Skeletal Muscle Development Research Articles

IGF2BP1-mediated the stability and protein translation of FGFR1 mRNA regulates myogenesis through the ERK signaling pathway

N6-methyladenosine (m6A) is the most prevalent post-transcriptional modification of RNAs and plays a key regulatory role in various biological processes. As a member of the insulin-like growth factor 2 mRNA-binding proteins (IGF2BPs) family, IGF2BP1 has recently demonstrated its ability to specifically bind m6A-modified sites within mRNAs and effectively regulate their mRNA stability. However, the precise roles of IGF2BP1 in mammalian skeletal muscle development, along with its downstream mRNA targets during myogenesis, have yet to be fully elucidated. Here, we observed that IGF2BP1 expression significantly decreased during myogenic differentiation. Knockdown of IGF2BP1 significantly inhibited myoblast proliferation while promoted myogenic differentiation. In contrast, IGF2BP1 overexpression robustly stimulated myoblast proliferation but suppressed their differentiation. Combined analysis of high-throughput sequencing and RNA stability assays revealed that IGF2BP1 can enhance fibroblast growth factor receptor 1 (FGFR1) mRNA stability and promote its translation in an m6A-dependent manner, thereby regulating its expression level and the Extracellular Signal-Regulated Kinase (ERK) pathway. Additionally, knockdown of FGFR1 rescued the phenotypic changes (namely increased cell proliferation and suppressed differentiation) induced by IGF2BP1 overexpression via attenuating ERK signaling. Taken together, our findings suggest that IGF2BP1 maintains the stability and translation of FGFR1 mRNA in an m6A-dependent manner, thereby inhibiting skeletal myogenesis through activation of the ERK signaling pathway. This study further enriches the understanding of the molecular mechanisms by which RNA methylation regulates myogenesis, providing valuable insights into the role of IGF2BP1-mediated post-transcriptional regulation in muscle development.

Regulatory role of lncMD1 in goat skeletal muscle satellite cell differentiation via miR-133a-3p and miR-361-3p targeting

Skeletal muscle satellite cells (SMSCs) are pivotal in skeletal muscle development and are influenced by numerous regulatory factors. This study focuses on the regulatory and functional mechanism roles of lncMD1, a muscle-specific long non-coding RNA, in the proliferation and differentiation of goat SMSCs. Employing in vitro cultured goat SMSCs, this study demonstrated that lncMD1, functions as a decoy for miR-133a-3p and miR-361-3p. This interaction competitively binds these microRNAs to modulate the expression of dynactin subunit 2 (DCTN2) and dynactin subunit 1 (DCTN1), thereby affects SMSCs proliferation and differentiation. These findings enhance the understanding of non-coding RNAs in goat SMSCs growth cycles and offer a theoretical foundation for exploring the molecular processes of goat skeletal muscle myogenic development.

Myonuclear position and blood vessel organization during skeletal muscle postnatal development.

Skeletal muscle development is a complex process involving myoblast fusion to generate multinucleated fibers. Myonuclei first align in the center of the myotubes before migrating to the periphery of the myofiber. Blood vessels (BVs) are important contributors to the correct development of skeletal muscle, and myonuclei are found next to BVs in adult muscle. Here, we show that most myonuclear migration to the periphery occurs between embryonic day 17.5 and postnatal day 1 in mouse. Furthermore, myonuclear accretion after postnatal day 7 does not result in centrally nucleated myofibers as observed in the embryo. Instead, myonuclei remain at the periphery of the myofiber without moving to the center. Finally, we show that hypovascularization of skeletal muscle alters the interaction between myonuclei and BVs, suggesting that BVs may contribute to myonuclear positioning during skeletal muscle postnatal development. Overall, this work provides a comprehensive analysis of skeletal muscle development during the highly dynamic postnatal period, bringing new insights about myonuclear positioning and its interaction with BVs.

