Myogenic Regulatory Factors Research Articles

6979 Effect of Orchiectomy on Muscle Growth and Differentiation Factors in Male Mice With Kidney Disease

Abstract Disclosure: O. Marks: None. J. Bruno: None. D. Wu: None. A. Patwardhan: None. Y. Komaru: None. G. Ling: None. E. Kefalogianni: None. R. Aurora: None. S.S. Dhindsa: None. A. Herrlich: None. Background: Sarcopenia and frailty are major clinical concerns in participants with chronic kidney disease (CKD). In males, an additional factor contributes to the development of sarcopenia. Around half of the men with CKD have subnormal serum testosterone (T) concentrations. The mechanisms that underlie the effect of low T concentrations on muscle growth in CKD are not known. In this study, we performed orchiectomy in male mice with kidney disease and evaluated the expression and protein content of the following muscle growth and differentiation factors. Methods: Forty mice were divided into 4 groups: 1) renal injury, induced by ischemia-induced reperfusion (IRI, N=11) at 2 months of age; 2) orchidectomy (ORX), performed 4 weeks after IRI (IRI + ORX, N=9); 3) sham-IRI and ORX (sham-ORX, N=8); and 4) sham-sham surgery (N=12). The mice were sacrificed when they were ∼7 months old (mean age 206±13 days). Gastrocnemius and levator ani muscles were harvested to measure expression by RT-qPCR of muscle catabolism markers (Atrogin-1 and ubiquitin ligase MuRF1), muscle growth inhibitors, myostatin and myogenic regulatory factor (Mrf4), fibroblast growth factor-2 (FGF2, a stimulator of satellite stem cells) and its receptor FGFR2, androgen receptor (AR) and IGF-1. Results: The mean BUN (blood urea nitrogen) at the time of sacrifice was higher in the IRI/ORX (32±4 mg/dl) and IRI/Sham (26±5 mg/dl) groups as compared to sham-sham (21±5 mg/dl, p<0.05 for both). There was a significant reduction (p<0.05) in the expression of myostatin (by 41%), Mrf4 (29%), atrogin-1 (51%) and MuRF1 (32%) in gastrocnemius muscle from the ORX-IRI and ORX-sham groups, while there was no difference in FGF2 and FGFR2, as compared to sham-sham group. Data from levator ani muscle (the most androgen sensitive muscle in mammals) showed increases in AR (64%) and Atrogin-1 (66%); and decreases in IGF-1 (88%) and myostatin (86%) in ORX-IRI and ORX-sham groups. Consistent with the mRNA expression, we found a drastic decline in protein content of myostatin in gastrocnemius (85%) in ORX-IRI and ORX-sham groups as compared to sham-sham (p=0.04). Conclusions: These data show that ORX in mice with renal injury is associated with changes in muscle growth and differentiation factors. Myostatin was the most affected gene, with 41-86% decline in mRNA and protein. These data are informative for attempts to develop treatments for sarcopenia in patients with kidney disease. Limitations: Our experimental model differs in some respects from hypogonadal men with CKD. ORX results in complete elimination of T, and thus much lower T concentrations than in men with kidney disease. There is a recovery of kidney function after IRI. We sacrificed the mice 5 months after renal injury was inflicted. Hence, the effect of IRI on muscle growth genes may have been different immediately following IRI. Thus, the impact of ORX was more apparent than of IRI in our study. Presentation: 6/1/2024

