissues and is an NADPH oxidoreductase. Interactome analysis and in vitro enzymatic assays revealed an essential role for RTN4IP1 in coenzyme Q (CoQ) biosynthesis by regulating the O-methylation activity of COQ3. Rtn4ip1-knockout myoblasts had markedly decreased CoQ levels and impaired cellular respiration. Furthermore, muscle-specific knockdown of dRtn4ip1 in flies resulted in impaired muscle function, which was reversed by dietary supplementation with soluble CoQ. Collectively, these results demonstrate that RTN4IP1 is a mitochondrial NAD(P)H oxidoreductase essential for supporting mitochondrial respiration activity in the muscle tissue.
Topics: Animals; Mice; Drosophila melanogaster; Mice, Transgenic; Mitochondria; Mitochondrial Proteins; Oxidoreductases; Proteome; Ubiquinone; Carrier Proteins
PubMed: 37884807
DOI: 10.1038/s41589-023-01452-w -
Journal of the International Society of... Dec 2024Non-steroidal anti-inflammatory drugs (NSAIDs) like ibuprofen, flurbiprofen, naproxen sodium, and indomethacin are commonly employed for their pain-relieving and...
BACKGROUND
Non-steroidal anti-inflammatory drugs (NSAIDs) like ibuprofen, flurbiprofen, naproxen sodium, and indomethacin are commonly employed for their pain-relieving and inflammation-reducing qualities. NSAIDs work by blocking COX-1 and/or COX-2, enzymes which play roles in inflammation, fever, and pain. The main difference among NSAIDs lies in their affinity to these enzymes, which in turn, influences prostaglandin secretion, and skeletal muscle growth and regeneration. The current study investigated the effects of NSAIDs on human skeletal muscle cells, focusing on myoblast proliferation, differentiation, and muscle protein synthesis signaling.
METHODS
Using human primary muscle cells, we examined the dose-response impact of flurbiprofen (25-200 µM), indomethacin (25-200 µM), ibuprofen (25-200 µM), and naproxen sodium (25-200 µM), on myoblast viability, myotube area, fusion, and prostaglandin production.
RESULTS
We found that supraphysiological concentrations of indomethacin inhibited myoblast proliferation (-74 ± 2% with 200 µM; -53 ± 3% with 100 µM; both < 0.05) compared to control cells and impaired protein synthesis signaling pathways in myotubes, but only attenuated myotube fusion at the highest concentrations (-18 ± 2% with 200 µM, < 0.05) compared to control myotubes. On the other hand, ibuprofen had no such effects. Naproxen sodium only increased cell proliferation at low concentrations (+36 ± 2% with 25 µM, < 0.05), and flurbiprofen exhibited divergent impacts depending on the concentration whereby low concentrations improved cell proliferation (+17 ± 1% with 25 µM, < 0.05) but high concentrations inhibited cell proliferation (-32 ± 1% with 200 µM, < 0.05).
CONCLUSION
Our findings suggest that indomethacin, at high concentrations, may detrimentally affect myoblast proliferation and differentiation via an AKT-dependent mechanism, and thus provide new understanding of NSAIDs' effects on skeletal muscle cell development.
Topics: Humans; Naproxen; Ibuprofen; Flurbiprofen; Indomethacin; Anti-Inflammatory Agents, Non-Steroidal; Muscle Fibers, Skeletal; Inflammation; Pain; Prostaglandins
PubMed: 38198469
DOI: 10.1080/15502783.2024.2302046 -
GeroScience Apr 2024As we age, the ability to regenerate and repair skeletal muscle damage declines, partially due to increasing dysfunction of muscle resident stem cells-satellite cells...
As we age, the ability to regenerate and repair skeletal muscle damage declines, partially due to increasing dysfunction of muscle resident stem cells-satellite cells (SC). Recent evidence implicates cellular senescence, which is the irreversible arrest of proliferation, as a potentiator of SC impairment during aging. However, little is known about the role of senescence in SC, and there is a large discrepancy in senescence classification within skeletal muscle. The purpose of this study was to develop a model of senescence in skeletal muscle myoblasts and identify how common senescence-associated biomarkers respond. Low-passage CC myoblasts were treated with bleomycin or vehicle and then evaluated for cytological and molecular senescence markers, proliferation status, cell cycle kinetics, and differentiation potential. Bleomycin treatment caused double-stranded DNA breaks, which upregulated p21 mRNA and protein, potentially through NF-κB and senescence-associated super enhancer (SASE) signaling (p < 0.01). Consequently, cell proliferation was abruptly halted due to G2/M-phase arrest (p < 0.01). Bleomycin-treated myoblasts displayed greater senescence-associated β-galactosidase staining (p < 0.01), which increased over several days. These myoblasts remained senescent following 6 days of differentiation and had significant impairments in myotube formation (p < 0.01). Furthermore, our results show that senescence can be maintained despite the lack of p16 gene expression in CC myoblasts. In conclusion, bleomycin treatment provides a valid model of damage-induced senescence that was associated with elevated p21, reduced myoblast proliferation, and aberrant cell cycle kinetics, while confirming that a multi-marker approach is needed for the accurate classification of senescence within skeletal muscle.
