Changes in protein fluxes and gene expression in non-injured muscle tissue distant from an acute myotoxic injury in male mice.

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.

Fast and slow myofiber nuclei, satellite cells, and size distribution with lifelong endurance exercise in men and women.

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.

Targeted expression of heme oxygenase-1 in satellite cells improves skeletal muscle pathology in dystrophic mice.

BACKGROUND: Adult muscle-resident myogenic stem cells, satellite cells (SCs), that play non-redundant role in muscle regeneration, are intrinsically impaired in Duchenne muscular dystrophy (DMD). Previously we revealed that dystrophic SCs express low level of anti-inflammatory and anti-oxidative heme oxygenase-1 (HO-1, HMOX1). Here we assess whether targeted induction of HMOX1 affect SC function and alleviates hallmark symptoms of DMD. METHODS: We generated double-transgenic mouse model (mdx;HMOX1Pax7Ind) that allows tamoxifen (TX)-inducible HMOX1 expression in Pax7 positive cells of dystrophic muscles. Mdx;HMOX1Pax7Ind and control mdx mice were subjected to 5-day TX injections (75 mg/kg b.w.) followed by acute exercise protocol with high-speed treadmill (12 m/min, 45 min) and downhill running to worsen skeletal muscle phenotype and reveal immediate effects of HO-1 on muscle pathology and SC function. RESULTS: HMOX1 induction caused a drop in SC pool in mdx;HMOX1Pax7Ind mice (vs. mdx counterparts), while not exaggerating the effect of physical exercise. Upon physical exercise, the proliferation of SCs and activated CD34- SC subpopulation, was impaired in mdx mice, an effect that was reversed in mdx;HMOX1Pax7Ind mice, however, both in vehicle- and TX-treated animals. This corresponded to the pattern of HO-1 expression in skeletal muscles. At the tissue level, necrotic events of selective skeletal muscles of mdx mice and associated increase in circulating levels of muscle damage markers were blunted in HO-1 transgenic animals which showed also anti-inflammatory cytokine profile (vs. mdx). CONCLUSIONS: Targeted expression of HMOX1 plays protective role in DMD and alleviates dystrophic muscle pathology.

Integrated ATAC-seq and RNA-seq Analysis of In Vitro Cultured Skeletal Muscle Satellite Cells to Understand Changes in Cell Proliferation.

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.

Daily Eicosapentaenoic Acid Infusion in IUGR Fetal Lambs Reduced Systemic Inflammation, Increased Muscle ADRß2 Content, and Improved Myoblast Function and Muscle Growth.

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.

A dual-color PAX7 and MYF5 in vivo reporter to investigate muscle stem cell heterogeneity in regeneration and aging.

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.

IFRD2, a target of miR-2400, regulates myogenic differentiation of bovine skeletal muscle satellite cells via decreased phosphorylation of ERK1/2 proteins.

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.

Biocomputational screening of natural compounds targeting 15-hydroxyprostaglandin dehydrogenase to improve skeletal muscle during aging.

Skeletal muscle (SM) contains a diverse population of muscle stem (or satellite) cells, which are essential for the maintenance of muscle tissue and positively regulated by prostaglandin E2 (PGE2). However, in aged SM, PGE2 levels are reduced due to increased prostaglandin catabolism by 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a negative regulator of SM tissue repair and regeneration. Screening of a library of 80,617 natural compounds in the ZINC database against 15-PGDH was conducted from PyRx. Further, drug-likeness rules, including those of Lipinski, Ghose, Veber, Egan, and Muegge were performed. The selected complex was forwarded for MD simulations up to 100ns. Based on free energy of binding obtained from docking revealed that ZINC14557836 and ZINC14638400 more potently inhibiting to 15-PGDH than SW033291 (the control and high-affinity inhibitor of 15-PGDH). The free energies of binding obtained from PyRx for 15-PGDH-ZINC14557836, 15-PGDH-ZINC14638400, and 15-PGDH-SW033291 complexes were - 10.30, -9.80, and - 8.0 kcal/mol, respectively. Root mean square deviations (RMSDs), root mean square fluctuations (RMSFs), radii of gyration (Rg), solvent-accessible surface areas (SASAs), and H-bond parameters obtained by 100 ns MD simulations predicted ZINC14557836 and ZINC14638400 more stably complexed with 15-PGDH than SW033291. The several parameters, including physicochemical properties and drug-likenesses, were within acceptable limits, and ZINC14557836 and ZINC14638400 also satisfied other drug-likeness rules, including those of Lipinski, Ghose, Veber, Egan, and Muegge. These findings suggest that ZINC14557836 and ZINC14638400 provide starting points for the development of medications that increase SM regeneration and muscle stem (or satellite) cell numbers by inhibiting 15-PGDH.