6979 Effect of Orchiectomy on Muscle Growth and Differentiation Factors in Male Mice With Kidney Disease

Abstract Disclosure: O. Marks: None. J. Bruno: None. D. Wu: None. A. Patwardhan: None. Y. Komaru: None. G. Ling: None. E. Kefalogianni: None. R. Aurora: None. S.S. Dhindsa: None. A. Herrlich: None. Background: Sarcopenia and frailty are major clinical concerns in participants with chronic kidney disease (CKD). In males, an additional factor contributes to the development of sarcopenia. Around half of the men with CKD have subnormal serum testosterone (T) concentrations. The mechanisms that underlie the effect of low T concentrations on muscle growth in CKD are not known. In this study, we performed orchiectomy in male mice with kidney disease and evaluated the expression and protein content of the following muscle growth and differentiation factors. Methods: Forty mice were divided into 4 groups: 1) renal injury, induced by ischemia-induced reperfusion (IRI, N=11) at 2 months of age; 2) orchidectomy (ORX), performed 4 weeks after IRI (IRI + ORX, N=9); 3) sham-IRI and ORX (sham-ORX, N=8); and 4) sham-sham surgery (N=12). The mice were sacrificed when they were ∼7 months old (mean age 206±13 days). Gastrocnemius and levator ani muscles were harvested to measure expression by RT-qPCR of muscle catabolism markers (Atrogin-1 and ubiquitin ligase MuRF1), muscle growth inhibitors, myostatin and myogenic regulatory factor (Mrf4), fibroblast growth factor-2 (FGF2, a stimulator of satellite stem cells) and its receptor FGFR2, androgen receptor (AR) and IGF-1. Results: The mean BUN (blood urea nitrogen) at the time of sacrifice was higher in the IRI/ORX (32±4 mg/dl) and IRI/Sham (26±5 mg/dl) groups as compared to sham-sham (21±5 mg/dl, p<0.05 for both). There was a significant reduction (p<0.05) in the expression of myostatin (by 41%), Mrf4 (29%), atrogin-1 (51%) and MuRF1 (32%) in gastrocnemius muscle from the ORX-IRI and ORX-sham groups, while there was no difference in FGF2 and FGFR2, as compared to sham-sham group. Data from levator ani muscle (the most androgen sensitive muscle in mammals) showed increases in AR (64%) and Atrogin-1 (66%); and decreases in IGF-1 (88%) and myostatin (86%) in ORX-IRI and ORX-sham groups. Consistent with the mRNA expression, we found a drastic decline in protein content of myostatin in gastrocnemius (85%) in ORX-IRI and ORX-sham groups as compared to sham-sham (p=0.04). Conclusions: These data show that ORX in mice with renal injury is associated with changes in muscle growth and differentiation factors. Myostatin was the most affected gene, with 41-86% decline in mRNA and protein. These data are informative for attempts to develop treatments for sarcopenia in patients with kidney disease. Limitations: Our experimental model differs in some respects from hypogonadal men with CKD. ORX results in complete elimination of T, and thus much lower T concentrations than in men with kidney disease. There is a recovery of kidney function after IRI. We sacrificed the mice 5 months after renal injury was inflicted. Hence, the effect of IRI on muscle growth genes may have been different immediately following IRI. Thus, the impact of ORX was more apparent than of IRI in our study. Presentation: 6/1/2024

Androgen receptor regulates the differentiation of myoblasts under cyclic mechanical stretch and its upstream and downstream signals

Our previous studies have demonstrated the important roles of androgen receptor (AR) in myoblast proliferation regulated by 15 % (mimic appropriate exercise) and 20 % (mimic excessive exercise) mechanical stretches. Except for myoblast proliferation, differentiation is also an important factor affecting muscle mass and strength. But the role of AR in stretch-regulated myoblast differentiation and AR's upstream and downstream signals remain unknown. In the present study, firstly the differences of myogenic differentiation between C2C12 (with AR expression) and L6 (without AR expression) myoblasts induced by 15 % and 20 % mechanical stretches were compared; secondly, AR antagonist flutamide and AR agonist GTx-007 were used in 15 % and 20 % stretched myoblasts respectively to confirm AR's roles in stretch-regulated myoblast differentiation; thirdly, RNA-seq, molecular dynamic simulation (MD) and co-immunoprecipitation were performed to screen the downstream and upstream molecules of AR during stretches. We found that (1) 15 % stretch increased while 20 % stretch decreased myotube number in differentiating C2C12 and L6 myoblasts, with more significant changes in C2C12 cells than L6 cells; (2) in stretched C2C12 myoblasts, AR antagonist flutamide inhibited 15 % stretch-promoted differentiation while AR agonist GTx-007 reversed 20 % stretch-inhibited differentiation (reflected by changes in myotube number, MHC contents of fast-twitch and slow-twitch fiber, and the levels of myogenic regulatory factors (MRFs) such as MyoD and myogenin); (3) KEGG analysis of RNA-seq showed that the differently expressed genes (DEGs) in C2C12 cells induced by 15 % stretch were enriched in FoxO and JAK-STAT signaling pathways, while DEGs by 20 % stretch were enriched in FoxO and MAPK signaling pathways; (4) MD and co-immunoprecipitation showed that β1 integrin could interact with AR and influence AR's activity in C2C12 cells. In conclusion, AR plays important roles in myoblast differentiation promoted by 15 % stretch while inhibited by 20 % stretch, which was fulfilled through FoxO-MRFs. In addition, α7β1 integrin may be a bridge linking mechanical stretch and AR. This study is beneficial to deeply understand the roles and mechanisms of AR in stretch-regulated muscle mass and strength; and reports firstly that myoblasts sense mechanical stimulus and transmit into intracellular AR via α7β1 integrin.

