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1.
Maturitas ; 188: 108066, 2024 Jul 14.
Article in English | MEDLINE | ID: mdl-39089047

ABSTRACT

Ageing populations globally are associated with increased musculoskeletal disease, including osteoporosis and sarcopenia. These conditions place a significant burden of disease on the individual, society and the economy. To address this, we need to understand the underpinning biological changes, including stem cell exhaustion, which plays a key role in the ageing of the musculoskeletal system. This review of the recent evidence provides an overview of the associated biological processes. The review utilised the PubMed/Medline, Science Direct, and Google Scholar databases. Mechanisms of ageing identified involve a reaction to the chronic inflammation and oxidative stress associated with ageing, resulting in progenitor cell senescence and adipogenic differentiation, leading to decreased mass and quality of both bone and muscle tissue. Although the mechanisms underpinning stem cell exhaustion are unclear, it remains a promising avenue through which to identify new strategies for prevention, detection and management.

2.
Methods Mol Biol ; 2835: 249-259, 2024.
Article in English | MEDLINE | ID: mdl-39105920

ABSTRACT

Cultivated meat, an alternative to conventional meat, holds great promise in alleviating environmental and ethical concerns. Skeletal muscle stem cell isolation is a critical phase in cultivated meat production, and efficiency is a major determinant in the final differentiated muscle cell yield. The conventional enzymatic dissociation method for cell isolation presents drawbacks, including added costs and the destruction of vital extracellular matrix components. We developed an alternative cell isolation technique, explant cell isolation, to isolate muscle stem cells from muscle tissue. The present protocol yields myogenic cell populations, mainly composed of skeletal muscle stem cells without the use of enzymes, and through a simplified process. Overall, the explant method allows for propagation of cells in their natural environment, preserving intricate cell-cell and cell-matrix interactions, resulting in both economic efficiency and consistent generation of high-quality cells.


Subject(s)
Cell Culture Techniques , Cell Separation , In Vitro Meat , Animals , Cell Culture Techniques/methods , Cell Differentiation , Cell Proliferation , Cell Separation/methods , Cells, Cultured , Muscle, Skeletal/cytology , Stem Cells/cytology
3.
Food Chem X ; 23: 101603, 2024 Oct 30.
Article in English | MEDLINE | ID: mdl-39100247

ABSTRACT

Animal cell culture technology in the production of slaughter-free meat offers ethical advantages with regards to animal welfare, rendering it a more socially acceptable approach for dog meat production. In this study, edible plant-derived scaffold was used as a platform for cell expansion to construct cell-cultured dog meat slices. Primary dog skeletal muscle satellite cells (MSCs) and adipose stem cells (ASCs) were isolated and cultured as seed cells, and 3D spheroid culture in vitro promoted MSCs and ASCs myogenic and adipogenic differentiation, respectively. Natural leaf veins (NLV) were produced as edible mesh scaffolds to create 3D engineered dog muscle and fat tissues. After MSCs and ASCs adhered, proliferated and differentiated on the NLV scaffolds, and muscle and fat slices were produced with cultured dog muscle fibers and adipocytes, respectively. These findings demonstrate the potential of plant-derived NLV scaffolds in the production of cultured dog meat.

