Expression Of Myogenic Regulatory Factors Research Articles

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.

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.

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.

Lactiplantibacillus plantarum LM1001 Improves Digestibility of Branched-Chain Amino Acids in Whey Proteins and Promotes Myogenesis in C2C12 Myotubes.

Lactiplantibacillus plantarum is a valuable potential probiotic species with various proven health-beneficial effects. L. plantarum LM1001 strain was selected among ten strains of L. plantarum based on proteolytic activity on whey proteins. L. plantarum LM1001 produced higher concentrations of total free amino acids and branched-chain amino acids (Ile, Leu, and Val) than other L. plantarum strains. Treatment of C2C12 myotubes with whey protein culture supernatant (1%, 2% and 3%, v/v) using L. plantarum LM1001 significantly increased the expression of myogenic regulatory factors, such as Myf-5, MyoD, and myogenin, reflecting the promotion of myotubes formation (p<0.05). L. plantarum LM1001 displayed β-galactosidase activity but did not produce harmful β-glucuronidase. Thus, the intake of whey protein together with L. plantarum LM1001 has the potential to aid protein digestion and utilization.

MiR-214 promotes the activation, proliferation and differentiation of skeletal muscle satellite cells by modulating Hedgehog signaling in Chinese perch

Fish exhibit indeterminate growth by recruiting new muscle fibers (hyperplasia) and increasing the size of already existing fibers (hypertrophy) to promote muscle growth. However, the molecular mechanism by which muscle fibers maintain hyperplasia and hypertrophy during the posthatching period in fish remains unclear. microRNAs have been reported to participate in the proliferation and differentiation of myoblasts in fish. In this study, we found that overexpression of miR-214 in Chinese perch (Siniperca chuatsi) by injecting agomiR-214 in vivo resulted in an increase in the diameter of myofibers, the number of satellite cells and proliferating myoblasts as well as the expression of Pax7, Myomaker and myogenic regulatory factors. However, inhibiting miR-214 by injecting antagomiR-214 in vivo significantly decreased the diameter of myofibers, the number of proliferating myoblasts and the expression of Myf5 and MyoD. In addition, inhibition of the Hedgehog pathway showed results similar to those of inhibition of miR-214. The in silico analysis and dual luciferase reporter assay revealed that Sufu, a negative regulator of the Hedgehog (Hh) signaling pathway, is a direct target of miR-214. Taken together, our results reveal that miR-214 induces the activation of satellite cells by regulating Hh signaling activity, and subsequently stimulates the proliferation, differentiation and fusion of myoblast, thus promoting hyperplasia and hypertrophy of skeletal muscle in Chinese perch.

Novel Perspective on Mechanism in Muscle Growth Inhibited by Ochratoxin A Associated with Ferroptosis: Model of Juvenile Grass Carp (Ctenopharyngodon idella) In Vivo and In Vitro Trials.

Ochratoxin A (OTA) is a common mycotoxin in food and feed that seriously harms human and animal health. This study investigated the effect of OTA on the muscle growth of juvenile grass carp (Ctenopharyngodon idella) and its possible mechanism in vitro. Our results have the following innovative findings: (1) Dietary OTA increased the expression of increasing phase I metabolic enzymes and absorbing transporters while reducing the expression of efflux transporters, thereby increasing their residue in muscles; (2) OTA inhibited the expressions of cell cycle and myogenic regulatory factors (MyoD, MyoG, and MyHC) and induced ferroptosis by decreasing the mRNA and protein expressions of FTH, TFR1, GPX4, and Nrf2 both in vivo and in vitro; and (3) the addition of DFO improved OTA-induced ferroptosis of grass carp primary myoblasts and promoted cell proliferation, while the addition of AKT improved OTA-inhibited myoblast differentiation and fusion, thus inhibiting muscle growth. Overall, this study provides a potential research target to further mitigate the myotoxicity of OTA.