Evaluation of enzymatic protocols to optimize efficiency of bovine adipose tissue-derived mesenchymal stromal cell isolation

Sustainable food provision for a continuously growing human population is one of the major challenges for the next decades. Cultured meat represents one of the alternatives which is currently extensively explored. Yet, the most appropriate cell type, capable of long-term proliferation and myogenic differentiation, remains to be identified. Bovine mesenchymal stromal cells (MSCs) are considered as a promising cell source. Within the context of cultured meat production, it is mandatory to maximize cell yield per tissue source. Although many enzymatic methods to isolate MSCs from adipose tissue (AT) have been described, cell yield has never been compared. In this study, we evaluate 32 isolation conditions including four enzyme mixtures (Collagenase type I, Collagenase type I + Trypsin, LiberaseTM and Collagenase type IV) at varying concentrations and incubation times, regarding their efficiency to isolate MSCs from bovine subcutaneous AT. The highest cell yield in combination with a low population doubling time was obtained using LiberaseTM at a concentration of 0.1% for 3 h. MSC identity of the cells was confirmed by tri-lineage differentiation potential and cell surface marker expression. Subsequently, isolated cells were myogenically differentiated using 5-aza-2’-deoxycytidine and galectin-1. mRNA levels of the myogenic regulatory factors (MRF) myogenic factor 5 (MYF5), myogenic differentiation 1 (MYOD1), MYF6, and myogenin (MYOG) were increased, while less paired box 3 (PAX3) mRNA expression was observed when compared to undifferentiated MSCs. The presence of desmin (DES), tropomyosin (TM), and myosin heavy chain (MyHC) in myogenically differentiated bovine AT-MSCs was confirmed using immunofluorescence stainings. When considering MSCs from bovine AT as potential cell source to produce cultured meat, it is recommended to use 0.1% LiberaseTM for 3 h to ensure a high cell yield.

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.

A change in cis-regulatory logic underlying obligate versus facultative muscle multinucleation in chordates.

Vertebrates and tunicates are sister groups that share a common fusogenic factor, Myomaker (Mymk), that drives myoblast fusion and muscle multinucleation. Yet they are divergent in when and where they express Mymk. In vertebrates, all developing skeletal muscles express Mymk and are obligately multinucleated. In tunicates, Mymk is expressed only in post-metamorphic multinucleated muscles, but is absent from mononucleated larval muscles. In this study, we demonstrate that cis-regulatory sequence differences in the promoter region of Mymk underlie the different spatiotemporal patterns of its transcriptional activation in tunicates and vertebrates. Although in vertebrates myogenic regulatory factors (MRFs) such as MyoD1 alone are required and sufficient for Mymk transcription in all skeletal muscles, we show that transcription of Mymk in post-metamorphic muscles of the tunicate Ciona requires the combinatorial activity of MRF, MyoD and Early B-cell Factor (Ebf). This macroevolutionary difference appears to be encoded in cis, likely due to the presence of a putative Ebf-binding site adjacent to predicted MRF binding sites in the Ciona Mymk promoter. We further discuss how Mymk and myoblast fusion might have been regulated in the last common ancestor of tunicates and vertebrates, for which we propose two models.

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.