4.
Methods Mol Biol ; 2835: 229-247, 2024.
Article in English | MEDLINE | ID: mdl-39105919

ABSTRACT

Skeletal muscle is a postmitotic tissue composed of contractile myofibers that are oriented and connected to different layers of connective tissue. Nevertheless, adult muscle fibers retain the capacity to regenerate in response to damage, activating the classical muscle stem cell compartment, namely, satellite cells (SCs), which are mitotically quiescent cells until required for growth or repair and are localized between the basal lamina and sarcolemma of myofibers. The transition of SCs from the quiescent state toward activation, commitment, and differentiation involves the genetic and epigenetic adaptation to novel biological functions, entailing dynamic changes in the protein expression profile. Interestingly, some of the activities and signaling regulating proliferation, commitment, differentiation, and survival/apoptosis of satellite cells have been also partially recapitulated in vitro, taking advantage of robust markers, reliable techniques, and reproducible protocols. Over the years, different techniques of muscular cell culture have been designed including primary cultures from embryonic or postnatal muscle, myogenic cell line, and three-dimensional (3D) skeletal muscle construct. Typical two-dimensional (2D) muscle cell culture cannot fully recapitulate the complexity of living muscle tissues, restricting their usefulness for physiological studies. The development of functional 3D culture models represents a valid alternative to overcome the limitations of already available in vitro model, increasing our understanding of the roles played by the various cell types and how they interact. In this chapter, the development of bidimensional and three-dimensional cell cultures have been described, improving the technical aspect of satellite cell isolation, the best culture-based conditions for muscle cell growth and differentiation, and the procedures required to develop a three-dimensional skeletal muscle construct.


Subject(s)
Cell Culture Techniques , Muscle, Skeletal , Satellite Cells, Skeletal Muscle , Animals , Satellite Cells, Skeletal Muscle/cytology , Satellite Cells, Skeletal Muscle/metabolism , Muscle, Skeletal/cytology , Muscle, Skeletal/metabolism , Muscle, Skeletal/growth & development , Cell Culture Techniques/methods , Cell Differentiation , Mice , Cell Culture Techniques, Three Dimensional/methods , Muscle Fibers, Skeletal/cytology , Muscle Fibers, Skeletal/metabolism , Muscle Development , Cell Proliferation , Cells, Cultured
5.
J Mol Histol ; 2024 Aug 06.
Article in English | MEDLINE | ID: mdl-39105942

ABSTRACT

Osteoporosis is a metabolic bone disease. ß-Catenin is associated with fractures. Jian-Pi-Bu-Shen (JPBS) can promote the healing of osteoporotic fractures (OPF). However, the mechanism of ß-catenin-mediated skeletal muscle satellite cells (SMSCs) in OPF by the JPBS is unclear. SMSCs were isolated and divided into five groups. The results showed that the survival rate of SMSCs was significantly higher in the low, medium, and high dose JPBS-containing serum groups after 7 days of incubation. The ALP activity and the number of SMSCs mineralized in the JPBS-containing serum intervention group were elevated. Axin, GSK-3ß, ß-catenin siRNAs were constructed and transfected into cells. Transfection of siRNAs reduced Axin, GSK-3ß, and ß-catenin expressions, respectively. ß-Catenin-siRNA reversed ALP activity, the number of SMSCs mineralized, and the expression of ß-catenin, BMP2, Runx2, COL-I, SP7/Ostrix, Osteocalcin, and BMP-7. Transcriptomic results suggested that the TNF signaling pathway associated with OPF was enriched. SD rats were subjected to the construction of OPF model by removing the ovaries. JPBS decreased the levels of PINP, ALP, CTX, and NTX through ß-catenin in OPF rats, while increasing Runx2, ß-catenin expressions through ß-catenin at the broken end of fractures. Moreover, JPBS decreased BMC, BMD, and BV/TV and improved pathological damage through ß-catenin in OPF rats. JPBS decreased the expression of Axin, GSK-3ß mRNA, and protein, but increased the expressions of ß-catenin, Pax7, COL-II, COL-II, BMP2, and Runx2 through ß-catenin in OPF rats. In conclusion, JPBS inhibits Axin/GSK-3ß expression, activates the ß-catenin signaling, and promotes the osteogenic differentiation of SMSCs.