Developmental and therapeutic implications of IL4ra expression for rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is a solid tumor whose metastatic progression can be accelerated through interleukin-4 receptor alpha (Il4ra) mediated interaction with normal muscle stem cells (satellite cells). To understand the function of Il4ra in this tumor initiation phase of RMS, we conditionally deleted Il4ra in genetically-engineered RMS mouse models. Nullizygosity of Il4ra altered the latency, site and/or stage distribution of RMS tumors compared to IL4RA intact models. Primary tumor cell cultures taken from the genetically-engineered models then used in orthotopic allografts further defined the interaction of satellite cells and RMS tumor cells in the context of tumor initiation: in alveolar rhabdomyosarcoma (ARMS), satellite cell co-injection was necessary for Il4ra null tumor cells engraftment, whereas in embryonal rhabdomyosarcoma (ERMS), satellite cell co-injection decreased latency of engraftment of Il4ra wildtype tumor cells but not Il4ra null tumor cells. When refocusing on Il4ra wildtype tumors by single cell sequencing and cytokine studies, we have uncovered a putative signaling interplay of Il4 from T-lymphocytes being received by Il4ra + rhabdomyosarcoma tumor cells, which in turn express Ccl2, the ligand for Ccr2 and Ccr5. Taken together, these results suggest that mutations imposed during tumor initiation have different effects than genetic or therapeutic intervention imposed once tumors are already formed. We also propose that CCL2 and its cognate receptors CCR2 and/or CCR5 are potential therapeutic targets in Il4ra mediated RMS progression.

Single-cell RNA-seq reveals novel interaction between muscle satellite cells and fibro-adipogenic progenitors mediated with FGF7 signalling.

BACKGROUND: Muscle satellite cells (MuSCs) exert essential roles in skeletal muscle adaptation to growth, injury and ageing, and their functions are extensively modulated by microenvironmental factors. However, the current knowledge about the interaction of MuSCs with niche cells is quite limited. METHODS: A 10× single-cell RNA sequencing (scRNA-seq) was performed on porcine longissimus dorsi and soleus (SOL) muscles to generate a single-cell transcriptomic dataset of myogenic cells and other cell types. Sophisticated bioinformatic analyses, including unsupervised clustering analysis, marker gene, gene set variation analysis (GSVA), AUCell, pseudotime analysis and RNA velocity analysis, were performed to explore the heterogeneity of myogenic cells. CellChat analysis was used to demonstrate cell-cell communications across myogenic cell subpopulations and niche cells, especially fibro-adipogenic progenitors (FAPs). Integrated analysis with human and mice datasets was performed to verify the expression of FGF7 across diverse species. The role of FGF7 on MuSC proliferation was evaluated through administering recombinant FGF7 to porcine MuSCs, C2C12, cardiotoxin (CTX)-injured muscle and d-galactose ( d-gal)-induced ageing model. RESULTS: ScRNA-seq totally figured out five cell types including myo-lineage cells and FAPs, and myo-lineage cells were further classified into six subpopulations, termed as RCN3+, S100A4+, ID3+, cycling (MKI67+), MYF6+ and MYMK+ satellite cells, respectively. There was a higher proportion of cycling and MYF6+ cells in the SOL population. CellChat analysis uncovered a particular impact of FAPs on myogenic cells mediated by FGF7, which was relatively highly expressed in SOL samples. Administration of FGF7 (10 ng/mL) significantly increased the proportion of EdU+ porcine MuSCs and C2C12 by 4.03 ± 0.81% (P < 0.01) and 6.87 ± 2.17% (P < 0.05), respectively, and knockdown of FGFR2 dramatically abolished the pro-proliferating effects (P < 0.05). In CTX-injured muscle, FGF7 significantly increased the ratio of EdU+/Pax7+ cells by 15.68 ± 5.45% (P < 0.05) and elevated the number of eMyHC+ regenerating myofibres by 19.7 ± 4.25% (P < 0.01). Under d-gal stimuli, FGF7 significantly reduced γH2AX+ cells by 17.19 ± 3.05% (P < 0.01) in porcine MuSCs, induced EdU+ cells by 4.34 ± 1.54% (P < 0.05) in C2C12, and restored myofibre size loss and running exhaustion in vivo (all P < 0.05). CONCLUSIONS: Our scRNA-seq reveals a novel interaction between muscle FAPs and satellite cells mediated by FGF7-FGFR2. Exogenous FGF7 augments the proliferation of satellite cells and thus benefits muscle regeneration and counteracts age-related myopathy.