Dietary Supplementation with Nano-Curcumin Improves the Meat Quality and Nutrition Value of Largemouth Bass (Micropterus salmoides) Fed with a High-Carbohydrate Diet

This study investigated the effects of curcumin nanoparticles on the flesh quality of largemouth bass on a high-carbohydrate diet. A total of 180 fish (11.01 ± 0.02 g) were fed three semi-purified diets: the Control group (LC) were supplemented with standard carbohydrate (10%), the experimental group (HC) were supplemented with 15% carbohydrate, and the experimental group were supplemented with 0.2% nano-curcumin added to 15% carbohydrate (HCN) for 6 weeks. Results showed that a* value, taurine, valine, isoleucine, histidine, cystine, fatty acids (C17:0 and C20:2n6), MDA (malondialdehyde) content, and SOD (Superoxide dismutase) activity were significantly elevated (p < 0.05) in HC. Muscle fibers showed significant increases in horizontal diameter, longitudinal diameter, and cross-sectional area (p < 0.05), as well as up-regulated expression levels of the MRFS (myogenic regulatory factor) family gene and MSTN (myostatin) (p < 0.05), and a significant decrease in C16:1n7 in HC (p < 0.05). Importantly, the HCN group enhanced the muscle quality of largemouth bass by elevating the L* value, valine, isoleucine, arginine and cystine, C20:2n6, decreasing (p < 0.05) refrigeration loss, chewability, firmness and hardness, then MDA content and SOD activity, and downregulating (p < 0.05) MSTN and MRFS family gene expression levels to improve largemouth bass muscle quality.

A myogenic regulatory factor is required for myogenesis during limb regeneration in the Chinese mitten crab

Myogenic regulatory factors (MRFs) are a group of transcription factors that regulate the activity of skeletal muscle cells during embryonic development and postnatal myogenesis in various vertebrate species. However, the role of MRFs in limb regeneration remains poorly understood in crustaceans. In this study, we identified a full-length cDNA encoding a myogenic regulatory factor from Eriocheir sinensis (EsMRF) and evaluated its mRNA expression profile during muscle development, growth, and regeneration. The expression of EsMRF was found to correlate with the onset of muscle formation during development and with the regeneration process following limb autotomy. To elucidate the function of MRF during limb regeneration in E. sinensis, we assessed regenerative efficiency using RNA interference (RNAi) targeting EsMRF. Our findings revealed that the blockade of MRF delayed limb regeneration by disrupting the proliferation and myogenesis of blastema cells at the basal growth stage. Furthermore, luciferase assays results demonstrated that EsMRF can transcriptionally activate target myogenic genes, either through direct binding to their promoters or by interacting with co-regulators such as EsHEB or EsMEF2. This study identifies a novel MRF in E. sinensis and elucidates its function during limb regeneration, thereby contributing to our understanding of muscle growth and regeneration mechanisms in crustaceans.

Inhibitor of Myom3 inhibits proliferation and promotes differentiation of sheep myoblasts

Skeletal muscle quality and yield are important production traits in livestock, and improving skeletal muscle quality while increasing its yield is an important goal of economic breeding. The proliferation and differentiation process of sheep myoblasts directly affects the growth and development of their muscles, thereby affecting the yield of mutton. Myomesin 3 (Myom3), as a functional gene related to muscle growth, currently lacks research on its function in myoblasts. This study aims to investigate the effect of the Myom3 gene on the proliferation and differentiation of sheep myoblasts and its potential molecular mechanisms. The results showed that inhibitor of Myom3 in the proliferation phase of myoblasts resulted in significant downregulation of the proliferation marker gene paired box 7 (Pax7) and myogenic regulatory factors (MRFs; Myf5, Myod1, Myog, P < 0.01), a significant decrease in the EdU-positive cell rate (P < 0.05), and a significant increase in the cell apoptosis rate (P < 0.01), which inhibited the proliferation of myoblasts and promoted their apoptosis. During the differentiation phase of myoblasts, the inhibitor of Myom3 resulted in significant downregulation of the Pax7 gene, upregulation of MRFs (Myod1, Myog, P < 0.05), and a significant increase in fusion index (P < 0.05), promoting the differentiation of myoblasts. Further transcriptome sequencing revealed that differentially expressed genes in the Myom3 interference group were mainly enriched in the MAPK signaling pathway, TNF signaling pathway, and IL-17 signaling pathway. In summary, the inhibitor of Myom3 inhibits myoblast proliferation and promotes myoblast differentiation. Therefore, Myom3 has a potential regulatory effect on the growth and development of sheep muscles, and in-depth functional research can be used for molecular breeding practices in sheep.