Changes in development and function of skeletal muscle in UCP-1 knockout mice

Changes in development and function of skeletal muscle in UCP-1 knockout mice

PSV-6 Effect of maternal undernutrition on gene expression related to immune system in spleen of Japanese Black (Wagyu) fetuses

Abstract Underdevelopment in the fetal period increases the risk of disease in the neonatal period, and there are concerns about the effects on later growth and fattening. Previous studies have indicated that poor nutrition during gestation affects not only the development of skeletal muscle and liver, but also the formation of the thymus gland and spleen, which are involved in immunity. The purpose of this study was to investigate the effects of maternal undernutrition on gene expression related to the immunity of the spleen. Multiparous Wagyu cattle (n = 12) were randomly assigned to two nutrition groups: the control group (CON: 120% of Japanese feeding standard for beef cattle; n = 6) and the maternal undernutrition group (MUN: 60% of Japanese feeding standard for beef cattle; n = 6) from conception to d 260 of gestation. Fetuses (n = 12) were harvested from the maternal cows and fetal spleens were collected. Total RNA was extracted from each spleen to synthesize cDNA. The quantitative RT-PCR (qRT-PCR) analysis was carried out to investigate the expression of 17 target genes [CD4, CD8a, CD8b, CD19, CD21, CD79a, CD205, interferon (IFN) -γ, NFk-β, toll-like receptor (TLR) 4, interleukin (IL) 2, IL4, IL6, IL7, IL10, IL18, and IFI16) related to the immune system. Ribosomal protein S18 was used as an internal control. The specificity of PCR products was confirmed by analyzing the dissociation curves. Data were analyzed by student t-test or Welch’s t-test after the Smirnov-Grubbs test and F-test to remove outliers and compare variances. The expression of CD8a (P < 0.1) and CD8b (P < 0.1) related to Cytotoxic T cell tended to be less in MUN than in CON (Figure 1). Other immunological cell marker genes expression (CD4, CD19, CD21, CD79a, CD205) did not show any significant differences between CON and MUN (P > 0.11). In genes related to immunological mechanisms (IFN-γ, NFk-β, TLR4, IL2, IL4, IL6, IL7, IL10, IL18, and IFI16), the expression of NFk-β was lless in MUN than in CON (P < 0.05). NFk-β was reported to regulate more than 400 genes including immune function. In addition, the expression of IL10 was less in MUN than in CON (P < 0.05). The cytokine of IL-10 has functions to suppress over-activated cytotoxic immunity by CD8+ T cell. These results might suggest that maternal undernutrition induces cytotoxic immune deactivation through NFk-β expression decline in the fetal spleen.

438 DNA methylation patterns and transcriptional regulation during pig fetal skeletal muscle development

Abstract Fetal development is controlled by a complex cascade of highly regulated and coordinated gene expression patterns. Epigenetic mechanisms have important roles in regulating development and differentiation. Among such mechanisms, DNA methylation exhibits context-specific associations with gene expression and has been shown to be highly dynamic during developmental processes. We performed whole-genome bisulfite sequencing (WGBS) to assess DNA methylation in pig longissimus dorsi muscle at 41- and 70-d gestation (dg), as well as RNA- and small RNA-sequencing to identify coordinated changes in methylation and expression between myogenic stages. We identified 45,739 differentially methylated regions (DMRs) between stages, and the majority (n = 34,232) were hypomethylated at 70 vs. 41 dg. Developmental DMRs exhibited feature-specific enrichment in gene regulatory regions, as well as in regions proximal to micro-RNAs (miRNAs) that have known roles in myogenesis. Integration of methylation and transcriptomic data revealed strong associations between differential gene methylation and transcript abundance. We surveyed myogenic regulatory factor (MRF) genes to determine if differential methylation was present in expected genomic regions. Within the MYF5 and MYF6 locus, MYF5 was significantly promoter-hypermethylated at 70 dg, whereas MYF6 was significantly hypomethylated upstream of its transcription start site. MYF5 is the earliest MRF to be expressed and primarily functions in myoblast proliferation and determination, while MYF6 functions in muscle cell differentiation. Thus, these patterns were consistent with expected downregulation of MYF5 and upregulation of MYF6 as muscle development progresses and demonstrate that differential methylation is evident at myogenic transcription factors. Differential miRNA methylation was significantly negatively correlated with abundance, and dynamic expression of assayed miRNAs persisted postnatally. Motif analysis revealed significant enrichment of myogenic regulatory factor motifs among hypomethylated regions, suggesting that DNA hypomethylation may function to increase accessibility of muscle-specific transcription factors. We also show that developmental DMRs are enriched for GWAS SNPs associated with muscle physiology and meat quality traits, demonstrating the potential for epigenetic processes to influence phenotypic diversity. Our results enhance understanding of DNA methylation dynamics in pig fetal skeletal muscle and reveal putative cis-regulatory elements governed by epigenetic processes during porcine myogenesis.