Topics: Bleomycin; Cell Line; Cellular Senescence; Cell Differentiation; Myoblasts; Biomarkers
PubMed: 37751045
DOI: 10.1007/s11357-023-00929-9 -
Communications Biology Nov 2023Skeletal muscle stem cells (MuSC) are crucial for tissue homoeostasis and repair after injury. Following activation, they proliferate to generate differentiating...
Skeletal muscle stem cells (MuSC) are crucial for tissue homoeostasis and repair after injury. Following activation, they proliferate to generate differentiating myoblasts. A proportion of cells self-renew, re-enter the MuSC niche under the basal lamina outside the myofiber and become quiescent. Quiescent MuSC have a primary cilium, which is disassembled upon cell cycle entry. Ex vivo experiments suggest cilia are important for MuSC self-renewal, however, their requirement for muscle regeneration in vivo remains poorly understood. Talpid3 (TA) is essential for primary cilia formation and Hedgehog (Hh) signalling. Here we use tamoxifen-inducible conditional deletion of TA in MuSC (iSC-KO) and show that regeneration is impaired in response to cytotoxic injury. Depletion of MuSC after regeneration suggests impaired self-renewal, also consistent with an exacerbated phenotype in TA mice after repeat injury. Single cell transcriptomics of MuSC progeny isolated from myofibers identifies components of several signalling pathways, which are deregulated in absence of TA, including Hh and Wnt. Pharmacological activation of Wnt restores muscle regeneration, while purmorphamine, an activator of the Smoothened (Smo) co-receptor in the Hh pathway, has no effect. Together, our data show that TA and primary cilia are important for MuSC self-renewal and pharmacological treatment can efficiently restore muscle regeneration.
Topics: Animals; Mice; Cells, Cultured; Cilia; Hedgehog Proteins; Muscles; Satellite Cells, Skeletal Muscle; Cell Cycle Proteins; Stem Cells
PubMed: 37925530
DOI: 10.1038/s42003-023-05503-9 -
Matrix Biology : Journal of the... May 2024Extracellular matrix (ECM) pathologic remodeling underlies many disorders, including muscular dystrophy. Tissue decellularization removes cellular components while...
The extracellular matrix differentially directs myoblast motility and differentiation in distinct forms of muscular dystrophy: Dystrophic matrices alter myoblast motility.
Extracellular matrix (ECM) pathologic remodeling underlies many disorders, including muscular dystrophy. Tissue decellularization removes cellular components while leaving behind ECM components. We generated "on-slide" decellularized tissue slices from genetically distinct dystrophic mouse models. The ECM of dystrophin- and sarcoglycan-deficient muscles had marked thrombospondin 4 deposition, while dysferlin-deficient muscle had excess decorin. Annexins A2 and A6 were present on all dystrophic decellularized ECMs, but annexin matrix deposition was excessive in dysferlin-deficient muscular dystrophy. Muscle-directed viral expression of annexin A6 resulted in annexin A6 in the ECM. C2C12 myoblasts seeded onto decellularized matrices displayed differential myoblast mobility and fusion. Dystrophin-deficient decellularized matrices inhibited myoblast mobility, while dysferlin-deficient decellularized matrices enhanced myoblast movement and differentiation. Myoblasts treated with recombinant annexin A6 increased mobility and fusion like that seen on dysferlin-deficient decellularized matrix and demonstrated upregulation of ECM and muscle cell differentiation genes. These findings demonstrate specific fibrotic signatures elicit effects on myoblast activity.
Topics: Animals; Myoblasts; Extracellular Matrix; Mice; Cell Differentiation; Sarcoglycans; Cell Movement; Dysferlin; Muscular Dystrophies; Dystrophin; Annexin A2; Decorin; Cell Line; Disease Models, Animal; Muscle, Skeletal
PubMed: 38582404
DOI: 10.1016/j.matbio.2024.04.001 -
Genes Jul 2023Myocyte enhancer factor 2A () is a member of the myocyte enhancer factor 2 family. is widely distributed in various tissues and organs and participates in various...
Myocyte enhancer factor 2A () is a member of the myocyte enhancer factor 2 family. is widely distributed in various tissues and organs and participates in various physiological processes. This study aimed to investigate the effect of expression on the proliferation and apoptosis of bovine myoblasts. CCK8, ELISA, cell cycle, and apoptosis analyses were conducted to assess cell status. In addition, the mRNA expression levels of genes associated with bovine myoblast proliferation and apoptosis were evaluated using RT-qPCR. The results showed that the upregulation of mRNA promoted the proliferation rate of myoblasts, shortened the cycle process, and increased the anti-apoptotic rate. Furthermore, the RT-qPCR results showed that the upregulation of mRNA significantly increased the cell proliferation factors and , cell cycle factors and , and the apoptotic factors and ( < 0.01). These results show that the gene can positively regulate myoblast proliferation and anti-apoptosis, providing a basis for the analysis of the regulatory mechanism of the gene on bovine growth and development.
Topics: Animals; Cattle; Cell Proliferation; Gene Expression; MEF2 Transcription Factors; Myoblasts; RNA, Messenger; Apoptosis
PubMed: 37510401
DOI: 10.3390/genes14071498