Integrated Transcriptomic and Metabolomic Analysis Reveals Gut Microbiota-Mediated Regulation of Muscle Fiber Composition and Metabolism in Germ-Free Pigs.

Simple SummaryOne of the traits considered in livestock production is the gain of muscle mass, which is one of the factors responsible for governing the growth of skeletal muscles. Muscle hypertrophy involves the proliferation and differentiation of muscle cells and the maturation of muscle fibers. Advances in animal genetics and breeding rely on identifying hub genes, understanding ontological features, and identifying pathways of gene activity. Therefore, the aim of this study was to identify the hub genes and mechanisms involved in skeletal muscle maturation and hypertrophy in livestock species (Bos taurus, Ovis aries, and Sus scrofa). The hub genes identified in this study can be used to better identify and improve the growth and maturation of skeletal muscles and, as a result, enhance meat quality characteristics for breeding purposes in the mentioned species.The aim of the current study was to identify the major genes and pathways involved in the process of hypertrophy and skeletal muscle maturation that is common for Bos taurus, Ovis aries, and Sus scrofa species. Gene expression profiles related to Bos taurus, Ovis aries, and Sus scrofa muscle, with accession numbers GSE44030, GSE23563, and GSE38518, respectively, were downloaded from the GEO database. Differentially expressed genes (DEGs) were screened out using the Limma package of R software. Genes with Fold Change > 2 and an adjusted p-value < 0.05 were identified as significantly different between two treatments in each species. Subsequently, gene ontology and pathway enrichment analyses were performed. Moreover, hub genes were detected by creating a protein–protein interaction network (PPI). The results of the analysis in Bos taurus showed that in the period of 280 dpc–3-months old, a total of 1839 genes showed a significant difference. In Ovis aries, however, during the period of 135dpc–2-months old, a total of 486 genes were significantly different. Additionally, in the 91 dpc–adult period, a total of 2949 genes were significantly different in Sus scrofa. The results of the KEGG pathway enrichment analysis and GO function annotation in each species separately revealed that in Bos taurus, DEGs were mainly enriched through skeletal muscle fiber development and skeletal muscle contraction, and the positive regulation of fibroblast proliferation, positive regulation of skeletal muscle fiber development, PPAR signaling pathway, and HIF-1 signaling pathway. In Ovis aries, DEGs were mainly enriched through regulating cell growth, skeletal muscle fiber development, the positive regulation of fibroblast proliferation, skeletal muscle cell differentiation, and the PI3K-Akt signaling, HIF-1 signaling, and Rap1 signaling pathways. In Sus scrofa, DEGs were mainly enriched through regulating striated muscle tissue development, the negative regulation of fibroblast proliferation and myoblast differentiation, and the HIF-1 signaling, AMPK signaling, and PI3K-Akt signaling pathways. Using a Venn diagram, 36 common DEGs were identified between Bos taurus, Ovis aries, and Sus scrofa. A biological pathways analysis of 36 common DEGs in Bos taurus, Ovis aries, and Sus scrofa allowed for the identification of common pathways/biological processes, such as myoblast differentiation, the regulation of muscle cell differentiation, and positive regulation of skeletal muscle fiber development, that orchestrated the development and maturation of skeletal muscle. As a result, hub genes were identified, including PPARGC1A, MYOD1, EPAS1, IGF2, CXCR4, and APOA1, in all examined species. This study provided a better understanding of the relationships between genes and their biological pathways in the skeletal muscle maturation process.

The relationship between muscle fibre composition and fibre conduction velocity was investigated in 19 male track athletes, 12 sprinters and 7 distance runners, aged 20-24 years, using needle biopsy samples from vastus lateralis. Cross sectional areas of the fast twitch (FT) and slow twitch (ST) fibres were determined by histochemical analysis. The percentage of FT fibre areas ranged from 22.6 to 93.6%. Sprinters had a higher percentage of FT fibres than distance runners. Muscle fibre conduction velocity was measured with a surface electrode array placed along the muscle fibres, and calculated from the time delay between 2 myoelectric signals recorded during a maximal voluntary contraction. The conduction velocity ranged from 4.13 to 5.20 m.s-1. A linear correlation between conduction velocity and the relative area of FT fibres was statistically significant (r = 0.84, p less than 0.01). This correlation indicates that muscle fibre composition can be estimated from muscle fibre conduction velocity measured noninvasively with surface electrodes.

Muscle fibers, as the fundamental units of muscle tissue, play a crucial role in determining skeletal muscle function through their growth, development, and composition. To investigate changes in muscle fiber types and their regulatory mechanisms in Mongolian horses (MG), Xilingol horses (XL), and Grassland-Thoroughbreds (CY), we conducted histological and bioinformatic analyses on the gluteus medius muscle of these three horse breeds. Immunofluorescence analysis revealed that Grassland-Thoroughbreds had the highest proportion of fast-twitch muscle fibers at 78.63%, while Mongolian horses had the lowest proportion at 57.54%. Whole-transcriptome analysis identified 105 differentially expressed genes (DEGs) in the CY vs. MG comparison and 104 DEGs in the CY vs. XL comparison. Time-series expression profiling grouped the DEGs into eight gene sets, with three sets showing significantly up-regulated or down-regulated expression patterns (p &lt; 0.05). Additionally, 280 differentially expressed long non-coding RNAs (DELs) were identified in CY vs. MG, and 213 DELs were identified in CY vs. XL. A total of 32 differentially expressed microRNAs (DEMIRs) were identified in CY vs. MG, while 44 DEMIRs were found in CY vs. XL. Functional enrichment analysis indicated that the DEGs were significantly enriched in essential biological processes, such as actin filament organization, muscle contraction, and protein phosphorylation. KEGG pathway analysis showed their involvement in key signaling pathways, including the mTOR signaling pathway, FoxO signaling pathway, and HIF-1 signaling pathway. Furthermore, functional variation-based analyses revealed associations between non-coding RNAs and mRNAs, with some non-coding RNAs targeting genes potentially related to muscle function regulation. These findings provide valuable insights into the molecular basis for the environmental adaptability, athletic performance, and muscle characteristics in horses, offering new perspectives for the breeding of Grassland-Thoroughbreds.