Circadian Clock in Muscle Disease Etiology and Therapeutic Potential for Duchenne Muscular Dystrophy.

Circadian clock and clock-controlled output pathways exert temporal control in diverse aspects of skeletal muscle physiology, including the maintenance of muscle mass, structure, function, and metabolism. They have emerged as significant players in understanding muscle disease etiology and potential therapeutic avenues, particularly in Duchenne muscular dystrophy (DMD). This review examines the intricate interplay between circadian rhythms and muscle physiology, highlighting how disruptions of circadian regulation may contribute to muscle pathophysiology and the specific mechanisms linking circadian clock dysregulation with DMD. Moreover, we discuss recent advancements in chronobiological research that have shed light on the circadian control of muscle function and its relevance to DMD. Understanding clock output pathways involved in muscle mass and function offers novel insights into the pathogenesis of DMD and unveils promising avenues for therapeutic interventions. We further explore potential chronotherapeutic strategies targeting the circadian clock to ameliorate muscle degeneration which may inform drug development efforts for muscular dystrophy.

Generation of Skeletal Muscle Organoids from Human Pluripotent Stem Cells.

Various protocols have been proven effective in the directed differentiation of mouse and human pluripotent stem cells into skeletal muscles and used to study myogenesis. Current 2D myogenic differentiation protocols can mimic muscle development and its alteration under pathological conditions such as muscular dystrophies. 3D skeletal muscle differentiation approaches can, in addition, model the interaction between the various cell types within the developing organoid. Our protocol ensures the differentiation of human embryonic/induced pluripotent stem cells (hESC/hiPSC) into skeletal muscle organoids (SMO) via cells with paraxial mesoderm and neuromesodermal progenitors' identity and further production of organized structures of the neural plate margin and the dermomyotome. Continuous culturing omits neural lineage differentiation and promotes fetal myogenesis, including the maturation of fibroadipogenic progenitors and PAX7-positive myogenic progenitors. The PAX7 progenitors resemble the late fetal stages of human development and, based on single-cell transcriptomic profiling, cluster close to adult satellite cells of primary muscles. To overcome the limited availability of muscle biopsies from patients with muscular dystrophy during disease progression, we propose to use the SMO system, which delivers a stable population of skeletal muscle progenitors from patient-specific iPSCs to investigate human myogenesis in healthy and diseased conditions. Key features • Development of skeletal muscle organoid differentiation from human pluripotent stem cells, which recapitulates myogenesis. • Analysis of early embryonic and fetal myogenesis. • Provision of skeletal muscle progenitors for in vitro and in vivo analysis for up to 14 weeks of organoid culture. • In vitro myogenesis from patient-specific iPSCs allows to overcome the bottleneck of muscle biopsies of patients with pathological conditions.

Hydrogel/microcarrier cell scaffolds for rapid expansion of satellite cells from large yellow croakers: Differential analysis between 2D and 3D cell culture.