138 Regulation of post protein translational modification in adipogenesis and fat accumulation in pigs

Abstract In this study, we explored the role of protein transcriptional modification (PTM) in the adipogenesis and fat accumulation of intramuscular adipocytes. Intramuscular fat (IMF) is mainly stored in adipocytes interspersed in the perimysial space or within fascicles, and its content is a key factor attributing to meat quality in farm animals, especially in terms of tenderness, flavor and juiciness. The early stage from the fetus to the neonate is crucial for skeletal muscle development involving myogenesis, adipogenesis, and fibrogenesis, and is susceptible to environmental factors. Previous studies have been subjected to explore the molecular mechanism involved in IMF development. However, it was obscured by muscular background of IMF adipocytes. Therefore, we investigated PTM dynamics from three aspects: 1) first analyzed PTM levels of lysine acetylation (Kac), 2-hydroxyisobutyrylation (Khib), crotonylation (Kcr) and succinylation (Ksu) in three distinct differentiation potenctial primary stem cells, including myogenic precursor cells (MPCs), fibro-adipogenic progenitors (FAPs) and MSCs isolated from neonatal longissimus dorsi muscle of pigs within 3-d of age; 2) Comparing the Khib levels of proliferative and differentiated FAPs; and 3) Exploring the role of Khib modification in the adipose tissue of obese- and lean-types of pigs. We found that only Khib level in FAPs was significantly greater than MSCs. During adipogenic differentiation of FAPs, the modification level of Khib was significantly increased quadratically in parallel with protein levels of acylation regulatory enzymes KAT5 (writer) and HDAC2 (Eraser). In comparison of Khib levels in FAPs in proliferation and in differentiation and Khib levels in backfat tissues from obese pigs (Laiwu pigs) and lean pigs (Duroc pigs), we also demonstrated that Khib modification level was positive correlated with adipogenic differentiation and fat accumulation. Accordingly, the expression patterns of KAT5 and HDAC2 were parallel to the degree of lipid accumulation in the backfat adipose tissue of finishing pigs. Furthermore, we showed that KAT5 knockdown in FAPs inhibited adipogenic differentiation, while HDAC2 knockdown enhanced adipogenic differentiation of FAPs. By employing C3H10T1/2 cells, we identified EON1 involved in Khib modification in regulation of adipo gesis and fat accumulation with analysis of Liquid chromatography-tandem mass spectrometry (LC-MS/MS) using the Tandem Mass Tag (TMT Through conditional loss- and gain-of-function mutations, we further demonstrated that khib modification of ENO1 is necessary for preadipocyte differentiation and subsequent fat accumulation by mediating cellular glycolysis. In conclusion, we are the first to reveal the role of Khib modification in adipogenesis and fat deposition in pigs, and provided new clues for the improvement of fat accumulation and distribution via genetic selection and nutritional strategy in pigs.

FHOD-1 and profilin protect sarcomeres against contraction-induced deformation in C. elegans.