6.
Methods Mol Biol ; 2024 Aug 21.
Article in English | MEDLINE | ID: mdl-39162976

ABSTRACT

Regeneration is a remarkable characteristic of the skeletal muscle. Triggered by common lesions, regeneration is stimulated resulting in muscle fiber repair and restoration of muscle homeostasis in normal muscle. In genetic dystrophic muscle, the cycle of degeneration/regeneration is an endless loop that leads to impaired regeneration and substitution of muscle fibers by connective and adipose tissue, causing muscle weakness. Identification and characterization of muscle regeneration steps can help discover potential therapy targets for muscle diseases and aging. Muscle regeneration markers such as the number of satellite cells in the muscle, the proportion of activated satellite cells, and the quantity of regenerating muscle fiber can be quantified using immunolabeling.Here we are presenting a quantitative method to measure muscle regeneration that can be applied to different proposals. To demonstrate the protocol applicability, we used models for acute and chronic muscle injuries. As model of acute degeneration, a wild-type C57BL6 mice with muscle injury induced by electroporation was used, and the muscle was analyzed after 5 and 10 days post-injury. DMDmdx mouse muscle was used as a model of chronic degeneration. The methodologies presented here are among the gold standard methodologies for muscle regeneration analysis and can be easily applied to any type of muscle regeneration study.

8.
Sci Rep ; 14(1): 16422, 2024 07 16.
Article in English | MEDLINE | ID: mdl-39013963

ABSTRACT

Interactions between tissues and cell types, mediated by cytokines or direct cell-cell exchanges, regulate growth. To determine whether mature adipocytes influence the in vitro growth of trout mononucleated muscle cells, we developed an indirect coculture system, and showed that adipocytes (5 × 106 cells/well) derived from perivisceral adipose tissue increased the proliferation (BrdU-positive cells) of the mononucleated muscle cells (26% vs. 39%; p < 0.001) while inhibiting myogenic differentiation (myosin+) (25% vs. 15%; p < 0.001). Similar effects were obtained with subcutaneous adipose tissue-derived adipocytes, although requiring more adipocytes (3 × 107 cells/well vs. 5 × 106 cells/well). Conditioned media recapitulated these effects, stimulating proliferation (31% vs. 39%; p < 0.001) and inhibiting myogenic differentiation (32 vs. 23%; p < 0.001). Adipocytes began to reduce differentiation after 24 h, whereas proliferation stimulation was observed after 48 h. While adipocytes did not change pax7+ and myoD1/2+ percentages, they reduced myogenin+ cells showing inhibition from early differentiation stage. Finally, adipocytes increased BrdU+ cells in the Pdgfrα+ population but not in the myoD+ one. Collectively, our results demonstrate that trout adipocytes promote fibro-adipocyte precursor proliferation while inhibiting myogenic cells differentiation in vitro, suggesting the key role of adipose tissue in regulating fish muscle growth.


Subject(s)
Adipocytes , Adipogenesis , Cell Differentiation , Cell Proliferation , Muscle Development , Animals , Adipocytes/cytology , Coculture Techniques , Cells, Cultured , Trout , Culture Media, Conditioned/pharmacology
9.
Eur J Appl Physiol ; 2024 Jul 24.
Article in English | MEDLINE | ID: mdl-39044030

ABSTRACT

BACKGROUND: Non-steroidal anti-inflammatory drugs (NSAIDs) possess analgesic and anti-inflammatory properties by inhibiting cyclooxygenase (COX) enzymes. Conflicting evidence exists on whether NSAIDs influence signaling related to muscle adaptations and exercise with some research finding a reduction in muscle protein synthesis signaling via the AKT-mTOR pathway, changes in satellite cell signaling, reductions in muscle protein degradation, and reductions in cell proliferation. In this study, we determined if a single maximal dose of flurbiprofen (FLU), celecoxib (CEL), ibuprofen (IBU), or a placebo (PLA) affects the short-term muscle signaling responses to plyometric exercise. METHODS: This was a block randomized, double-masked, crossover design, where 12 participants performed four plyometric exercise bouts consisting of 10 sets of 10 plyometric jumps at 40% 1RM. Two hours before exercise, participants consumed a single dose of celecoxib (CEL 200 mg), IBU (800 mg), FLU (100 mg) or PLA with food. Muscle biopsy samples were collected before and 3-h after exercise from the vastus lateralis. Data were analyzed using a repeated measures (RM) ANOVA, ANOVA, or a Friedman test. Significance was considered at p < 0.05. RESULTS: We found no treatment effects on the mRNA expression of PTSG1, PTSG2, MYC, TBP, RPLOP, MYOD1, Pax7, MYOG, Atrogin-1, or MURF1 (all, p > 0.05). We also found no treatment effects on AKT-mTOR signaling or MAPK signaling measured through the phosphorylation status of mTORS2441, mTORS2448, RPS6 235/236, RPS 240/244, 4EBP1, ERK1/2, p38 T180/182 normalized to their respective total abundance (all, p > 0.05). However, we did find a significant difference between MNK1 T197/202 in PLA compared to FLU (p < .05). CONCLUSION: A single, maximal dose of IBU, CEL, or FLU taken prior to exercise did not affect the signaling of muscle protein synthesis, protein degradation, or ribosome biogenesis three hours after a plyometric training bout.