Myosin heavy chain-perinatal regulates skeletal muscle differentiation, oxidative phenotype and regeneration.

Myosin heavy chain-perinatal (MyHC-perinatal) is one of two development-specific myosin heavy chains expressed exclusively during skeletal muscle development and regeneration. The specific functions of MyHC-perinatal are unclear, although mutations are known to lead to contracture syndromes such as Trismus-pseudocamptodactyly syndrome. Here, we characterize the functions of MyHC-perinatal during skeletal muscle differentiation and regeneration. Loss of MyHC-perinatal function leads to enhanced differentiation characterized by increased expression of myogenic regulatory factors and differentiation index as well as reduced reserve cell numbers in vitro. Proteomic analysis revealed that loss of MyHC-perinatal function results in a switch from oxidative to glycolytic metabolism in myofibers, suggesting a shift from slow type I to fast type IIb fiber type, also supported by reduced mitochondrial numbers. Paracrine signals mediate the effect of loss of MyHC-perinatal function on myogenic differentiation, possibly mediated by non-apoptotic caspase-3 signaling along with enhanced levels of the pro-survival apoptosis regulator Bcl2 and nuclear factor kappa-B (NF-κB). Knockdown of MyHC-perinatal during muscle regeneration in vivo results in increased expression of the differentiation marker myogenin (MyoG) and impaired differentiation, evidenced by smaller myofibers, elevated fibrosis and reduction in the number of satellite cells. Thus, we find that MyHC-perinatal is a crucial regulator of myogenic differentiation, myofiber oxidative phenotype and regeneration.

Overexpression of Syndecan-4 inhibits myogenesis by regulating the expression of myogenic regulatory factors

Overexpression of Syndecan-4 inhibits myogenesis by regulating the expression of myogenic regulatory factors

Preventive effect of fermented whey protein mediated by Lactobacillus gasseri IM13 via the PI3K/AKT/FOXO pathway in muscle atrophy

This study investigated the preventive effects of whey protein fermented with Lactobacillus gasseri IM13 (F-WP) against dexamethasone (DEX)-induced muscle atrophy. C2C12 muscle cells were treated with F-WP followed by DEX treatment. Dexamethasone treatment inhibited myotube formation and the expression of myogenic regulatory factors; however, pretreatment with F-WP attenuated DEX-induced damage. The F-WP significantly activated the phosphorylation of the IGF-1/PI3K/AKT pathway and improved muscle homeostasis suppressed by DEX. Moreover, F-WP alleviated the phosphorylation of mTOR, S6K1, and 4E-BP1 and enhanced muscle protein synthesis. Muscle-specific ubiquitin ligases and autophagy lysosomes, which were activated by the dephosphorylation of FOXO3a by DEX treatment, were significantly attenuated by F-WP pretreatment of myotubes. For peptidomic analysis, F-WP was fractionated using preparative HPLC (prep-HPLC), and the AA sequences of 11 peptides were identified using MALDI-TOF/MS/MS. In conclusion, fermentation of whey protein by the specific probiotic strain IM13 produced bioactive peptides with high antioxidant and anti-sarcopenic-sarcopenic effects, which markedly enhanced myogenesis and muscle protein synthesis while diminishing muscle protein degradation compared with intact whey protein.

Implications of vitamin D for flesh quality of grass carp (Ctenopharyngodon idella): antioxidant ability, nutritional value, sensory quality, and myofiber characteristics

