Chicken Skeletal Muscle Research Articles

Lysosomes accumulate at the perinuclear region of muscle cells during chick myogenesis.

Lysosomes are involved in a myriad of cellular functions, such as degradation of macromolecules, endocytosis and exocytosis, modulation of several signaling pathways, and regulation of cell metabolism. To fulfill these diverse functions, lysosomes can undergo several dynamic changes in their content, size, pH, and location within cells. Here, we studied some of these parameters during embryonic chick skeletal muscle cells. We used an anti-lysosome-associated membrane protein 2 (LAMP2) antibody to specifically determine the intracellular localization of lysosomes in these cells. Our data shows that lysosomes are highly enriched in the perinuclear region of chick embryonic muscle cells. We also showed that the wingless signaling pathway (Wnt)/β-catenin signaling pathway can modulate the location of LAMP2 in chick myogenic cells. Our results highlight the role of lysosomes during muscle differentiation and particularly the presence of a subcellular population of lysosomes that are concentrated in the perinuclear region of muscle cells.

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

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

Asparagine synthetase regulates the proliferation and differentiation of chicken skeletal muscle satellite cells.

Asparagine synthetase (ASNS) is an aminotransferase responsible for the biosynthesis of aspartate by using aspartic acid and glutamine. ASNS is highly expressed in fast-growing broilers, but few studies have reported the regulatory role of ASNS in muscle development. To explore the function of ASNS in chicken muscle development, the expression of ASNS in different chicken breeds and tissues were first performed by real-time quantitative reverse transcription polymerase chain reaction (RT-PCR). Then, using real-time quantitative RT-PCR, western blot, EdU assay, cell cycle assay and immunofluorescence, the effects of ASNS on the proliferation and differentiation of chicken skeletal muscle satellite cell (SMSC) were investigated. Finally, potential mechanisms by which ASNS influences chicken muscle fiber differentiation were identified through RNA-Seq. The mRNA expression pattern of ASNS in muscles mirrors trends in muscle fiber cross-sectional area, average daily weight gain, and muscle weight across different breeds. ASNS knockdown inhibited SMSC proliferation, while overexpression showed the opposite. Moreover, ASNS attenuated SMSC differentiation by activating the Adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) pathway. Additionally, 5-aminoimidazole-4-carboxamide1-β-D-ribofuranoside (AICAR) treatment suppressed the cell differentiation induced by siRNA-ASNS. RNA-Seq identified 1968 differentially expressed genes (DEGs) during chicken SMSC differentiation when overexpression ASNS. Gene Ontology (GO) enrichment analysis revealed that these DEGs primarily participated in 8 biological processes, 8 cellular components, and 4 molecular functions. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis identified several significantly enriched signaling pathways, such as the JAK-STAT signaling pathway, TNF signaling pathway, Toll-like receptor signaling pathway, and PI3K-Akt signaling pathway. ASNS promotes proliferation while inhibits the differentiation of chicken skeletal muscle satellite cells. This study provides a theoretical basis for studying the role of ASNS in muscle development.

Transcriptome sequencing reveals the potential mechanisms of dietary lutein regulation on chicken leg muscle development

Lutein is an antioxidant that can indicate the oxidative status of organisms through its coloration and may be involved in the development process of chicken skeletal muscle. In this study, after feeding Nanhai Yellow Chickens with lutein-containing feed for 21 d, the lutein group significantly increased the muscle fiber diameter and decreased the fiber density in the chicken's leg muscles compared to the control group. To elucidate the potential regulatory mechanisms by which lutein is involved in muscle development, RNA-seq was used to detect changes in gene expression in chicken leg muscle tissue. After data analysis, a total of 249 significantly differentially expressed genes (DEG) were identified, including TGF-β superfamily (MSTN and TGFB1) and nonreceptor tyrosine kinase c-Src (SRC). Results from GO and KEGG analysis showed that the DEGs were enriched in GO terms such as positive regulation of the ERK1/ERK2 cascade and negative regulation of myoblast differentiation, as well as signaling pathways including the Toll-like receptor signaling pathway and the MAPK signaling pathway. These significantly enriched GO terms and pathways are closely related to muscle development, suggesting that lutein may play an important role in the process of chicken muscle development. This study provides insights into the regulatory mechanisms of dietary lutein on chicken muscle development.