10.
Data Brief ; 55: 110576, 2024 Aug.
Article in English | MEDLINE | ID: mdl-39006349

ABSTRACT

HnRNPK, a prominent member of the heterogeneous nuclear ribonucleoprotein (hnRNP) family, is widely expressed in mammalian tissues and plays a crucial role in animal development. Despite its well-established functions, limited information is available regarding its role in skeletal muscle development and regeneration. To elucidate the functional role of hnRNPK in skeletal muscle, we utilized Pax7CreER; HnrnpkLoxP/LoxP (Hnrnpk pKO) mice as a model, isolated primary mouse skeletal muscle satellite cells (MuSCs), and induced hnRNPK knockout using 4-OTH. Transcriptome sequencing was performed on four distinct groups: Hnrnpk pKO MuSCs undergoing proliferation for 24 h (ethanol 24 h) and 48 h (ethanol 48 h) after treatment with ethanol as the control, as well as Hnrnpk pKO MuSCs undergoing proliferation for 24 h (4-OHT 24 h) and 48 h (4-OHT 48 h) after treatment with 4-OHT as the hnRNPK-induced knockout group. The RNA sequencing data was generated using the Illumina HiSeq 2000/2500 sequencing platform. The raw data files have been archived in the Sequence Read Archive at the China National Center for Bioinformation (CNCB) under the accession number CRA015864. This data article is related to the research paper "Deletion of heterogeneous nuclear ribonucleoprotein K in satellite cells leads to inhibited skeletal muscle regeneration in mice, Genes & Diseases 11: 101,062, DOI: 10.1016/j.gendis.2023.06.031".

11.
Biomolecules ; 14(7)2024 Jul 22.
Article in English | MEDLINE | ID: mdl-39062592

ABSTRACT

Amyotrophic lateral sclerosis (ALS) is a complex neuromuscular disease characterized by progressive motor neuron degeneration, neuromuscular junction dismantling, and muscle wasting. The pathological and therapeutic studies of ALS have long been neurocentric. However, recent insights have highlighted the significance of peripheral tissue, particularly skeletal muscle, in disease pathology and treatment. This is evidenced by restricted ALS-like muscle atrophy, which can retrogradely induce neuromuscular junction and motor neuron degeneration. Moreover, therapeutics targeting skeletal muscles can effectively decelerate disease progression by modulating muscle satellite cells for muscle repair, suppressing inflammation, and promoting the recovery or regeneration of the neuromuscular junction. This review summarizes and discusses therapeutic strategies targeting skeletal muscles for ALS treatment. It aims to provide a comprehensive reference for the development of novel therapeutics targeting skeletal muscles, potentially ameliorating the progression of ALS.


Subject(s)
Amyotrophic Lateral Sclerosis , Muscle, Skeletal , Amyotrophic Lateral Sclerosis/metabolism , Amyotrophic Lateral Sclerosis/pathology , Amyotrophic Lateral Sclerosis/therapy , Humans , Muscle, Skeletal/metabolism , Muscle, Skeletal/pathology , Animals , Neuromuscular Junction/metabolism , Neuromuscular Junction/pathology , Motor Neurons/metabolism , Motor Neurons/pathology
12.
Physiol Rep ; 12(13): e16052, 2024 Jul.
Article in English | MEDLINE | ID: mdl-38987200