BackgroundMuscle represents a unique and complex system with many components and comprises the major edible part of animals. Vitamin D is a critical nutrient for animals and is known to enhance calcium absorption and immune response. In recent years, dietary vitamin D supplementation in livestock has received increased attention due to biological responses including improving shear force in mammalian meat. However, the vitamin D acquisition and myofiber development processes in fish differ from those in mammals, and the effect of vitamin D on fish flesh quality is poorly understood. Here, the influence of dietary vitamin D on fillet quality, antioxidant ability, and myofiber development was examined in grass carp (Ctenopharyngodon idella).MethodsA total of 540 healthy grass carp, with an initial average body weight of 257.24 ± 0.63 g, were allotted in 6 experimental groups with 3 replicates each, and respectively fed corresponding diets with 15.2, 364.3, 782.5, 1,167.9, 1,573.8, and 1,980.1 IU/kg vitamin D for 70 d.ResultsSupplementation with 1,167.9 IU/kg vitamin D significantly improved nutritional value and sensory quality of fillets, enhancing crude protein, free amino acid, lipid, and collagen contents; maintaining an ideal pH; and reducing lactate content, shear force, and cooking loss relative to respective values in the control (15.2 IU/kg) group. Average myofiber diameter and the frequency of myofibers > 50 μm in diameter increased under supplementation with 782.5–1,167.9 IU/kg vitamin D. Levels of oxidative damage biomarkers decreased, and the expression of antioxidant enzymes and nuclear factor erythroid 2-related factor 2 signaling molecules was upregulated in the 1,167.9 IU/kg vitamin D treatment compared to respective values in the control group. Furthermore, vitamin D supplementation activated cell differentiation by enhancing the expression of myogenic regulatory factors and myocyte enhancer factors compared to that in the control group. In addition, supplementation with 1,167.9 IU/kg vitamin D improved protein deposition associated with protein synthesis molecule (target of rapamycin) signaling and vitamin D receptor paralogs, along with inhibition of protein degradation (forkhead box protein 1) signaling.ConclusionsOverall, the results demonstrated that vitamin D strengthened antioxidant ability and myofiber development, thereby enhancing nutritional value and sensory quality of fish flesh. These findings suggest that dietary vitamin D supplementation is conducive to the production of nutrient-rich, high quality aquaculture products.

Hexavalent chromium inhibits myogenic differentiation and induces myotube atrophy

Hexavalent chromium [Cr(VI)] is extensively used in many industrial processes. Previous studies reported that Cr(VI) exposures during early embryonic development reduced body weight with musculoskeletal malformations in rodents while exposures in adult mice increased serum creatine kinase activity, a marker of muscle damage. However, the impacts of Cr(VI) on muscle differentiation remain largely unknown. Here, we report that acute exposures to Cr(VI) in mouse C2C12 myoblasts inhibit myogenic differentiation in a dose-dependent manner. Exposure to 2 μM of Cr(VI) resulted in delayed myotube formation, as evidenced by a significant decrease in myotube formation and expression of muscle-specific markers, such as muscle creatine kinase (Mck), Myocyte enhancer factor 2 (Mef2), Myomaker (Mymk) and Myomixer (Mymx). Interestingly, exposure to 5 μM of Cr(VI) completely abolished myotube formation in differentiating C2C12 cells. Moreover, the expression of key myogenic regulatory factors (MRFs) including myoblast determination protein 1 (MyoD), myogenin (MyoG), myogenic factor 5 (Myf5), and myogenic factor 6 (Myf6) were significantly altered in Cr(VI)-treated cells. The inhibitory effect of Cr(VI) on myogenic differentiation was further confirmed in freshly isolated mouse satellite cells, a stem cell population essential for adult skeletal muscle regeneration. Furthermore, Cr(VI) exposure to fully differentiated C2C12 myotubes resulted in a decrease in myotube diameter, which was exacerbated upon co-treatment with dexamethasone. Together, our results demonstrate that Cr(VI) inhibits myogenic differentiation and induces myotube atrophy in vitro.

FLII Modulates the Myogenic Differentiation of Progenitor Cells via Actin Remodeling-Mediated YAP1 Regulation.