Formin HOmology Domain 2-containing (FHOD) proteins are a subfamily of actin-organizing formins important for striated muscle development in many animals. We showed previously that absence of the sole FHOD protein, FHOD-1, from Caenorhabditis elegans results in thin body wall muscles with misshapen dense bodies that serve as sarcomere Z-lines. We demonstrate here that mutations predicted to specifically disrupt actin polymerization by FHOD-1 similarly disrupt muscle development, and that FHOD-1 cooperates with profilin PFN-3 for dense body morphogenesis, and with profilins PFN-2 and PFN-3 to promote body wall muscle growth. We further demonstrate that dense bodies in worms lacking FHOD-1 or PFN-2/PFN-3 are less stable than in wild type animals, having a higher proportion of dynamic protein, and becoming distorted by prolonged muscle contraction. We also observe accumulation of actin and actin depolymerization factor/cofilin homolog UNC-60B in body wall muscle of these mutants. Such accumulations may indicate targeted disassembly of thin filaments dislodged from unstable dense bodies, possibly accounting for the abnormally slow growth and reduced body wall muscle strength in fhod-1 mutants. Overall, these results implicate FHOD protein-mediated actin assembly in forming stable sarcomere Z-lines, and identify profilin as a new contributor to FHOD activity in striated muscle development.

MBNL splicing factors regulate the microtranscriptome of skeletal muscles.

Muscleblind like splicing regulators (MBNLs) govern various RNA-processing steps, including alternative splicing, polyadenylation, RNA stabilityand mRNA intracellular localization. In myotonic dystrophy type 1 (DM1), the most common muscular dystrophy in adults, MBNLs are sequestered on toxic RNA containing expanded CUG repeats, which leads to disruption of MBNL-regulated processes and disease features of DM1. Herein, we show the significance of MBNLs in regulating microtranscriptome dynamics during the postnatal development of skeletal muscles and in microRNA (miRNA) misregulation observed in mouse models and patients with DM1. We identify multiple miRNAs sensitive to MBNL proteins insufficiency and reveal that many of them were postnatally regulated, which correlates with increases in the activity of these proteins during this process. In adult Mbnl1-knockout mice, miRNA expression exhibited an adult-to-newborn shift. We hypothesize that Mbnl1 deficiency influences miRNA levels through a combination of mechanisms. First, the absence of Mbnl1 protein results in alterations to the levels of pri-miRNAs. Second, MBNLs affect miRNA biogenesis by regulating the alternative splicing of miRNA primary transcripts. We propose that the expression of miR-23b, miR-27b and miR-24-1, produced from the same cluster, depends on the MBNL-sensitive inclusion of alternative exons containing miRNA sequences. Our findings suggest that MBNL sequestration in DM1 is partially responsible for altered miRNA activity. This study provides new insights into the biological roles and functions of MBNL proteins as regulators of miRNA expression in skeletal muscles.

SRSF2 is a key player in orchestrating the directional migration and differentiation of MyoD progenitors during skeletal muscle development

SRSF2 plays a dual role, functioning both as a transcriptional regulator and a key player in alternative splicing. The absence of Srsf2 in MyoD + progenitors resulted in perinatal mortality in mice, accompanied by severe skeletal muscle defects. SRSF2 deficiency disrupts the directional migration of MyoD progenitors, causing them to disperse into both muscle and non-muscle regions. Single-cell RNA-sequencing analysis revealed significant alterations in Srsf2-deficient myoblasts, including a reduction in extracellular matrix components, diminished expression of genes involved in ameboid-type cell migration and cytoskeleton organization, mitosis irregularities, and premature differentiation. Notably, one of the targets regulated by Srsf2 is the serine/threonine kinase Aurka. Knockdown of Aurka led to reduced cell proliferation, disrupted cytoskeleton, and impaired differentiation, reflecting the effects seen with Srsf2 knockdown. Crucially, the introduction of exogenous Aurka in Srsf2-knockdown cells markedly alleviated the differentiation defects caused by Srsf2 knockdown. Furthermore, our research unveiled the role of Srsf2 in controlling alternative splicing within genes associated with human skeletal muscle diseases, such as BIN1, DMPK, FHL1, and LDB3. Specifically, the precise knockdown of the Bin1 exon17-containing variant, which is excluded following Srsf2 depletion, profoundly disrupted C2C12 cell differentiation. In summary, our study offers valuable insights into the role of SRSF2 in governing MyoD progenitors to specific muscle regions, thereby controlling their differentiation through the regulation of targeted genes and alternative splicing during skeletal muscle development.

SRSF2 is a key player in orchestrating the directional migration and differentiation of MyoD progenitors during skeletal muscle development.