ABSTRACT

We previously observed lifelong endurance exercise (LLE) influenced quadriceps whole-muscle and myofiber size in a fiber-type and sex-specific manner. The current follow-up exploratory investigation examined myofiber size regulators and myofiber size distribution in vastus lateralis biopsies from these same LLE men (n = 21, 74 ± 1 years) and women (n = 7, 72 ± 2 years) as well as old, healthy nonexercisers (OH; men: n = 10, 75 ± 1 years; women: n = 10, 75 ± 1 years) and young exercisers (YE; men: n = 10, 25 ± 1 years; women: n = 10, 25 ± 1 years). LLE exercised ~5 days/week, ~7 h/week for the previous 52 ± 1 years. Slow (myosin heavy chain (MHC) I) and fast (MHC IIa) myofiber nuclei/fiber, myonuclear domain, satellite cells/fiber, and satellite cell density were not influenced (p > 0.05) by LLE in men and women. The aging groups had ~50%-60% higher proportion of large (>7000 µm2) and small (<3000 µm2) myofibers (OH; men: 44%, women: 48%, LLE; men: 42%, women: 42%, YE; men: 27%, women: 29%). LLE men had triple the proportion of large slow fibers (LLE: 21%, YE: 7%, OH: 7%), while LLE women had more small slow fibers (LLE: 15%, YE: 8%, OH: 9%). LLE reduced by ~50% the proportion of small fast (MHC II containing) fibers in the aging men (OH: 14%, LLE: 7%) and women (OH: 35%, LLE: 18%). These data, coupled with previous findings, suggest that myonuclei and satellite cell content are uninfluenced by lifelong endurance exercise in men ~60-90 years, and this now also extends to septuagenarian lifelong endurance exercise women. Additionally, lifelong endurance exercise appears to influence the relative abundance of small and large myofibers (fast and slow) differently between men and women.


Subject(s)
Exercise , Muscle Fibers, Fast-Twitch , Muscle Fibers, Slow-Twitch , Physical Endurance , Satellite Cells, Skeletal Muscle , Humans , Female , Male , Satellite Cells, Skeletal Muscle/physiology , Satellite Cells, Skeletal Muscle/cytology , Adult , Physical Endurance/physiology , Exercise/physiology , Aged , Muscle Fibers, Fast-Twitch/physiology , Muscle Fibers, Fast-Twitch/cytology , Muscle Fibers, Slow-Twitch/physiology , Muscle Fibers, Slow-Twitch/cytology , Cell Nucleus/physiology , Myosin Heavy Chains/metabolism , Quadriceps Muscle/cytology , Quadriceps Muscle/physiology , Aging/physiology , Young Adult
13.
J Physiol ; 602(15): 3661-3691, 2024 Aug.
Article in English | MEDLINE | ID: mdl-38968395

ABSTRACT

The response to acute myotoxic injury requires stimulation of local repair mechanisms in the damaged tissue. However, satellite cells in muscle distant from acute injury have been reported to enter a functional state between quiescence and active proliferation. Here, we asked whether protein flux rates are altered in muscle distant from acute local myotoxic injury and how they compare to changes in gene expression from the same tissue. Broad and significant alterations in protein turnover were observed across the proteome in the limb contralateral to injury during the first 10 days after. Interestingly, mRNA changes had almost no correlation with directly measured protein turnover rates. In summary, we show consistent and striking changes in protein flux rates in muscle tissue contralateral to myotoxic injury, with no correlation between changes in mRNA levels and protein synthesis rates. This work motivates further investigation of the mechanisms, including potential neurological factors, responsible for this distant effect. KEY POINTS: Previous literature demonstrates that stem cells of uninjured muscle respond to local necrotic muscle tissue damage and regeneration. We show that muscle tissue that was distant from a model of local necrotic damage had functional changes at both the gene expression and the protein turnover level. However, these changes in distant tissue were more pronounced during the earlier stages of tissue regeneration and did not correlate well with each other. The results suggest communication between directly injured tissue and non-affected tissues that are distant from injury, which warrants further investigation into the potential of this mechanism as a proactive measure for tissue regeneration from damage.