IGF2 promotes the differentiation of chicken embryonic myoblast by regulating mitochondrial remodeling.

Skeletal muscle is crucial for animal movement and posture maintenance, and it serves as a significant source of meat in the livestock and poultry industry. The number of muscle fibers differentiated from myoblast in the embryonic stage is one of the factors determining the content of skeletal muscle. Insulin-like growth factor 2 (IGF2), a well-known growth-promoting hormone, is crucial for embryonic and skeletal muscle growth and development. However, the specific molecular mechanism underlying its impact on chicken embryonic myoblast differentiation remains unclear. To elucidate the molecular mechanism by which IGF2 regulates chicken myoblast differentiation, we manipulated IGF2 expression in chicken embryonic myoblast. The results demonstrated that IGF2 was upregulated during chicken skeletal muscle development and myoblast differentiation. On the one hand, we found that IGF2 promotes mitochondrial biogenesis through the PGC1/NRF1/TFAM pathway, thereby enhancing mitochondrial membrane potential, oxidative phosphorylation, and ATP synthesis during myoblast differentiation. This process is mediated by the PI3K/AKT pathway. On the other hand, IGF2 regulates BNIP3-mediated mitophagy, clearing dysfunctional mitochondria. Collectively, our findings confirmed that IGF2 cooperatively regulates mitochondrial biogenesis and mitophagy to remodel the mitochondrial network and enhance mitochondrial function, ultimately promoting myoblast differentiation.

Novel circular RNA Sestrin1 promotes chicken myoblast proliferation and differentiation via circSesn1/miR-16-5p/SESN1 pathway

ABSTRACT 1. The development of chicken skeletal muscle is directly relevant to poultry husbandry production. Numerous studies have suggested that circular RNA play pivotal roles in muscle development. However, the functions and mechanisms of most circRNA in chicken myogenesis remain largely unknown. 2. This study identified a novel circSESN1 based on existing sequencing data and examined its authenticity and subcellular localisation by enzyme digestion and RNA fluorescence in situ hybridisation. Additionally, there was a positive correlation between the expression levels of circSESN1 and the developmental stage of chicken muscle. 3. Mechanistically, knockdown or overexpression of circSESN1 was performed in primary myoblasts to validate its function. The interactions between circSESN1, miR-16-5p, and the target gene sestrin 1 (SESN1) were investigated using bioinformatics analysis and a dual fluorescein reporter system. Real-time qPCR, a cell proliferation assay, and immunofluorescence staining techniques were used to investigate the promotion effect of circSESN1 on myoblast proliferation and differentiation by miR-16-5p/SESN1 pathway. 4. The results demonstrated that the newly identified chicken circSESN1 directly sponges gga-miR-16-5p to regulate SESN1 gene expression, promoting myoblast proliferation and differentiation.

Divergent Regulatory Roles of Transcriptional Variants of the Chicken LDB3 Gene in Muscle Shaping.

LIM domain binding 3 (LDB3) serves as a striated muscle-specific Z-band alternatively spliced protein that plays an important role in mammalian skeletal muscle development, but its regulatory role and molecular mechanism in avian muscle development are still unclear. In this study, we reanalyzed RNA sequencing data sets of 1415 samples from 21 chicken tissues published in the NCBI GEO database. First, three variants (LDB3-X, LDB3-XN1, and LDB3-XN2) generated by alternative splicing of the LDB3 gene were identified in chicken skeletal muscle, among which LDB3-XN1 and LDB3-XN2 are novel variants. LDB3-X and LDB3-XN1 are derived from exon skipping in chicken skeletal muscle at the E18-D7 stage and share three LIM domains, but LDB3-XN2 lacks a LIM domain. Our results preliminarily suggest that the formation of three variants of LDB3 is regulated by RBM20. The three splice isomers have divergent functions in skeletal muscle according to in vitro and in vivo assays. Finally, we identified the mechanism by which different variants play different roles through interactions with IGF2BP1 and MYHC, which promote the proliferation and differentiation of chicken myoblasts, in turn regulating chicken myogenesis. In conclusion, this study revealed the divergent roles of three LDB3 variants in chicken myogenesis and muscle remodeling and demonstrated their regulatory mechanism through protein-protein interactions.