Alpha-linolenic acid/linoleic acid ratio can effectively promote muscle growth and protein deposition in grass carp (Ctenopharyngodon idella)

Polyunsaturated fatty acids (PUFAs) promoted the muscle structure (muscle fiber cross-sectional area and diameter, as well as thickness of the connective tissue surrounding muscle fibers-endomysium) and taste (fishy) of fillets. As members of the omega-3 and omega-6 PUFA family, alpha-linolenic acid (ALA) and linoleic acid (LNA) have important functions. This study explores influences of ALA/LNA ratio on muscle growth and protein deposition and its mechanisms. Six different diets containing ALA/LNA ratios (0.03, 0.47, 0.92, 1.33, 1.69, and 2.15) were fed to grass carp (Ctenopharyngodon idella) for 9 weeks. Results showed that according to the quadratic regression model, the optimal ALA/LNA ratios of grass carp were 1.12 and 1.22 using malondialdehyde content and muscle fiber diameter as indicators, respectively. Optimum ALA/LNA ratio promoted hypertrophy of myofibers, which might be attributed to genes related to myofiber development (myogenic determinant, myogenin, myosin heavy chain, myogenic regulatory factor 4 and myogenic factor 5). Optimum ALA/LNA ratio enhanced protein deposition, which might be associated with the promotion of protein synthesis (insulin-like growth factors-1, phosphoinositide 3-kinase, target of rapamycin), and inhibition of the ubiquitin-proteasome system and autophagy-lysosome system to inhibit protein degradation. In addition, optimum ALA/LNA ratio enhanced muscle amino acid composition and antioxidant capacity of fish.

Effect of precursor amino acids for carnosine synthesis on breast fiber microstructures and myofiber differentiation-related gene expression in slow-growing chicken.

: The effects of carnosine synthesis on the structural and microstructural determinants of meat quality have not been studied to date. Therefore, this study aimed to investigate the effect of supplementation with carnosine synthesis precursors on the characteristics and microstructure of breast muscle fibers in slow-growing Korat chickens (KR). : Slow-growing KR were fed a non-supplemented commercial diet (control group) or a commercial diet supplemented with 1.0% β-alanine, 0.5% L-histidine, or a combination of both 1.0% β-alanine and 0.5% L-histidine. At 10 weeks, KR were slaughtered, and the breast muscle was collected. Samples were fixed and extracted to study the microstructure, fat level, and porosity of the meat using X-ray and scanning electron microscopy, and real-time polymerase chain reaction was performed to analyze the expression of genes related to myofiber differentiation. : L-histidine supplementation significantly altered myofiber diameter and muscle fiber density and compactness by regulating muscle fiber-type differentiation via carnosine synthase (CARNS1) and myocyte enhancer factor 2C (MEF2C) expression, as well as myogenic differentiation antigen (MyoD) and myogenic regulatory factor 5 (Myf5) expression. While excess L-histidine potentially stimulated CARNS1 to modify muscle fiber arrangement and tenderness in breast meat, dietary β-alanine supplementation alone or in combination with L-histidine supplementation induced a relatively less remarkable but not significant (p<0.05) effect on the breast meat characteristics studied. : Interestingly, the combination of β-alanine and L-histidine supplementation had no effect on meat microstructure, meat porosity, and fat content in comparison with the control group. Thus, this combination had the best selectivity for improving meat quality. However, further studies are required to clarify the effects of carnosine levels on meat processing.

Transcriptomics and gut microbiome analysis of the edible herb Bidens pilosa as a functional feed additive to promote growth and metabolism in tilapia (Oreochromis spp.)