Subject(s)
Mice, Inbred C57BL , Muscle, Skeletal , Animals , Male , Muscle, Skeletal/metabolism , Muscle, Skeletal/injuries , Mice , Gene Expression , Muscle Proteins/metabolism , Muscle Proteins/genetics , RNA, Messenger/metabolism , RNA, Messenger/genetics
14.
Cytometry A ; 105(8): 580-594, 2024 Aug.
Article in English | MEDLINE | ID: mdl-38995093

ABSTRACT

Senescence is an irreversible arrest of the cell cycle that can be characterized by markers of senescence such as p16, p21, and KI-67. The characterization of different senescence-associated phenotypes requires selection of the most relevant senescence markers to define reliable cytometric methodologies. Mass cytometry (a.k.a. Cytometry by time of flight, CyTOF) can monitor up to 40 different cell markers at the single-cell level and has the potential to integrate multiple senescence and other phenotypic markers to identify senescent cells within a complex tissue such as skeletal muscle, with greater accuracy and scalability than traditional bulk measurements and flow cytometry-based measurements. This article introduces an analysis framework for detecting putative senescent cells based on clustering, outlier detection, and Boolean logic for outliers. Results show that the pipeline can identify putative senescent cells in skeletal muscle with well-established markers such as p21 and potential markers such as GAPDH. It was also found that heterogeneity of putative senescent cells in skeletal muscle can partly be explained by their cell type. Additionally, autophagy-related proteins ATG4A, LRRK2, and GLB1 were identified as important proteins in predicting the putative senescent population, providing insights into the association between autophagy and senescence. It was observed that sex did not affect the proportion of putative senescent cells among total cells. However, age did have an effect, with a higher proportion observed in fibro/adipogenic progenitors (FAPs), satellite cells, M1 and M2 macrophages from old mice. Moreover, putative senescent cells from muscle of old and young mice show different expression levels of senescence-related proteins, with putative senescent cells of old mice having higher levels of p21 and GAPDH, whereas putative senescent cells of young mice had higher levels of IL-6. Overall, the analysis framework prioritizes multiple senescence-associated proteins to characterize putative senescent cells sourced from tissue made of different cell types.


Subject(s)
Biomarkers , Cellular Senescence , Flow Cytometry , Muscle, Skeletal , Animals , Cellular Senescence/physiology , Mice , Muscle, Skeletal/cytology , Muscle, Skeletal/metabolism , Flow Cytometry/methods , Biomarkers/metabolism , Female , Male , Mice, Inbred C57BL , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Single-Cell Analysis/methods
15.
J Cell Sci ; 137(15)2024 Aug 01.
Article in English | MEDLINE | ID: mdl-39037211

ABSTRACT

Muscle stem cells (MuSCs) play an indispensable role in postnatal muscle growth and hypertrophy in adults. MuSCs also retain a highly regenerative capacity and are therefore considered a promising stem cell source for regenerative therapy for muscle diseases. In this study, we identify tumor-suppressor protein Tob1 as a Pax7 target protein that negatively controls the population expansion of MuSCs. Tob1 protein is undetectable in the quiescent state but is upregulated during activation in MuSCs. Tob1 ablation in mice accelerates MuSC population expansion and boosts muscle regeneration. Moreover, inactivation of Tob1 in MuSCs ameliorates the efficiency of MuSC transplantation in a murine muscular dystrophy model. Collectively, selective targeting of Tob1 might be a therapeutic option for the treatment of muscular diseases, including muscular dystrophy and age-related sarcopenia.