Cell culture meat is based on the scaled-up expansion of seed cells. The biological differences between seed cells from large yellow croakers in the two-dimensional (2D) and three-dimensional (3D) culture systems have not been explored. Here, satellite cells (SCs) from large yellow croakers (Larimichthys crocea) were grown on cell climbing slices, hydrogels, and microcarriers for five days to analyze the biological differences of SCs on different cell scaffolds. The results exhibited that SCs had different cell morphologies in 2D and 3D cultures. Cell adhesion receptors (Itgb1andsdc4) and adhesion spot markervclof the 3D cultures were markedly expressed. Furthermore, myogenic decision markers (Pax7andmyod) were significantly enhanced. However, the expression of myogenic differentiation marker (desmin) was significantly increased in the microcarrier group. Combined with the transcriptome data, this suggests that cell adhesion of SCs in 3D culture was related to the integrin signaling pathway. In contrast, the slight spontaneous differentiation of SCs on microcarriers was associated with rapid cell proliferation. This study is the first to report the biological differences between SCs in 2D and 3D cultures, providing new perspectives for the rapid expansion of cell culture meat-seeded cells and the development of customized scaffolds.

Effect of LncRNA LOC106505926 on myogenesis and Lipogenesis of porcine primary cells.

BACKGROUND: Skeletal muscle development and fat deposition have important effects on meat quality. The study of regulating skeletal muscle development and fat deposition is of great significance in improving the quality of carcass and meat. In the present study, whole transcriptome sequencing (including RNA-Seq and miRNA-Seq) was performed on the longissimus dorsi muscle (LDM) of Jinfen White pigs at 1, 90, and 180 days of age. RESULTS: The results showed that a total of 245 differentially expressed miRNAs were screened in any two comparisons, which may be involved in the regulation of myogenesis. Among them, compared with 1-day-old group, miR-22-5p was significantly up-regulated in 90-day-old group and 180-day-old group. Functional studies demonstrated that miR-22-5p inhibited the proliferation and differentiation of porcine skeletal muscle satellite cells (PSCs). Pearson correlation coefficient analysis showed that long non-coding RNA (lncRNA) LOC106505926 and CXXC5 gene had strong negative correlations with miR-22-5p. The LOC106505926 and CXXC5 were proven to promote the proliferation and differentiation of PSCs, as opposed to miR-22-5p. In terms of mechanism, LOC106505926 functions as a molecular sponge of miR-22-5p to modulate the expression of CXXC5, thereby inhibits the differentiation of PSCs. In addition, LOC106505926 regulates the differentiation of porcine preadipocytes through direct binding with FASN. CONCLUSIONS: Collectively, our results highlight the multifaceted regulatory role of LOC106505926 in controlling skeletal muscle and adipose tissue development in pigs and provide new targets for improving the quality of livestock products by regulating skeletal muscle development and fat deposition.

Leucine promotes energy metabolism and stimulates slow-twitch muscle fibers expression through AMPK/mTOR signaling in equine skeletal muscle satellite cells.