The dynamic rearrangement of the actin cytoskeleton plays an essential role in myogenesis, which is regulated by diverse mechanisms, such as mechanotransduction, modulation of the Hippo signaling pathway, control of cell proliferation, and the influence of morphological changes. Despite the recognized importance of actin-binding protein Flightless-1 (FLII) during actin remodeling, the role played by FLII in the differentiation of myogenic progenitor cells has not been explored. Here, we investigated the roles of FLII in the proliferation and differentiation of myoblasts. FLII was found to be enriched in C2C12 myoblasts, and its expression was stable during the early stages of differentiation but down-regulated in fully differentiated myotubes. Knockdown of FLII in C2C12 myoblasts resulted in filamentous actin (F-actin) accumulation and inhibited Yes-associated protein 1 (YAP1) phosphorylation, which triggers its nuclear translocation from the cytoplasm. Consequently, the expressions of YAP1 target genes, including PCNA, CCNB1, and CCND1, were induced, and the cell cycle and proliferation of myoblasts were promoted. Moreover, FLII knockdown significantly inhibited the expression of myogenic regulatory factors, i.e., MyoD and MyoG, thereby impairing myoblast differentiation, fusion, and myotube formation. Thus, our findings demonstrate that FLII is crucial for the differentiation of myoblasts via modulation of the F-actin/YAP1 axis and suggest that FLII is a putative novel therapeutic target for muscle wasting.

Comparative evaluation of soybean meal vs. extruded soybean meal as a replacer for fishmeal in diets of olive flounder (Paralichthys olivaceus): Effects on growth performance and muscle quality

A 56-day feeding trial was performed to compare the impacts of replacement of dietary fishmeal by soybean meal (SM) or extruded soybean meal (ESM) on the growth performance and muscle quality of olive flounder (Paralichthys olivaceus) (initial weight: 6.32 ± 0.01 g). Nine isonitrogenous and isolipidic diets were prepared by substituting 0% (FM, control), 12% (SM12/ESM12), 24% (SM24/ESM24), 36% (SM36/ESM36) and 48% (SM48/ESM48) of fishmeal with SM or ESM respectively. Results indicated that contents of anti-nutritional factors (trypsin inhibitors, glycinin and β-conglycinin) in ESM were significantly lower than those in SM. The weight gain rate, specific growth rate, feed efficiency ratio, protein efficiency ratio, activities of intestinal trypsin and lipase, as well as expressions of myogenic regulatory factors (myf5, myod and mrf4) and protein synthesis-related genes (akt, pi3k, tor and s6) in fish muscle decreased linearly and quadratically with increasing replacement levels of dietary fishmeal by SM or ESM. Expressions of mstn and protein degradation-related genes (foxo1, murf-1, mafbx, capn2 and ctsl) in muscle showed a trend opposite to growth parameters responding to dietary SM or ESM levels. Significant differences in all the above parameters were observed in the SM36, SM48 and ESM48 groups, accompanied by the declines in contents of most muscle free amino acids. The increase of dietary fishmeal replacement level by SM linearly and quadratically decreased muscle hardness, water holding capacity (WHC), pH and total collagen content. Markedly lower values were found in the SM36 and SM48 groups. However, ESM could replace up to 36% of dietary fishmeal without significantly impacts on above muscle quality parameters. Additionally, reduced myofiber density and sarcomere length were observed in the SM36, SM48, ESM36 and ESM48 groups. However, at the same substitution level, dietary ESM relieved the effects of SM on myofiber morphology to some extent. In conclusion, according to broken-line regression on SGR, the optimal replacing level of fishmeal by SM and ESM for olive flounder was 22.07% and 34.39% respectively. The improved replacement level of fishmeal by ESM, in terms of fish growth, muscle hardness and WHC, may be due to the fact that dietary ESM alleviated the adverse impacts of SM on digestive enzyme activity, muscle growth-related genes expression, muscle pH, collagen content and myofiber morphology of fish.

Regulation of HDAC11 gene expression in early myogenic differentiation.