SRSF2 plays a dual role, functioning both as a transcriptional regulator and a key player in alternative splicing. The absence of Srsf2 in MyoD + progenitors resulted in perinatal mortality in mice, accompanied by severe skeletal muscle defects. SRSF2 deficiency disrupts the directional migration of MyoD progenitors, causing them to disperse into both muscle and non-muscle regions. Single-cell RNA-sequencing analysis revealed significant alterations in Srsf2-deficient myoblasts, including a reduction in extracellular matrix components, diminished expression of genes involved in ameboid-type cell migration and cytoskeleton organization, mitosis irregularities, and premature differentiation. Notably, one of the targets regulated by Srsf2 is the serine/threonine kinase Aurka. Knockdown of Aurka led to reduced cell proliferation, disrupted cytoskeleton, and impaired differentiation, reflecting the effects seen with Srsf2 knockdown. Crucially, the introduction of exogenous Aurka in Srsf2-knockdown cells markedly alleviated the differentiation defects caused by Srsf2 knockdown. Furthermore, our research unveiled the role of Srsf2 in controlling alternative splicing within genes associated with human skeletal muscle diseases, such as BIN1, DMPK, FHL1, and LDB3. Specifically, the precise knockdown of the Bin1 exon17-containing variant, which is excluded following Srsf2 depletion, profoundly disrupted C2C12 cell differentiation. In summary, our study offers valuable insights into the role of SRSF2 in governing MyoD progenitors to specific muscle regions, thereby controlling their differentiation through the regulation of targeted genes and alternative splicing during skeletal muscle development.

Crosstalk among canonical Wnt and Hippo pathway members in skeletal muscle and at the neuromuscular junction.

Skeletal muscles are essential for locomotion, posture, and metabolic regulation. To understand physiological processes, exercise adaptation, and muscle-related disorders, it is critical to understand the molecular pathways that underlie skeletal muscle function. The process of muscle contraction, orchestrated by a complex interplay of molecular events, is at the core of skeletal muscle function. Muscle contraction is initiated by an action potential and neuromuscular transmission requiring a neuromuscular junction. Within muscle fibers, calcium ions play a critical role in mediating the interaction between actin and myosin filaments that generate force. Regulation of calcium release from the sarcoplasmic reticulum plays a key role in excitation-contraction coupling. The development and growth of skeletal muscle are regulated by a network of molecular pathways collectively known as myogenesis. Myogenic regulators coordinate the differentiation of myoblasts into mature muscle fibers. Signaling pathways regulate muscle protein synthesis and hypertrophy in response to mechanical stimuli and nutrient availability. Several muscle-related diseases, including congenital myasthenic disorders, sarcopenia, muscular dystrophies, and metabolic myopathies, are underpinned by dysregulated molecular pathways in skeletal muscle. Therapeutic interventions aimed at preserving muscle mass and function, enhancing regeneration, and improving metabolic health hold promise by targeting specific molecular pathways. Other molecular signaling pathways in skeletal muscle include the canonical Wnt signaling pathway, a critical regulator of myogenesis, muscle regeneration, and metabolic function, and the Hippo signaling pathway. In recent years, more details have been uncovered about the role of these two pathways during myogenesis and in developing and adult skeletal muscle fibers, and at the neuromuscular junction. In fact, research in the last few years now suggests that these two signaling pathways are interconnected and that they jointly control physiological and pathophysiological processes in muscle fibers. In this review, we will summarize and discuss the data on these two pathways, focusing on their concerted action next to their contribution to skeletal muscle biology. However, an in-depth discussion of the non-canonical Wnt pathway, the fibro/adipogenic precursors, or the mechanosensory aspects of these pathways is not the focus of this review.