To reduce the use of antibiotics and chemicals in aquaculture, an edible herb, Bidens pilosa, has been selected as a multifunctional feed additive. Although there has been considerable research into the effects of B. pilosa on poultry, the wider effects of B. pilosa, particularly on the growth and gut microbiota of fish, remain largely unexplored. We aimed to investigate the interactive effects between the host on growth and the gut microbiota using transcriptomics and the gut microbiota in B. pilosa-fed tilapia. In this study, we added 0.5% and 1% B. pilosa to the diet and observed that the growth performance of tilapia significantly increased over 8 weeks of feeding. Comparative transcriptome analysis was performed on RNA sequence profiles obtained from liver and muscle tissues. Functional enrichment analysis revealed that B. pilosa regulates several pathways and genes involved in amino acid metabolism, lipid metabolism, carbohydrate metabolism, endocrine system, signal transduction, and metabolism of other amino acids. The expression of the selected growth-associated genes was validated by qRT–PCR. The qRT–PCR results indicated that B. pilosa may enhance growth performance by activating the expression of the liver igf1 and muscle igf1rb genes and inhibiting the expression of the muscle negative regulator mstnb. Both the enhancement of liver endocrine IGF1/IGF1Rb signaling and the suppression of muscle autocrine/paracrine MSTN signaling induced the expression of myogenic regulatory factors (MRFs), myod1, myog and mrf4 in muscle to promote muscle growth in tilapia. The predicted function of the gut microbiota showed several significantly different pathways that overlapped with the KEGG enrichment results of differentially expressed genes in the liver transcriptomes. This finding suggested that the gut microbiota may influence liver metabolism through the gut–liver axis in B. pilosa-fed tilapia. In conclusion, dietary B. pilosa can regulate endocrine IGF1 signaling and autocrine/paracrine MSTN signaling to activate the expression of MRFs to promote muscle growth and alter the composition of gut bacteria, which can then affect liver amino acid metabolism, carbohydrate metabolism, endocrine system, lipid metabolism, metabolism of other amino acids, and signal transduction in the host, ultimately enhancing growth performance. Our results suggest that B. pilosa has the potential to be a functional additive that can be used as an alternative to reduce antibiotic use as a growth promoter in aquaculture.

The SPARC-related modular calcium binding 1 (Smoc1) regulated by androgen is required for mouse gubernaculum development and testicular descent.

Testicular descent occurs in two consecutive stages: the transabdominal stage and the inguinoscrotal stage. Androgens play a crucial role in the second stage by influencing the development of the gubernaculum, a structure that pulls the testis into the scrotum. However, the mechanisms of androgen actions underlying many of the processes associated with gubernaculum development have not been fully elucidated. To identify the androgen-regulated genes, we conducted large-scale gene expression analyses on the gubernaculum harvested from luteinizing hormone/choriogonadotropin receptor knockout (Lhcgr KO) mice, an animal model of inguinoscrotal testis maldescent resulting from androgen deficiency. We found that the expression of secreted protein acidic and rich in cysteine (SPARC)-related modular calcium binding 1 (Smoc1) was the most severely suppressed at both the transcript and protein levels, while its expression was the most dramatically induced by testosterone administration in the gubernacula of Lhcgr KO mice. The upregulation of Smoc1 expression by testosterone was curtailed by the addition of an androgen receptor antagonist, flutamide. In addition, in vitro studies demonstrated that SMOC1 modestly but significantly promoted the proliferation of gubernacular cells. In the cultures of myogenic differentiation medium, both testosterone and SMOC1 enhanced the expression of myogenic regulatory factors such as paired box 7 (Pax7) and myogenic factor 5 (Myf5). After short-interfering RNA-mediated knocking down of Smoc1, the expression of Pax7 and Myf5 diminished, and testosterone alone did not recover, but additional SMOC1 did. These observations indicate that SMOC1 is pivotal in mediating androgen action to regulate gubernaculum development during inguinoscrotal testicular descent.

EPA and DHA promote cell proliferation and enhance activity of the Akt-TOR-S6K anabolic signaling pathway in primary muscle cells of turbot (Scophthalmus maximus L.).

Fish growth and health are predominantly governed by dietary nutrient supply. Although the beneficial effects of omega-3 polyunsaturated fatty acids supplementation have been shown in a number of fish species, the underlying mechanisms are still mostly unknown. In this study, we conducted an investigation into the effects of EPA and DHA on cell proliferation, nutrient sensing signaling, and branched-chain amino acids (BCAA) transporting in primary turbot muscle cells. The findings revealed that EPA and DHA could stimulate cell proliferation, promote protein synthesis and inhibit protein degradation through activation of target of rapamycin (TOR) signaling pathway, a pivotal nutrient-sensing signaling cascade. While downregulating the expression of myogenin and myostatin, EPA and DHA increased the level of myogenic regulatory factors, such as myoD and follistatin. Furthermore, we observed a significant increase in the concentrations of intracellular BCAAs following treatment with EPA or DHA, accompanied by an upregulation of the associated amino acid transporters. Our study providing valuable insights into the mechanisms underlying the growth-promoting effects of omega-3 fatty acids in fish.