Histone acetylation and deacetylation affect the patterns of gene expression in cellular differentiation, playing pivotal roles in tissue development and maintenance. For example, the intrinsic histone acetyltransferase activity of transcriptional coactivator p300 is especially required for the expression of myogenic regulatory factors including Myf5 and MyoD, and consequently for skeletal myogenesis. On the other hand, histone deacetylases (HDACs) remove the acetyl group from histones, which is critical for gene repression in stem cell fate transition. Through integrative omic analyses, we found that while some HDACs were differentially expressed at the early stage of skeletal myoblast differentiation, Hdac11 gene expression was significantly enhanced by nuclear receptor signaling. In addition, p300 and MyoD control Hdac11 expression in milieu of normal and signal-enhanced myoblast differentiation. Thus, HDAC11 may be essential to differential gene expression at the onset of myoblast differentiation.

Indoxyl sulfate inhibits muscle cell differentiation via Myf6/MRF4 and MYH2 downregulation.

Chronic kidney disease (CKD) is associated with a significant decrease in muscle strength and mass, possibly related to muscle cell damage by uremic toxins. Here, we studied in vitro and in vivo the effect of indoxyl sulfate (IS), an indolic uremic toxin, on myoblast proliferation, differentiation and expression of myogenic regulatory factors (MRF)-myoblast determination protein 1 (MyoD1), myogenin (Myog), Myogenic Factor 5 (Myf5) and myogenic regulatory factor 4 (Myf6/MRF4)-and expression of myosin heavy chain, Myh2. C2C12 myoblasts were cultured in vitro and differentiated in myotubes for 7 days in the presence of IS at a uremic concentration of 200µM. Myocytes morphology and differentiation was analyzed after hematoxylin-eosin staining. MRF genes' expression was studied using reverse transcription polymerase chain reaction in myocytes and 5/6th nephrectomized mice muscle. Myf6/MRF4 protein expression was studied using enzyme-linked immunosorbent assay; MYH2 protein expression was studied using western blotting. The role of Aryl Hydrocarbon Receptor (AHR)-the cell receptor of IS-was studied by adding an AHR inhibitor into the cell culture milieu. In the presence of IS, the myotubes obtained were narrower and had fewer nuclei than control myotubes. The presence of IS during differentiation did not modify the gene expression of the MRFs Myf5, MyoD1 and Myog, but induced a decrease in expression of Myf6/MRF4 and MYH2 at the mRNA and the protein level. AHR inhibition by CH223191 did not reverse the decrease in Myf6/MRF4 mRNA expression induced by IS, which rules out the implication of the ARH genomic pathway. In 5/6th nephrectomized mice, the Myf6/MRF4 gene was down-regulated in striated muscles. In conclusion, IS inhibits Myf6/MRF4 and MYH2 expression during differentiation of muscle cells, which could lead to a defect in myotube structure. Through these new mechanisms, IS could participate in muscle atrophy observed in CKD.

Dietary zinc supplementation in breeding pigeons improves the carcass traits of squabs through regulating antioxidant capacity and myogenic regulatory factor expression

The purpose of this experiment was to explore the effects of zinc supplementation in breeding pigeons diet on carcass traits, meat quality, antioxidant capacity and mRNA expressions of myogenic regulatory factors of squabs. A total of 120 healthy White King pigeons were randomly assigned to 5 treatments, each involving 8 replicates. The experiment lasted for 46 d (18-d incubation period of eggs and 28-d growth period of squabs). The 5 groups were 0, 30, 60, 90, and 120 mg/kg zinc addition. Results showed that the 28-d body weight, breast muscle yield, zinc content in crop milk and myogenic factor 6 (MyF6) abundance of breast muscle were linearly increased (P < 0.050), but the abdominal fat yield linearly decreased (P=0.040) with increasing dietary zinc supplementation. Both the linear (P < 0.050) and quadratic responses (P < 0.001) were observed in copper zinc superoxide dismutase (Cu-Zn SOD), total antioxidant capacity (T-AOC) and malondialdehyde (MDA) contents in liver and breast muscle. The 28-d body weight was increased by 90 mg/kg zinc supplementation (P < 0.05), and there is no significant difference between 90 and 120 mg/kg zinc addition. The breast muscle yield, Cu-Zn SOD and T-AOC contents in breast muscle and liver, zinc contents in crop milk and breast muscle, MyF6 mRNA expression in breast muscle were higher (P < 0.05) in the group supplemented with 120 mg/kg zinc than the control. The abdominal fat yield was numerically lowest, and MDA contents in breast muscle and liver were significantly lowest in the group fed 120 mg/kg zinc (P < 0.05). However, the meat quality traits were not affected (P > 0.05) by zinc supplementation, except for shear force. It should be stated dietary zinc supplementation at the level of 120 mg/kg for breeding pigeons increased body weight and breast muscle yield of squabs, which may be associated with the up-regulating MyF6 mRNA expression and antioxidant capacity in liver and breast muscle.