Establishment and analysis of immortalized chicken skeletal muscle satellite cell lines1

Skeletal muscle satellite cells are stem cells that are known for their multipotency and ability to proliferate in vitro. However, primary skeletal muscle satellite cells have limited proliferative capacity in vitro, which hinders their study in poultry skeletal muscle. The emergence of immortalization techniques for cells has provided a useful tool to overcome this limitation and explore the functions of skeletal muscle satellite cells. In this study, we achieved the immortalization of chicken skeletal muscle satellite cells by transducing primary cells with TERT (Telomerase reverse transcriptase) amplified from chicken (chTERT) using a lentiviral vector through reconstitution of telomerase activity. The cells successfully bypassed replicative senescence but did not achieve true immortalization. Preliminary functional characterization of the established cell line revealed that the proliferative characteristics and cell cycle profile of the immortalized chicken skeletal muscle satellite cell lines (ICMS) were similar to those of chicken primary muscle satellite cells (CPMSCs). Serum dependency analysis and soft agar assays indicated that ICMS did not undergo malignant transformation. Induced differentiation results demonstrated that ICMS retained their capacity for differentiation. The cell lines established in this study provide an important basis for the establishment of immortalized poultry cell lines and a cell model for the study of poultry skeletal muscle-related functional genes.

RRM2 promotes the proliferation of chicken myoblasts, inhibits their differentiation and muscle regeneration

During myogenesis and regeneration, the proliferation and differentiation of myoblasts play key regulatory roles and may be regulated by many genes. In this study, we analyzed the transcriptomic data of chicken primary myoblasts at different periods of proliferation and differentiation with protein‒protein interaction network, and the results indicated that there was an interaction between cyclin-dependent kinase 1 (CDK1) and ribonucleotide reductase regulatory subunit M2 (RRM2). Previous studies in mammals have a role for RRM2 in skeletal muscle development as well as cell growth, but the role of RRM2 in chicken is unclear. In this study, we investigated the effects of RRM2 on skeletal muscle development and regeneration in chickens in vitro and in vivo. The interaction between RRM2 and CDK1 was initially identified by co-immunoprecipitation and mass spectrometry. Through a dual luciferase reporter assay and quantitative real-time PCR, we identified the core promoter region of RRM2, which is regulated by the SP1 transcription factor. In this study, through cell counting kit-8 assays, 5-ethynyl-2'-deoxyuridine incorporation assays, flow cytometry, immunofluorescence staining, and Western blot analysis, we demonstrated that RRM2 promoted the proliferation and inhibited the differentiation of myoblasts. In vivo studies showed that RRM2 reduced the diameter of muscle fibers and slowed skeletal muscle regeneration. In conclusion, these data provide preliminary insights into the biological functions of RRM2 in chicken muscle development and skeletal muscle regeneration.

MiR-460b-5p promotes proliferation and differentiation of chicken myoblasts and targets RBM19 gene