Subject(s)
Muscle, Skeletal , PAX7 Transcription Factor , Regeneration , Stem Cells , Animals , Mice , Muscle, Skeletal/metabolism , PAX7 Transcription Factor/metabolism , PAX7 Transcription Factor/genetics , Stem Cells/metabolism , Stem Cells/cytology , Intracellular Signaling Peptides and Proteins/metabolism , Intracellular Signaling Peptides and Proteins/genetics , Cell Proliferation , Mice, Inbred C57BL
16.
Stem Cell Reports ; 19(7): 1024-1040, 2024 Jul 09.
Article in English | MEDLINE | ID: mdl-38876109

ABSTRACT

Increasing evidence suggests that the muscle stem cell (MuSC) pool is heterogeneous. In particular, a rare subset of PAX7-positive MuSCs that has never expressed the myogenic regulatory factor MYF5 displays unique self-renewal and engraftment characteristics. However, the scarcity and limited availability of protein markers make the characterization of these cells challenging. Here, we describe the generation of StemRep reporter mice enabling the monitoring of PAX7 and MYF5 proteins based on equimolar levels of dual nuclear fluorescence. High levels of PAX7 protein and low levels of MYF5 delineate a deeply quiescent MuSC subpopulation with an increased capacity for asymmetric division and distinct dynamics of activation, proliferation, and commitment. Aging primarily reduces the MYF5Low MuSCs and skews the stem cell pool toward MYF5High cells with lower quiescence and self-renewal potential. Altogether, we establish the StemRep model as a versatile tool to study MuSC heterogeneity and broaden our understanding of mechanisms regulating MuSC quiescence and self-renewal in homeostatic, regenerating, and aged muscles.


Subject(s)
Aging , Genes, Reporter , Myogenic Regulatory Factor 5 , PAX7 Transcription Factor , Regeneration , Animals , PAX7 Transcription Factor/metabolism , PAX7 Transcription Factor/genetics , Myogenic Regulatory Factor 5/metabolism , Myogenic Regulatory Factor 5/genetics , Mice , Aging/metabolism , Stem Cells/metabolism , Stem Cells/cytology , Cell Proliferation , Muscle, Skeletal/metabolism , Muscle, Skeletal/cytology , Cell Differentiation , Mice, Transgenic , Cell Self Renewal
17.
Tissue Eng Part A ; 2024 Jul 18.
Article in English | MEDLINE | ID: mdl-38874526

ABSTRACT

Volumetric muscle loss (VML) is a clinical state that results in impaired skeletal muscle function. Engineered skeletal muscle can serve as a treatment for VML. Currently, large biopsies are required to achieve the cells necessary for the fabrication of engineered muscle, leading to donor-site morbidity. Amplification of cell numbers using cell passaging may increase the usefulness of a single muscle biopsy for engineering muscle tissue. In this study, we evaluated the impact of passaging cells obtained from donor muscle tissue by analyzing characteristics of in vitro cellular growth and tissue-engineered skeletal muscle unit (SMU) structure and function. Human skeletal muscle cell isolates from three separate donors (P0-Control) were compared with cells passaged once (P1), twice (P2), or three times (P3) by monitoring SMU force production and determining muscle content and structure using immunohistochemistry. Data indicated that passaging decreased the number of satellite cells and increased the population doubling time. P1 SMUs had slightly greater contractile force and P2 SMUs showed statistically significant greater force production compared with P0 SMUs with no change in SMU muscle content. In conclusion, human skeletal muscle cells can be passaged twice without negatively impacting SMU muscle content or contractile function, providing the opportunity to potentially create larger SMUs from smaller biopsies, thereby producing clinically relevant sized grafts to aid in VML repair.

18.
Article in English | MEDLINE | ID: mdl-38896394

ABSTRACT

The proliferation and differentiation of skeletal muscle satellite cells is a complex physiological process involving various transcription factors and small RNA molecules. This study aimed to understand the regulatory mechanisms underlying these processes, focusing on interferon-related development factor 2 (IFRD2) as a target gene of miRNA-2400 in bovine skeletal MuSCs (MuSCs). IFRD2 was identified as a target gene of miRNA-2400 involved in regulating the proliferation and differentiation of bovine skeletal MuSCs. Our results indicate that miR-2400 can target binding the 3'UTR of IFRD2 and inhibit its translation. mRNA and protein expression levels of IFRD2 increased significantly with increasing days of differentiation. Moreover, overexpression of the IFRD2 gene inhibited proliferation and promoted differentiation of bovine MuSCs. Conversely, the knockdown of the gene had the opposite effect. Overexpression of IFRD2 resulted in the inhibition of ERK1/2 phosphorylation levels in bovine MuSCs, which in turn promoted differentiation. In summary, IFRD2, as a target gene of miR-2400, crucially affects bovine skeletal muscle proliferation and differentiation by precisely regulating ERK1/2 phosphorylation.