Curcumin-activated Wnt5a pathway mediates Ca2+ channel opening to affect myoblast differentiation and skeletal muscle regeneration.

Skeletal muscle injury is one of the most common sports injuries; if not properly treated or not effective rehabilitation treatment after injury, it can be transformed into chronic cumulative injury. Curcumin, an herbal ingredient, has been found to promote skeletal muscle injury repair and regeneration. The Wnt5a pathway is related to the expression of myogenic regulatory factors, and Ca2+ promotes the differentiation and fusion process of myoblasts. This study explored the effect and mechanism of curcumin on myoblast differentiation during the repair and regeneration of injured skeletal muscle and its relationship with the Wnt5a pathway and Ca2+ channel. Myogenic differentiation of C2C12 cells was induced with 2% horse serum, and a mouse (male, 10weeks old) model of acute skeletal muscle injury was established using cardiotoxin (20μL). In addition, we constructed a Wnt5a knockdown C2C12 cell model and a Wnt5a knockout mouse model. Besides, curcumin was added to the cell culture solution (80mg/L) and fed to the mice (50mg/kg). Fluorescence microscopy was used to determine the concentration of Ca2+. Western blot and RT-qPCR were used to detect the protein and mRNA levels of Wnt5a, CaN, NFAT2, MyoD, Myf5, Pax7, and Myogenin. The expression levels of MyoD, Myf5, Myogenin, MHC, Desmin, and NFAT2 were detected using immunofluorescence techniques. In addition, MyoD expression was observed using immunohistochemistry, and morphological changes in mouse muscle tissue were observed using HE staining. During myoblast differentiation and muscle regeneration, Wnt5a expression was upregulated (P<0.001) and the Wnt5a signalling pathway was activated. Wnt5a overexpression promoted the expression of MyoD, Myf5, Myogenin, MHC, and Desmin (P<0.05), and conversely, knockdown of Wnt5a inhibited their expression (P<0.001). The Wnt5a pathway mediated the opening of Ca2+ channels, regulated the expression levels of CaN, NFAT2, MyoD, Myf5, Myogenin, MHC, and Desmin (P<0.01) and promoted the differentiation of C2C12 myoblasts and the repair and regeneration of injured skeletal muscle. The expression of Wnt5a, CaN, NFAT2, MyoD, Myogenin, Myf5, and MHC in C2C12 myoblast was significantly increased after curcumin intervention (P<0.05); however, their expression decreased significantly after knocking down Wnt5a on the basis of curcumin intervention (P<0.05). Similarly, in Wnt5a knockout mice, the promotion of muscle regeneration by curcumin was significantly attenuated. Curcumin can activate the Wnt5a signalling pathway and mediate the opening of Ca2+ channels to accelerate the myogenic differentiation of C2C12 cells and the repair and regeneration of injured skeletal muscle.

Dual-specificity phosphatases 13 and 27 as key switches in muscle stem cell transition from proliferation to differentiation.

Muscle regeneration depends on muscle stem cell (MuSC) activity. Myogenic regulatory factors, including myoblast determination protein 1 (MyoD), regulate the fate transition of MuSCs. However, the direct target of MYOD in the process is not completely clear. Using previously established MyoD knock-in (MyoD-KI) mice, we revealed that MyoD targets dual-specificity phosphatase (Dusp) 13 and Dusp27. In Dusp13:Dusp27 double knock-out mice, the ability for muscle regeneration after injury was reduced. Moreover, single-cell RNA sequencing of MyoD-high expressing MuSCs from MyoD-KI mice revealed that Dusp13 and Dusp27 are expressed only in specific populations within MyoD-high MuSCs, which also express Myogenin. Overexpressing Dusp13 in MuSCs causes premature muscle differentiation. Thus, we propose a model where DUSP13 and DUSP27 contribute to the fate transition of MuSCs from proliferation to differentiation during myogenesis.