The meat production of broilers is crucial to economic benefits of broiler industries, while the slaughter performance of broilers is directly determined by skeletal muscle development. Hence, the broiler breeding for growth traits shows a great importance. As a kind of small noncoding RNA, microRNA (miRNA) can regulate the expression of multiple genes and perform a wide range of regulation in organisms. Currently, more and more studies have confirmed that miRNAs are closely associated with skeletal muscle development of chickens. Based on our previous miR-seq analysis (accession number: PRJNA668199), miR-460b-5p was screened as one of the key miRNAs probably involved in the growth regulation of chickens. However, the regulatory effect of miR-460b-5p on the development of chicken skeletal muscles is still unclear. Therefore, miR-460b-5p was further used for functional validation at the cellular level in this study. The expression pattern of miR-460b-5p was investigated in proliferation and differentiation stages of chicken primary myoblasts. It was showed that the expression level of miR-460b-5p gradually decreased from the proliferation stage (GM 50%) to the lowest at 24 h of differentiation. As differentiation proceeded, miR-460b-5p expression increased significantly, reaching the highest and stabilizing at 72 h and 96 h of differentiation. Through mRNA quantitative analysis of proliferation marker genes, CCK-8 and Edu assays, miR-460b-5p was found to significantly facilitate the transition of myoblasts from G1 to S phase and promote chicken myoblast proliferation. mRNA and protein quantitative analysis of differentiation marker genes, as well as the indirect immunofluorescence results of myotubes, revealed that miR-460b-5p significantly stimulated myotube development and promote chicken myoblast differentiation. In addition, the target relationship was validated for miR-460b-5p according to the dual-luciferase reporter assay and mRNA quantitative analysis, which indicates that miR-460b-5p was able to regulate RBM19 expression by specifically binding to the 3' UTR of RBM19. In summary, miR-460b-5p has positive regulatory effects on the proliferation and differentiation of chicken myoblasts, and RBM19 is a target gene of miR-460b-5p.

Exploration of Potential Target Genes of miR-24-3p in Chicken Myoblasts by Transcriptome Sequencing Analysis.

Broiler skeletal muscle growth is significantly influenced by miRNAs. Our earlier research demonstrated that miR-24-3p significantly suppressed the proliferation of chicken myoblasts while promoting their differentiation. The purpose of this study is to investigate miR-24-3p potential target genes in chickens. We collected myoblasts of Jinghai yellow chicken and transfected four samples with mimics of miR-24-3p and another four samples with mimic NC (negative control) for RNA-seq. We obtained 54.34 Gb of raw data in total and 50.79 Gb of clean data remained after filtering. Moreover, 11,635 genes were found to be co-expressed in these two groups. The mimic vs. NC comparison group contained 189 DEGs in total, 119 of which were significantly up-regulated and 70 of which were significantly down-regulated. Important biological process (BP) terminology such as nuclear chromosomal segregation, reproduction, and nuclear division were discovered by GO enrichment analysis for DEGs in the mimic vs. NC comparison group. KEGG pathway analysis showed that focal adhesion, cytokine-cytokine receptor interaction, the TGF-β signaling pathway, and the MAPK signaling pathway were enriched in the top 20. Variation site analysis illustrated the SNP (single nucleotide polymorphisms) and INDEL (insertion-deletion) in the tested samples. By comparing the target genes predicted by miRDB (MicroRNA target prediction database) and TargetScan with the 189 DEGs found by the transcriptome sequencing, we discovered two up-regulated DEGs (NEURL1 and IQSEC3) and two down-regulated DEGs (REEP1 and ST6GAL1). Finally, we carried out qPCR experiments on eight DEGs and discovered that the qPCR results matched the sequencing outcomes. These findings will aid in identifying potential miR-24-3p target genes in chicken skeletal muscle and offer some new directions for upcoming research on broiler breeding.

CircLRRFIP1 promotes the proliferation and differentiation of chicken skeletal muscle satellite cells by sponging the miR-15 family via activating AKT3-mTOR/p70S6K signaling pathway

Skeletal muscle is important for animal meat production, regulating movements, and maintaining homeostasis. Circular RNAs (circRNAs) have been founded to play vital role in myogenesis. However, the effects of the numerous circRNAs on growth and development of the skeletal muscle are yet to be uncovered. Herein, we identified circLRRFIP1, which is a novel circular RNA that is preferentially expressed in the skeletal muscle. To study the role of circLRRFIP1 in the skeletal muscle, the skeletal muscle satellite cells (SMSCs) was used to silenced or overexpressed circLRRFIP1. The results obtained in this study showed that circLRRFIP1 play a positive role in the proliferation and differentiation of SMSCs. The SMSCs were generated with stable knockdown and overexpression of circLRRFIP1, and the results showed that circLRRFIP1 exerts a stimulatory effect on the proliferation and differentiation of SMSCs. We further generated SMSCs with stable knockdown and overexpression of circLRRFIP1, and the results revealed that circLRRFIP1 exerts a stimulatory effect on the proliferation and differentiation of SMSCs. Mechanistically, circLRRFIP1 targets the myogenic inhibitory factor-miR-15 family to release the suppression of the miR-15 family to AKT3. The knockdown of AKT inhibits SMSC differentiation through the mTOR/p70S6K pathway. Taken together, the results obtained in this present study revealed the important role and the regulatory mechanisms of circLRRFIP1 in the development of chicken skeletal muscle. Therefore, this study provides an attractive target for molecular breeding to enhance meat production in the chicken industry.