19.
Cells ; 13(12)2024 Jun 13.
Article in English | MEDLINE | ID: mdl-38920660

ABSTRACT

Skeletal muscle satellite cells, the resident stem cells in pig skeletal muscle, undergo proliferation and differentiation to enable muscle tissue repair. The proliferative and differentiative abilities of these cells gradually decrease during in vitro cultivation as the cell passage number increases. Despite extensive research, the precise molecular mechanisms that regulate this process are not fully understood. To bridge this knowledge gap, we conducted transcriptomic analysis of skeletal muscle satellite cells during in vitro cultivation to quantify passage number-dependent changes in the expression of genes associated with proliferation. Additionally, we explored the relationships between gene transcriptional activity and chromatin accessibility using transposase-accessible chromatin sequencing. This revealed the closure of numerous open chromatin regions, which were primarily located in intergenic regions, as the cell passage number increased. Integrated analysis of the transcriptomic and epigenomic data demonstrated a weak correlation between gene transcriptional activity and chromatin openness in expressed genic regions; although some genes (e.g., GNB4 and FGD5) showed consistent relationships between gene expression and chromatin openness, a substantial number of differentially expressed genes had no clear association with chromatin openness in expressed genic regions. The p53-p21-RB signaling pathway may play a critical regulatory role in cell proliferation processes. The combined transcriptomic and epigenomic approach taken here provided key insights into changes in gene expression and chromatin openness during in vitro cultivation of skeletal muscle satellite cells. These findings enhance our understanding of the intricate mechanisms underlying the decline in cellular proliferation capacity in cultured cells.


Subject(s)
Cell Proliferation , RNA-Seq , Satellite Cells, Skeletal Muscle , Satellite Cells, Skeletal Muscle/metabolism , Satellite Cells, Skeletal Muscle/cytology , Animals , Cell Proliferation/genetics , Cells, Cultured , Swine , Chromatin/metabolism , Transcriptome/genetics , Gene Expression Regulation , Chromatin Immunoprecipitation Sequencing
20.
Metabolites ; 14(6)2024 Jun 18.
Article in English | MEDLINE | ID: mdl-38921474

ABSTRACT

Intrauterine growth-restricted (IUGR) fetuses exhibit systemic inflammation that contributes to programmed deficits in myoblast function and muscle growth. Thus, we sought to determine if targeting fetal inflammation improves muscle growth outcomes. Heat stress-induced IUGR fetal lambs were infused with eicosapentaenoic acid (IUGR+EPA; n = 9) or saline (IUGR; n = 8) for 5 days during late gestation and compared to saline-infused controls (n = 11). Circulating eicosapentaenoic acid was 42% less (p < 0.05) for IUGR fetuses but was recovered in IUGR+EPA fetuses. The infusion did not improve placental function or fetal O2 but resolved the 67% greater (p < 0.05) circulating TNFα observed in IUGR fetuses. This improved myoblast function and muscle growth, as the 23% reduction (p < 0.05) in the ex vivo differentiation of IUGR myoblasts was resolved in IUGR+EPA myoblasts. Semitendinosus, longissimus dorsi, and flexor digitorum superficialis muscles were 24-39% lighter (p < 0.05) for IUGR but not for IUGR+EPA fetuses. Elevated (p < 0.05) IL6R and reduced (p < 0.05) ß2 adrenoceptor content in IUGR muscle indicated enhanced inflammatory sensitivity and diminished ß2 adrenergic sensitivity. Although IL6R remained elevated, ß2 adrenoceptor deficits were resolved in IUGR+EPA muscle, demonstrating a unique underlying mechanism for muscle dysregulation. These findings show that fetal inflammation contributes to IUGR muscle growth deficits and thus may be an effective target for intervention.

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