Previous research has shown that leucine (Leu) can stimulate and enhance the proliferation of equine skeletal muscle satellite cells (SCs). The gene expression profile associated with Leu-induced proliferation of equine SCs has also been documented. However, the specific role of Leu in regulating the expression of slow-twitch muscle fibers (slow-MyHC) and mitochondrial function in equine SCs, as well as the underlying mechanism, remains unclear. During this investigation, equine SCs underwent culturing in differentiation medium and were subjected to varying concentrations of Leu (0 mM, 0.5 mM, 1 mM, 2 mM, 5 mM, and 10 mM) over a span of 3 days. AMP-activated protein kinase (AMPK) inhibitor Compound C and mammalian target of rapamycin complex (mTOR) inhibitor Rapamycin were utilized to explore its underlying mechanism. Here we showed that the expression of slow-MyHC at 2 mM Leu level was significantly higher than the concentration levels of 0 mM,0.5 mM and 10 mM (P <0.01), and there was no significant difference compared to other groups (P > 0.05); the basal respiration, maximum respiration, standby respiration and the expression of slow-MyHC, PGC-1α, Cytc, ND1, TFAM, and COX1 were significantly increased with Leu supplementation (P < 0.01). We also found that Leu up-regulated the expression of key proteins on AMPK and mTOR signaling pathways, including LKB1, p-LKB1, AMPK, p-AMPK, S6, p-S6, 4EBP1, p-4EBP1, mTOR and p-mTOR (P < 0.05 or P < 0.01). Notably, when we treated the equine SCs with the AMPK inhibitor Compound C and the mTOR inhibitor Rapamycin, we observed a reduction in the beneficial effects of Leu on the expression of genes related to slow-MyHC and signaling pathway-related gene expressions. This study provides novel evidence that Leu promotes slow-MyHC expression and enhances mitochondrial function in equine SCs through the AMPK/mTOR signaling pathways, shedding light on the underlying mechanisms involved in these processes for the first time.

Molecular Regulation of Porcine Skeletal Muscle Development: Insights from Research on CDC23 Expression and Function.

Cell division cycle 23 (CDC23) is a component of the tetratricopeptide repeat (TPR) subunit in the anaphase-promoting complex or cyclosome (APC/C) complex, which participates in the regulation of mitosis in eukaryotes. However, the regulatory model and mechanism by which the CDC23 gene regulates muscle production in pigs are largely unknown. In this study, we investigated the expression of CDC23 in pigs, and the results indicated that CDC23 is widely expressed in various tissues and organs. In vitro cell experiments have demonstrated that CDC23 promotes the proliferation of myoblasts, as well as significantly positively regulating the differentiation of skeletal muscle satellite cells. In addition, Gene Set Enrichment Analysis (GSEA) revealed a significant downregulation of the cell cycle pathway during the differentiation process of skeletal muscle satellite cells. The protein-protein interaction (PPI) network showed a high degree of interaction between genes related to the cell cycle pathway and CDC23. Subsequently, in differentiated myocytes induced after overexpression of CDC23, the level of CDC23 exhibited a significant negative correlation with the expression of key factors in the cell cycle pathway, suggesting that CDC23 may be involved in the inhibition of the cell cycle signaling pathway in order to promote the differentiation process. In summary, we preliminarily determined the function of CDC23 with the aim of providing new insights into molecular regulation during porcine skeletal muscle development.

Murine skeletal muscle satellite cells isolation and preliminary study on regulation in immune microenvironment during nurse cells formation of Trichinella spiralis infection.

As an intracellular parasitic nematode, Trichinella spiralis (T. spiralis) can induce the formation of nurse cells (NC) in host muscles and keep it to survive within the NC for an extended period. The formation of NC is similar to muscle cell injury and repair which lead to the arrest of satellite cells in the G2/M phase and build a suitable parasitic environment for the muscle larvae of T. spiralis. However, the molecular mechanisms involved in skeletal muscle repair through skeletal muscle satellite cells (SMSC) and the host immune response during T. spiralis infection have not been fully elucidated. In this study, histopathological examination revealed that the severity of damage increased as the infection progressed in the soleus muscle. SMSCs were isolated from BALB/c mice infected with T. spiralis at 4, 21 and 35 days post-infection (dpi). The immunological characteristics of these cells were analyzed by real-time PCR and flow cytometry (FCM). FCM analysis revealed a notable increase in the expression of B7 homolog 1 (B7-H1) in SMSCs following T. spiralis infection, while conversely, the expression of inducible costimulatory ligand (ICOSL) significantly decreased. Furthermore, real-time PCR results showed that toll like receptor 3 (TLR3) expression in SMSCs of the infected mice was upregulated at 21 dpi. The expression levels of three subtypes (PPARα, PPARß and PPARγ) of peroxisome proliferator-activated receptors (PPARs) also increased in the cells. This study highlights the immunological regulation significance of SMSCs host during T. spiralis infection and suggests that SMSCs actively participant in the local immune response to T. spiralis by regulating the interaction between the parasite and the host.