Transcriptome-Based Identification of the Muscle Tissue-Specific Expression Gene CKM and Its Regulation of Proliferation, Apoptosis and Differentiation in Chicken Primary Myoblasts.

Skeletal muscle is an essential tissue in meat-producing animals, and meat-producing traits have been a hot topic in chicken genetic breeding research. Current research shows that creatine kinase M-type-like (CKM) is one of the most abundant proteins in skeletal muscle and plays an important role in the growth and development of skeletal muscle, but its role in the development of chicken skeletal muscle is still unclear. Via RNA sequencing (RNA-seq), we found that CKM was highly expressed in chicken breast muscle tissue. In this study, the expression profile of CKM was examined by quantitative real-time PCR (qPCR), and overexpression and RNA interference techniques were used to explore the functions of CKM in the proliferation, apoptosis and differentiation of chicken primary myoblasts (CPMs). It was shown that CKM was specifically highly expressed in breast muscle and leg muscle and was highly expressed in stage 16 embryonic muscle, while CKM inhibited proliferation, promoted the apoptosis and differentiation of CPMs and was involved in regulating chicken myogenesis. Transcriptome sequencing was used to identify genes that were differentially expressed in CPMs after CKM disruption, and bioinformatics analysis showed that CKM was involved in regulating chicken myogenesis. In summary, CKM plays an important role in skeletal muscle development during chicken growth and development.

The VGLL2 gene participates in muscle development in Gushi chickens1

Previous studies have shown that VGLL2, a member of the VGLL family, plays important roles in the growth and development of animal skeletal muscle, but its specific role in the development of chicken skeletal muscle is unclear. The main goal of this study was to explore the biological functions of VGLL2 in the development of chicken skeletal muscle and the proliferation and differentiation of skeletal muscle cells in vitro. In this study, we detected the effect of VGLL2 on the proliferation of myoblasts by CCK8, EdU and flow cytometry analyses after overexpressing and interfering with VGLL2. Indirect immunofluorescence was used to detect the effect of VGLL2 on the differentiation of myoblasts. qRT-PCR and hematoxylin and eosin (H&E) staining were used to evaluate the effects of VGLL2 overexpression on the growth rate and muscle fiber structure of chicken skeletal muscle. The results showed that VGLL2 inhibited the proliferation of primary cultured chicken myoblasts and promoted the differentiation of these cells. Interestingly, food intake and muscle fiber development were significantly enhanced by the overexpression of VGLL2 in chickens. Taken together, these data demonstrate that the VGLL2 gene may be a useful marker for improving muscle mass in poultry.

Gellan gum-gelatin scaffolds with Ca2+ crosslinking for constructing a structured cell cultured meat model

As an emerging technology to obtain protein by culturing animal-derived cells in vitro, it is crucial to construct 3D edible scaffolds to prepare structured cell cultured meat products. In this study, a scaffold based on gellan gum (GG)-gelatin (Gel) was prepared and further cross-linked with Ca2+. FTIR confirmed the electrostatic interaction between GG and Gel and the ionic cross-linking of Ca2+ and carboxyl groups, and SEM images showed the porous structure of the scaffolds. The staining results showed that scaffolds with high concentrations of Ca2+ had higher biocompatibility than scaffolds with low concentrations of Ca2+ and non-crosslinked scaffolds, and scaffolds Ca2+-GG2-Gel3-0.5 adhered to more cells and were more conducive to cell spreading. The immunofluorescence staining, SEM images, Western blot, and RT-qPCR showed that the scaffolds supported the proliferation and myogenic differentiation of chicken skeletal muscle satellite cells (CSMSCs) and myotubes were formed on the scaffolds. Finally, the scaffolds were stained and fried after culturing. The results of the textural and chromatic analysis showed that the texture and color of the scaffolds were similar to fresh meat and meat products. These results showed that ionically crosslinked GG-Gel scaffolds are biocompatible and stable for structured cell cultured meat models.