In ovo betaine injection improves breast muscle growth in newly hatched goslings through FXR/IGF-2 pathway

Betaine has been shown to enhance growth performance and increase breast muscle yield in ducks and broilers through various mechanisms, including the modification of DNA methylation. However, the impact of in ovo betaine injection on muscle growth in newly hatched goslings remains unclear. In this study, fifty eggs were injected with saline or betaine at 7.5 mg/egg prior to incubation, and the subsequent effects on breast muscle growth in the newly hatched goslings were investigated. Betaine significantly increased (P < 0.05) the hatch weight, breast muscle weight, and breast muscle index, accompanied by an augmentation in muscle bundle cross-sectional area. Concurrently, betaine significantly upregulated (P < 0.05) the expression levels of myogenic regulatory factors, including myogenin (MyoG) and paired box 7 (Pax7) both mRNA and protein, while downregulating (P < 0.05) the mRNA and protein levels of myostatin (MSTN). Histological analysis revealed a higher abundance of proliferating cell nuclear antigen (PCNA) and Pax7 immune-positive cells in the breast muscle of the betaine group, consistent with elevated PCNA and Pax7 mRNA and protein levels. Additionally, significantly increased (P < 0.05) contents of insulin-like growth factor 1 (IGF-1) and insulin-like growth factor 2 (IGF-2) were observed in the breast muscle of the betaine group, so was mRNA expression of IGF-1, IGF-2, and insulin-like growth factor 1 receptor (IGF-1R). Betaine also significantly in8creased (P < 0.05) global DNA methylation of the breast muscle, accompanied by enhanced mRNA and protein levels of methionine cycle and DNA methylation-related enzymes, Interestingly, the promoter regions of IGF-1, IGF-2, and IGF-1R genes were significantly hypomethylated (P < 0.05). Moreover, in ovo betaine injection significantly upregulated (P < 0.05) the protein level of farnesoid X receptor (FXR) in breast muscle and FXR binding to the promoter of IGF-2 gene. These findings suggest that in ovo betaine injection promotes breast muscle growth during embryonic development in goslings through the FXR-mediated IGF-2 pathway, ultimately improving hatch weight and breast muscle weight.

Myogenic Regulator Genes Responsible For Muscle Development in Farm Animals

Farm animals especially poultry species play important roles in meeting the worldwide demand for meat and are genetically improved to increase the efficiency of meat production. Improving muscle growth and development is crucial to meet customers' demands for high-quality meat. Myogenesis, fibrogenesis, and adipogenesis are components of fetal skeletal muscle formation. Myogenesis is controlled by a complex network of intrinsic and extrinsic components, usually divided into two or three stages and regulated by kinase-encoding genes and myogenic regulatory factor genes. The basic family of helix-loop-helix transcription factors, which includes MYF5, MYOD, myogenin, and MRF4, regulates the specification and differentiation of skeletal muscle cells throughout embryogenesis and postnatal myogenesis. With the discovery of the myogenic regulatory factor family, which includes the transcription factors MYF5, MYOD, Myogenin, and MRF4, it has become possible to determine the skeletal muscle lineage and understand the regulation of myogenic differentiation during development. These elements also play a role in determining the muscle satellite cell lineage that develops into the resident stem cell compartment in adult skeletal muscle. Although MYF5, MYOD, Myogenin and MRF4 play minor roles in mature muscle, they once again play an important role in directing satellite cell activity to regenerate skeletal muscle, linking the genetic regulation of development and regeneration myogenesis. An important step towards the goal of increasing meat yield and improving meat quality is the understanding and identification of these genes. This in-depth analysis discusses the function of myogenic regulatory factors in the specification of satellite cells, maturation and regeneration of skeletal muscle.

Effect of Glycyrrhiza uralensis crude water extract on the expression of Nitric Oxide Synthase 2 gene during myogenesis

Glycyrrhiza uralensis is a traditional herbal medicine with significant bioactivity. This study investigated the effect of G. uralensis crude water extract (GU-CWE) on nitric oxide synthase 2 (NOS2) expression during myogenesis. GU-CWE treatment increased myoblast differentiation by downregulating NOS2 and upregulating myogenic regulatory factors (MYOD, MYOG, and MYH). Notably, this effect was supported by an observed decrease in NOS2 expression in the gastrocnemius tissues of mice treated with GU-CWE. In addition, GU-CWE treatment and NOS2 knockdown were associated with reductions in reactive oxygen species levels. We further elucidate the role of the NOS2 gene in myoblast differentiation, demonstrating that its role was expression dependent, being beneficial at low expression but detrimental at high expression. High NOS2 gene expression induced oxidative stress, whereas its low expression impaired myotube formation. These findings highlight that the modulation of NOS2 expression by G. uralensis can potentially be use for managing muscle wasting disorders.