Anti-apoptotic protein Bcl-2 contributes to the determination of reserve cells during myogenic differentiation of C2C12 cells.

Skeletal muscle's regenerative ability is vital for maintaining muscle function, but chronic diseases like Duchenne muscular dystrophy can deplete this capacity. Muscle satellite cells, quiescent in normal situations, are activated during muscle injury, expressing myogenic regulatory factors, and producing myogenic progenitor cells. It was reported that muscle stem cells in primary culture and reserve cells in C2C12 cells express anti-apoptotic protein Bcl-2. Although the role of Bcl-2 expressed in myogenic cells has been thought to be to enhance cell viability, we hypothesized that Bcl-2 may promote the formation of reserve cells. The expression pattern analysis showed the expression of Bcl-2 in undifferentiated mononucleated cells, emphasizing its usefulness as a reserve cell marker and reminding us that cells expressing Bcl-2 have low proliferative potential. Silencing of Bcl-2 by transfection with siRNA decreased cell viability and the number of reserve cells, while overexpression of Bcl-2 not only increases cell viability but also inhibits muscle differentiation and proliferation. These results emphasize dual roles of Bcl-2 in protecting cells from apoptosis and contributing to reserve cell formation by regulating myoblast proliferation and/or differentiation. Overall, the study sheds light on the multifaceted role of Bcl-2 in the maintenance of skeletal muscle regeneration.

Establishment and Characterization of SV40 T-Antigen Immortalized Porcine Muscle Satellite Cell.

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-ß-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.

Identification of mulberry leaf flavonoids and evaluating their protective effects on H2O2-induced oxidative damage in equine skeletal muscle satellite cells.

Introduction: Horses are susceptible to oxidative stress during strenuous endurance exercise, leading to muscle fatigue and damage. Mulberry leaf flavonoids (MLFs) possess significant antioxidant properties. However, the antioxidant efficacy of MLFs can be influenced by the extraction process, and their impact on H2O2-induced oxidative stress in equine skeletal muscle satellite cells (ESMCs) remains unexplored. Methods: Our study employed three extraction methods to obtain MLFs: ultrasound-assisted extraction (CEP), purification with AB-8 macroporous resin (RP), and n-butanol extraction (NB-EP). We assessed the protective effects of these MLFs on H2O2-induced oxidative stress in ESMCs and analyzed the MLF components using metabolomics. Results: The results revealed that pre-treatment with MLFs dose-dependently protected ESMCs against H2O2-induced oxidative stress. The most effective concentrations were 0.8 mg/mL of CEP, 0.6 mg/mL of RP, and 0.6 mg/mL of NB-EP, significantly enhancing EMSC viability (p < 0.05). These optimized MLF concentrations promoted the GSH-Px, SOD and T-AOC activities (p < 0.05), while reducing MDA production (p < 0.05) in H2O2-induced ESMCs. Furthermore, these MLFs enhanced the gene expression, including Nrf2 and its downstream regulatory genes (TrxR1, GPX1, GPX3, SOD1, and SOD2) (p < 0.05). In terms of mitochondrial function, ESMCs pre-treated with MLFs exhibited higher basal respiration, spare respiratory capacity, maximal respiration, ATP-linked respiration compared to H2O2-induced ESMCs (p < 0.05). Additionally, MLFs enhanced cellular basal glycolysis, glycolytic reserve, and maximal glycolytic capacity (p < 0.05). Metabolomics analysis results revealed significant differences in mulberrin, kaempferol 3-O-glucoside [X-Mal], neohesperidin, dihydrokaempferol, and isobavachalcone among the three extraction processes (p < 0.05). Discussion: Our study revealed that MLFs enhance antioxidant enzyme activity, alleviate oxidative damage in ESMCs through the activation of the Nrf2 pathway, and improve mitochondrial respiration and cell energy metabolism. Additionally, we identified five potential antioxidant flavonoid compounds, suggesting their potential incorporation into the equine diet as a strategy to alleviate exercise-induced oxidative stress.