Gga-miRNA-181-5p family facilitates chicken myogenesis via targeting TGFBR1 to block TGF-β signaling

MicroRNAs (miRNAs) have been demonstrated to control chicken skeletal muscle growth, however, the potential function of the miR-181-5p family in chicken myogenesis remains largely unknown. Here, our study identified the two chicken (Gallus gallus; Gga) miR-181-5p family members widely expressed in various tissues, specifically miR-181a-5p and miR-181b-5p. Besides, the breast muscles of fast-growing broilers expressed higher levels of miR-181a-5p and miR-181b-5p than those of slow-growing layers. Functionally, miR-181a-5p and miR-181b-5p both promote the expression level of myogenic factors including myogenin (MyoG), myogenic differentiation 1 (MyoD1), and myosin heavy chain (MyHC), meanwhile accelerating the myotube formation of skeletal muscle satellite cells (SMSCs). Mechanistically, miR-181a-5p and miR-181b-5p directly bind to the 3´ untranslated region (UTR) of the transforming growth factor beta receptor 1 (TGFBR1) mRNA, further reducing the expression of TGFBR1. TGFBR1 is a key Transforming growth factor beta (TGF-β) signaling transduction receptor and had a negative function in muscle cell differentiation. Furthermore, knockdown of TGFBR1 facilitated the expression of chicken myogenic factors, boosted myotube formation, and decreased the SMAD family member 2/3 (SMAD2/3) phosphorylation in chicken SMSCs. SMAD2/3 are downstream of TGF-β signaling, and miR-181a-5p and miR-181b-5p could reduce the expression of TGFBR1 to further diminish the SMAD2/3 phosphorylation. Our findings revealed that the miR-181-5p family targets TGFBR1 to break the TGF-β signaling transduction, which resulted in promoting chicken skeletal muscle development.

Microplastic exposure induces muscle growth but reduces meat quality and muscle physiological function in chickens

Microplastic (MP) pollution has become one of the global environmental concerns, but the contamination and effect of MP on chicken skeletal muscle are scarcely researched. Here, we found MP contamination in the chicken skeletal muscles, which were directly collected from a large-scale chicken farm. Using Pyrolysis-Gas Chromatography-Mass Spectrometry and Agilent 8700 laser direct infrared imaging spectrometer, we found that polystyrene (PS) and polyamide are the significant type of MPs detected in chicken skeletal muscle. Constant PS-MP oral feeding for >21 days increases the content of MP deposited in chicken breast muscle, but the MP content in the leg muscle was gradually decreased. Surprisingly, the chicken's body and skeletal muscle weight was increased after constant PS-MP feeding. Physiological results showed that PS-MP exposure inhibited energy and lipid metabolism, induced oxidative stress, and potential for neurotoxicity in the skeletal muscle. Metabolomic analysis of the liquid chromatography-tandem mass spectrometry and gas chromatography coupled with the mass spectrometer results showed that PS-MP exposure changed the metabolomic profile and reduced meat quality. In vitro, experimental results showed that PS-MP exposure induced chicken primary myoblasts proliferation and apoptosis but decreased myoblasts differentiation. Transcriptome analysis of the skeletal muscle indicates that PS-MP exposure affects skeletal muscle function by regulating genes involved in neural function and muscle development. Considering that chicken is one of the most important meat foods in the world, this study will provide an essential reference for protecting meat food safety.

Regulation of autophagy in chick myotube cultures: Effect of uncoupling mitochondrial oxidative phosphorylation.