IGFBP7 promotes the proliferation and differentiation of primary myoblasts and intramuscular preadipocytes in chicken

Though it is well known that insulin-like growth factor (IGF) binding protein 7 (IGFBP7) plays an important role in myogenesis and adipogenesis in mammals, its impact on the proliferation, differentiation, and lipid deposition in chicken primary myoblasts (CPM) and intramuscular preadipocytes remains unexplored. In the present study, we firstly examined the correlation between SNPs within the genomic sequence of the IGFBP7 gene and carcass and blood chemical traits in a F2 resource population by genetic association analysis, and found that a significant correlation between the SNP (4_49499525) located in the intron region of IGFBP7 and serum high-density lipoproteins (HDL). We then examined the expression patterns of IGFBP7 across different stages of proliferation and differentiation in CPMs and intramuscular preadipocytes via qPCR, and explored the biological functions of IGFBP7 through gain- and loss-of-function experiments and a range of techniques including qPCR, CCK-8, EdU, flow cytometry, Western blot, immunofluorescence, and Oil Red O staining to detect the proliferation, differentiation, and lipid deposition in CPMs and intramuscular preadipocytes. We ascertained that the expression levels of the IGFBP7 gene increased as cell differentiation progresses in CPMs and intramuscular preadipocytes, and that IGFBP7 promotes the proliferation and differentiation of these cells, as well as facilitates intracellular lipid deposition. Furthermore, we investigated the regulatory mechanism of IGFBP7 expression by using co-transfection strategy and dual-luciferase reporter assay, and discovered that the myogenic transcription factors (MRF), myoblast determination factor (MyoD) and myogenin (MyoG), along with the adipocyte-specific transcription factor (TF) CCAAT/enhancer-binding protein α (C/EBPα), can bind to the core transcription activation region of the IGFBP7 promoter located 500 bp upstream from the transcription start site, thereby promoting IGFBP7 transcription and expression. Taken together, our study underscores the role of IGFBP7 as a positive regulator for myogenesis and adipogenesis, while also elucidating the functional and transcriptional regulatory mechanisms of IGFBP7 in chicken skeletal muscle development and intramuscular adipogenesis.

Functional study of the ST6GAL2 gene regulating skeletal muscle growth and development

ST6GAL2, a member of the sialoglycosyltransferase family, primarily localizes within the cellular Golgi apparatus. However, the role of the ST6GAL2 gene in skeletal muscle growth and development remains elusive. In this study, the impact of the ST6GAL2 gene on the proliferation, differentiation, and apoptosis of primary chicken myoblasts at the cellular level was investigated. Quantitative fluorescent PCR was used to measure the expression levels of genes. Subsequently, using gene knockout mice, we assessed its effects on skeletal muscle growth and development in vivo. Our findings reveal that the ST6GAL2 gene promotes the expression of cell cycle and proliferation-related genes, including CCNB2 and PCNA, and apoptosis-related genes, such as Fas and Caspase-9. At the individual level, double knockout of ST6GAL2 inhibited the formation of both fast and slow muscle fibers in the quadriceps, extensor digitorum longus, and tibial anterior muscle, while promoting their formation in the gastrocnemius and soleus. These results collectively demonstrate that the ST6GAL2 gene facilitates the proliferation, apoptosis, and fusion processes of primary chicken myoblasts. Additionally, it promotes the enlargement of cross-sectional muscle fiber areas and regulates the formation of fast and slow muscle fibers at the individual level, albeit inhibiting muscle fusion. This study provides valuable insights into the role of the ST6GAL2 gene in promoting proliferation of skeletal muscle.

Selenium promotes broiler myoblast proliferation through the ROS/PTEN/PI3K/AKT signaling axis

Selenium (Se), an indispensable trace element in broiler chickens, is closely associated with the growth and development of skeletal muscles. However, the role of Se in the proliferation of broiler myoblasts and its specific biological mechanisms have not been elucidated. In the present study, an in vitro growth model of broiler pectoral myoblasts cultured with Se (Na2SeO3) for 24 h was established. Using light microscopy, Cell Counting Kit-8 (CCK-8) assay, and flow cytometry, we found that compared to the control (Con) group, Se supplemental level obviously promoted myoblast proliferation and prevented cell cycle arrest from the G1 phase to the S + G2 phase. Through intracellular reactive oxygen species (ROS) generation detection, western blotting, and quantitative reverse transcription-polymerase chain reaction (qRT-PCR), the study showed that the reduced ROS production caused by Se supplementation significantly decreased PTEN expression and activated the PI3K/AKT signaling pathway in myoblasts, thereby promoting the P53/P21/CyclinD1-regulated cell cycle progression, as well as the expression of proliferation-related myogenic regulatory factors (MRF). Our findings support the potential of Se to maintain the proliferative capacity of chicken myoblasts and emphasize the importance of Se intake in regulating skeletal muscle growth and development in poultry.