Functional characterization of Labeo rohita muscle cell line for in vitro research.

Labeo rohita represents the most dominant fish species in Indian aquaculture and the fish cell lines have been used as an excellent in vitro platform for performing variousbiological research. The LRM cell culture developed from the muscle tissue of L. rohita was used to study the in vitro applications. The developed muscle cells were maintained in a Leibovitz's-15 (L-15) supplemented with 10% FBS (Fetal Bovine Serum) and 10ng/ml bFGFat 28 oC temperature. The LRM cells showed fibroblastic-like morphology and was authenticated by sequencing mitochondrial gene 16S rRNA. The expression of myogenic regulatory factors (MRFs) was studied in different stages of LRM cells; however, the expression patterns varied at different passages. The MEF2A, Mrf-4, and Myogenin expressions were higher in passage 25, while the expression of MyoD was maximum in passage 15, and the expression of Myf-5 was highest in passage 1.The transfection efficiency of LRM cells revealed 14% of the GFP expression with a pmaxGFP vector DNA. The LRM cells were susceptible to the extracellular products prepared from Aeromonas hydrophilla and Edwardsiella tarda. The acute cytotoxicity of six heavy metals (Hg, Cd, Zn, Cu, Pb, Ni) was assessed in LRM cells by a dose-dependent manner in comparison to IC50 values obtained from MTT and NR assays. A revival rate of 70-75% was achieved when the LRM cells were cryopreserved at - 196°C using liquid nitrogen. The developed muscle cells serve as an functional in vitro tool for toxicological and biotechnological studies.

Acute, next-generation AMPK activation initiates a disease-resistant gene expression program in dystrophic skeletal muscle.

Duchenne muscular dystrophy (DMD) is a life-limiting neuromuscular disorder characterized by muscle weakness and wasting. Previous proof-of-concept studies demonstrate that the dystrophic phenotype can be mitigated with the pharmacological stimulation of AMP-activated protein kinase (AMPK). However, first-generation AMPK activators have failed to translate from bench to bedside due to either their lack of potency or toxic, off-target effects. The identification of safe and efficacious molecules that stimulate AMPK in dystrophic muscle is of particular importance as it may broaden the therapeutic landscape for DMD patients regardless of their specific dystrophin mutation. Here, we demonstrate that a single dose of the next generation, orally-bioactive AMPK agonist MK-8722 (MK) to mdx mice evoked skeletal muscle AMPK and extensive downstream stimulation within 12h post-treatment. Specifically, MK elicited a gene expression profile indicative of a more disease-resistant slow, oxidative phenotype including increased peroxisome proliferator-activated receptor ɣ coactivator-1⍺ activity and utrophin levels. In addition, we observed augmented autophagy signaling downstream of AMPK, as well as elevations in critical autophagic genes such as Map1lc3 and Sqstm1 subsequent to the myonuclear accumulation of the master regulator of the autophagy gene program, transcription factor EB. Lastly, we show that pharmacological AMPK stimulation normalizes the expression of myogenic regulatory factors and amends activated muscle stem cell content in mdx muscle. Our results indicate that AMPK activation via MK enhances disease-mitigating mechanisms in dystrophic muscle and prefaces further investigation on the chronic effects of novel small molecule AMPK agonists.