Abstracts: Skeletal muscles have a high demand for ATP, which is met largely through mitochondria oxidative phosphorylation. Autophagy is essential for the maintenance of skeletal muscle mass under catabolic conditions. This study investigated the effect of uncoupling mitochondrial oxidative phosphorylation on autophagy in chicken skeletal muscle. Chick myotubes were incubated with the mitochondrial uncoupler carbonyl cyanide m-chlorophenyl hydrazone (CCCP) at 25 μM for 3h. CCCP prevented the phosphorylation of p70 ribosomal S6 kinase 1 (Thr389), S6 ribosomal protein (Ser240/244), and eukaryotic translation initiation factor 4E-binding protein 1 (Thr37/46), which are the measures of the mechanistic target of rapamycin complex 1 (mTORC1) activity. CCCP significantly increased cytoplasmic and mitochondrial LC3-II content, which act as indices of index for autophagosome formation and mitophagy, respectively, but did not influence the expression of autophagy-related genes LC3B, GABARAPL1, and ATG12. Finally, surface sensing of translation method revealed that protein synthesis, a highly energy consuming process, was significantly decreased upon CCCP treatment. These results indicate that the uncoupling of mitochondrial oxidative phosphorylation stimulates autophagy and inhibits protein synthesis through mTORC1 signaling in chick myotube cultures.

Mining of chicken muscle growth genes and the function of important candidate gene RPL3L in muscle development

The birth weight of chickens does not significantly affect the weight at slaughter, while the different growth rate after birth was one of the important reasons for the difference in slaughter weight. Also, the increase in chickens' postnatal skeletal muscle weight is the main cause of the slaughter weight gain, but which genes are involved in this biological process is still unclear. In this study, by integrating four transcriptome datasets containing chicken muscles at different developmental times or different chicken tissues in public databases, a total of nine candidate genes that may be related to postnatal muscle development in chickens were obtained, including RPL3L, FBP2, ASB4, ASB15, CKMT2, PGAM1, YIPF7, PFKM, and LDHA. One of these candidate genes is RPL3L, whose 42bp insertion/deletion (indel) mutation significantly correlated with multiple carcass traits in the F2 resource population from Xinghua chickens crossing with White Recessive Rock (WRR) chickens, including live weight, carcass weight, half eviscerated weight, eviscerated weight, breast meat weight, wing weight, leg muscle shear force, and breast muscle shear force. Also, there was a very significant difference between different genotypes of the RPL3L 42bp indel mutation in these trains. Further experiments showed that RPL3L was highly expressed in chicken skeletal muscle, and its overexpression could promote the proliferation and inhibit the differentiation of chicken myoblasts by regulating ASB4 and ASB15 expression. Our findings demonstrated that the RPL3L 42bp indel may be one of the molecular markers of chicken weight-related traits.

CircRNAs Related to Breast Muscle Development and Their Interaction Regulatory Network in Gushi Chicken.

Circular RNAs (circRNAs) play a significant regulatory role during skeletal muscle development. To identify circRNAs during postnatal skeletal muscle development in chickens, we constructed 12 cDNA libraries from breast muscle tissues of Chinese Gushi chickens at 6, 14, 22, and 30 weeks and performed RNA sequencing. In total, 2112 circRNAs were identified, and among them 79.92% were derived from exons. CircRNAs are distributed on all chromosomes of chickens, especially chromosomes 1-9 and Z. Bioinformatics analysis showed that each circRNA had an average of 38 miRNA binding sites, 61.32% of which have internal ribosomal entry site (IRES) elements. Furthermore, in total 543 differentially expressed circRNAs (DE-circRNAs) were identified. Functional enrichment analysis revealed that DE-circRNAs source genes are engaged in biological processes and muscle development-related pathways; for example, cell differentiation, sarcomere, and myofibril formation, mTOR signaling pathway, and TGF-β signaling pathway, etc. We also established a competitive endogenous RNA (ceRNA) regulatory network associated with skeletal muscle development. The results in this report indicate that circRNAs can mediate the development of chicken skeletal muscle by means of a complex ceRNA network among circRNAs, miRNAs, genes, and pathways. The findings of this study might help increase the number of known circRNAs in skeletal muscle tissue and offer a worthwhile resource to further investigate the function of circRNAs in chicken skeletal muscle development.