Glucosamine inhibits myoblast proliferation and differentiation, and stimulates myotube atrophy through distinct signal pathways

Glucosamine (GlcN) is one of the dietary supplements used in the treatment of osteoarthritis. Endogenously, GlcN is synthesized from glucose through the hexosamine pathway. In addition to ameliorating arthritis, several biological functions of GlcN have been reported, including insulin resistance in skeletal muscle. However, the regulatory role of GlcN in skeletal muscle development is not clear. We therefore investigated the effect of GlcN on myoblast proliferation, differentiation, and myotube development and their underlying mechanisms in C2C12 cells. Myoblast proliferation was measured by MTT assay. The expressions of MyoD, myogenin (MyoG), and myosin heavy chain (MyHC) were identified as determinants of myoblast differentiation. Expressions of atrogin-1 and muscle RING-finger protein-1 (MuRF-1) were identified as markers of myotube atrophy. The results show that treatment with GlcN significantly reduced myoblast proliferation and phosphorylation of Stat3 and S6K. These findings suggest that GlcN can inhibit growth of myoblasts through inhibiting phosphorylation of Stat3 and S6K. In addition, GlcN significantly suppressed the expression of MyoD, MyoG, and MyHC, as well as myotube formation. Pretreatment of C2C12 myoblast cells with ER stress inhibitors significantly blocked GlcN-inhibited MyHC expression and myotube formation. It can be concluded that GlcN suppressed myogenic differentiation via a pathway that involved ER stress. Moreover, GlcN decreased myotube diameter and expression of MyHC, as well as increased MuRF-1 in C2C12 myotubes. Meanwhile, GlcN also reduced the expressions of phosphorylated Akt and mTOR were stimulated after GlcN treatment in C2C12 myotubes. Thus, GlcN induced skeletal muscle atrophy by inhibiting the protein synthesis pathway. Chronic GlcN infusion also caused skeletal muscle atrophy in mice. In conclusion, GlcN regulated important stages of skeletal muscle development through different signaling pathways.

Effect of Lactational Low-Protein Diet on Skeletal Muscle during Adulthood and Ageing in Male and Female Mouse Offspring.

Sarcopenia is characterised by the loss of skeletal muscle mass and function, which leads to a high risk of increased morbidity and mortality. Maternal malnutrition has been linked to impaired development of skeletal muscle of the offspring; however, there are limited studies that report the long-term effect of a maternal low-protein diet during lactation on the ageing of skeletal muscles. This study aimed to examine how a maternal low-protein diet (LPD) during lactation affects skeletal muscle ageing in the offspring. Pups born from control mothers were lactated by mothers fed with an LPD. Post-weaning, mice were either maintained on an LPD or switched to a control, normal-protein diet (NPD). In males, an LPD mainly affected the size of the myofibres without a major effect on fibre number and led to reduced grip strength in ageing mice (24 months). Female mice from mothers on an LPD had a lower body and muscle weight at weaning but caught up with control mice at 3 months. During ageing, the muscle weight, myofibre number and survival rate of female pups were significantly affected. These findings highlight the effect of an LPD during lactation on skeletal muscle ageing, the lifespan of offspring and the importance of sexual dimorphism in response to dietary challenges.