The Maintenance of AMPK Activity Eliminates Abnormally Accelerated Differentiation of Primary Myoblasts Isolated from Atrophied Rat Soleus Muscle

Mechanical unloading of skeletal muscles leads to the development of atrophic processes and a decrease in the total number of satellite cells (SCs) that are involved in muscle regeneration. In vitro studies revealed an increased differentiation of myoblasts derived from rat soleus muscle after an unloading-induced decrease in AMP-activated protein kinase (AMPK). AMPK is necessary for the activation of SCs and also participates in the regulation of myoblast proliferation and differentiation. It can be assumed that a decrease in the activity of AMPK after mechanical unloading can contribute to the acceleration of myoblast differentiation. The main purpose of this study was to elucidate a possible role of AMPK in the regulation of differentiation of myoblasts isolated from rat soleus muscle after mechanical unloading. To test this hypothesis, a specific AMPK activator, AICAR, was used to prevent a decrease in AMPK activity during differentiation of myoblasts isolated from rat soleus muscle after 7-day unloading. Immunocytochemistry, PCR-RT and Western blotting were used to assess changes during myoblast differentiation. In differentiating myoblasts derived from the unloaded soleus muscle there was a significant decrease in AMPK (Thr172) and ACC (Ser 79) phosphorylation levels, an increase in myotube differentiation index, myoblast fusion factors and the expression of myogenic regulatory factors (MRF). Furthermore, there was a decrease in the expression of slow myosin heavy chains (MyHC) and an increase in the expression of fast MyHC isoforms. AICAR treatment of differentiating myoblasts obtained from the unloaded soleus muscle prevented a decrease in AMPK and ACC phosphorylation, returned the expression levels of MRF and fast isoforms of MyHC to the control levels as well as maintained the expression of slow MyHC. Thus, abnormally accelerated differentiation of myoblasts isolated from atrophied rat soleus muscle can be compensated by maintaining the control levels of AMPK activity using AICAR.

Linoleic acid-induced ANGPTL4 inhibits C2C12 skeletal muscle differentiation by suppressing Wnt/β-catenin

Skeletal muscle differentiation is an essential process in embryonic development as well as regeneration and repair throughout the lifespan. It is well-known that dietary fat intake impacts biological and physiological function in skeletal muscle, however, understanding of the contribution of nutritional factors in skeletal muscle differentiation is limited. Therefore, the objective of the current study was to evaluate the effects of free fatty acids (FFAs) on skeletal muscle differentiation in vitro. We used C2C12 murine myoblasts and treated them with various FFAs, which revealed a unique response of angiopoietin-like protein-4 (ANGPTL4) with linoleic acid (LA) treatment that was associated with reduced differentiation. LA significantly inhibited myotube formation and lowered the protein expression of myogenic regulatory factors, including MyoD and MyoG and increased Pax7 during cell differentiation. Next, recombinant ANGPTL4 protein or siRNA knockdown of ANGPTL4 was employed to examine its role in skeletal muscle differentiation, and we confirmed that ANGPTL4 knockdown at day two and six of differentiation restored myotube formation in the presence of LA. RNA-sequencing analysis revealed that ANGPTL4-mediated inhibition of skeletal muscle differentiation at day two as well as LA at day two or -6 led to a reduction in Wnt/β-catenin signaling pathways. We confirmed that LA reduced Wnt11 and Axin2 while increasing expression of the Wnt inhibitor, Dkk2. ANGPTL4 knockdown increased β-catenin protein in the nucleus in response to LA and increased Axin2 and Wnt11 expression. Taken together, these results demonstrate that LA induced ANGPTL4 inhibits C2C12 differentiation by suppressing Wnt/β-catenin signaling.