Promoting and accelerating muscle regeneration through cell therapy in a mouse model

ObjectivesSkeletal muscle injuries and disorders are universal clinical challenges with direct and indirect mechanisms and notable residual effects, such as prolonged, intense pain and physical disability. Stem cells, an innovative tool for cell therapy for musculoskeletal disorders, specifically promote skeletal muscle regeneration. This study was aimed at investigating the use of mesenchymal stem cells (MSCs) and their differentiated myocytes as a cell-based therapy to promote regeneration in damaged or diseased skeletal muscle. MethodsBone marrow mesenchymal stem cells (BM-MSCs) were isolated from the bone marrow of adult mice and grown in tissue culture flasks. The BM-MSCs were positive for CD90 and CD105, and negative for CD45 and CD34. These cells were induced with specific differentiation medium in vitro to differentiate into a skeletal muscle cell lineage over 7 days. Skeletal muscle differentiation was characterized according to morphology through hematoxylin and eosin staining, and scanning electron microscopy. Immunostaining for Myf-6, myosin heavy chain (MHC), and desmin—specific factors for skeletal muscle development—was performed to confirm skeletal muscle differentiation. An in vivo study in a muscle injury model was used to evaluate cell therapy based on naïve stem cells and differentiated myocytes. ResultsCultured mouse BM-MSCS were positive for CD90 and CD105, and negative for CD45 and CD34. These cells developed into skeletal muscle with strong skeletal muscle differentiation potential, as confirmed by immunohistochemistry for the markers Myf6, MHC, and desmin. The differentiated myocytes showed better repair enhancement than undifferentiated stem cells after transplantations into a mouse model of skeletal muscle atrophy. ConclusionsMyocytes derived from BM-MSCs may be incorporated into muscular atrophy treatment as a biological strategy for managing skeletal muscle diseases and injuries, thus advancing cell-based clinical treatments.

Spatial Transcriptomics Analysis: Maternal Obesity Impairs Myogenic Cell Migration and Differentiation during Embryonic Limb Development.

Limb muscle is responsible for physical activities and myogenic cell migration during embryogenesis is indispensable for limb muscle formation. Maternal obesity (MO) impairs prenatal skeletal muscle development, but the effects of MO on myogenic cell migration remain to be examined. C57BL/6 mice embryos were collected at E13.5. The GeoMx DSP platform was used to customize five regions along myogenic cell migration routes (myotome, dorsal/ventral limb, limb stroma, limb tip), and data were analyzed by GeomxTools 3.6.0. A total of 2224 genes were down-regulated in the MO group. The GO enrichment analysis showed that MO inhibited migration-related biological processes. The signaling pathways guiding myogenic migration such as hepatocyte growth factor signaling, fibroblast growth factor signaling, Wnt signaling and GTPase signaling were down-regulated in the MO E13.5 limb tip. Correspondingly, the expression levels of genes involved in myogenic cell migration, such as Pax3, Gab1, Pxn, Tln2 and Arpc, were decreased in the MO group, especially in the dorsal and ventral sides of the limb. Additionally, myogenic differentiation-related genes were down-regulated in the MO limb. MO impedes myogenic cell migration and differentiation in the embryonic limb, providing an explanation for the impairment of fetal muscle development and offspring muscle function due to MO.

Evaluation of Reference Gene Stability in Goat Skeletal Muscle Satellite Cells during Proliferation and Differentiation Phases.

The process of skeletal muscle development is intricate and involves the regulation of a diverse array of genes. Accurate gene expression profiles are crucial for studying muscle development, making it essential to choose the right reference genes for real-time quantitative PCR (RT-qPCR). In the present study, eight candidate reference genes were identified from our previous transcriptome sequencing analysis of caprine skeletal muscle satellite cells (MuSCs), and two traditional reference genes (ACTB and GAPDH) were assessed. The quantitative levels of the candidate reference genes were determined through the RT-qPCR technique, while the stability of their expression was evaluated utilizing the GeNorm, NormFinder, BestKeeper, and RefFinder programs. Furthermore, the chosen reference genes were utilized for the normalization of the gene expression levels of PCNA and Myf5. It was determined that conventional reference genes, including ACTB and GAPDH, were not appropriate for normalizing target gene expression. Conversely, RPL14 and RPS15A, identified through RNA sequencing analysis, exhibited minimal variability and were identified as the optimal reference genes for normalizing gene expression during the proliferation and differentiation of goat MuSCs. Our research offers a validated panel of optimal reference genes for the detection of differentially expressed genes in goat muscle satellite cells using RT-qPCR.