The purpose of the present study was to determine whether the fast-twitch (FT) fiber composition in muscle was a) correlated with performance capacities, b) related to the trainability of the subjects, and c) whether the FT fiber composition could be predicted with standard laboratory tests. From twenty-four young women (ages 24.3 +/- 3.0 yrs) muscle biopsies were obtained from the vastus lateralis of both the left and right legs. In addition, each subject completed four laboratory tests: i) a maximal oxygen intake test on the cycle ergometer, ii) a high-kp power test, iii) a low-kp power test, and iv) a Sargent jump. Prior to training the correlations between %FT fibers and each of the tests (r equal to or less than 0.19) were not significant (P less than 0.05). After training anaerobically (N = 20) significant improvements (P less than 0.05) occurred in a) the high-kp test (delta = + 0.12 +/- 0.02 kpm/sec.kg) b) the low-kp test (delta = + 0.10 +/- 0.01 kpm/sec.kg) and c) the Sargent jump (delta = + 2.5 +/- 1.1 cm). However, these training-induced changes (delta) were not correlated with %FT fiber composition (r equal to or less than 0.35), nor were the post-training performances on the tests correlated with the %FT fibers (r equal to or less than 0.12). Performance capacities before and after training were not significantly different in groups with a low %FT fiber composition (35.8 +/- 1.6%) or a high %FT fiber composition (63.6 +/- 2.2%). None of the performance tests either singly, or in combination in a multiple regression equation, provided a suitable prediction of the FT muscle fiber composition. The results of this study indicate that athletic performances and/or susceptibility for training cannot be determined a priori from simple measurements of muscle fiber composition.

Clear cell renal cell carcinoma (ccRCC) is the most common pathological type of renal cancer, and is associated with a high mortality rate, which is related to high rates of tumor recurrence and metastasis. The aim of the present study was to identify reliable molecular biomarkers with high specificity and sensitivity for ccRCC. A total of eight ccRCC-related expression profiles were downloaded from Gene Expression Omnibus for integrated bioinformatics analysis to screen for significantly differentially expressed genes (DEGs). Reverse transcription-quantitative (RT-q)PCR, western blotting and immunohistochemistry staining assays were performed to evaluate the expression levels of candidate biomarkers in ccRCC tissues and cell lines. In total, 255 ccRCC specimens and 165 adjacent normal kidney specimens were analyzed, and 344 significant DEGs, consisting of 115 upregulated DEGs and 229 downregulated DEGs, were identified. The results of Gene Ontology analysis suggested a significant enrichment of DEGs in 'organic anion transport' and 'small molecule catabolic process' in biological processes, in 'apical plasma membrane' and 'apical part of the cell' in cell components, and in 'anion transmembrane transporter activity' and 'active transmembrane transporter activity' in molecular functions. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis indicated that the DEGs were significantly enriched in the 'phagosome', the 'PPAR signaling pathway', 'complement and coagulation cascades', the 'HIF-1 signaling pathway' and 'carbon metabolism'. Next, 7 hub genes (SUCNR1, CXCR4, VCAN, CASR, ATP6V0A4, VEGFA and SERPINE1) were identified and validated using The Cancer Genome Atlas database. Survival analysis showed that low expression of ATP6V0A4 was associated with a poor prognosis in patients with ccRCC. Additionally, received operating characteristic curves indicated that ATP6V0A4 could distinguish ccRCC samples from normal kidney samples. Furthermore, RT-qPCR, western blotting and immunohistochemistry staining results showed that ATP6V0A4 was significantly downregulated in ccRCC tissues and cell lines. In conclusion, ATP6V0A4 may be involved in tumor progression and regarded as a potential therapeutic target for the recurrence and metastasis of ccRCC.

It is generally accepted that muscle fibre composition may influence physical performance. The α-actinin-3 (ACTN3) gene R577X polymorphism is suspected to be one of the contributing gene variations in the determination of muscle fibre type composition and athletic status. In the present study, we examined the dependence of average preferred racing distance (PRD) on muscle fibre type composition of the vastus lateralis muscle in 34 subelite Russian speed skaters (20 men and 14 women) who competed in races of different length (500-10,000 m). We also investigated the association between the ACTN3 polymorphism and muscle fibre characteristics in 94 subjects (60 physically active healthy men and 34 speed skaters), as well as the relationship between PRD and ACTN3 genotype in 115 subelite and elite speed skaters. In addition, ACTN3 genotype and allele frequencies of the 115 speed skaters were compared with 1301 control subjects. The ACTN3 XX genotype frequency was significantly lower in sprinters (n = 39) compared with control subjects (2.6 versus 14.5%; P = 0.034). We observed a positive relationship between PRD and the proportion of slow-twitch muscle fibres that was close to linear, but better fitted a logarithmic curve (r = 0.593, P < 0.0005). The ACTN3 R577X polymorphism was associated with muscle fibre composition (slow-twitch fibres: RR genotype, 51.7 (12.8)%; RX, 57.4 (13.2)%; XX 61.5 (16.3)%; = 0.215; P = 0.049) in the overall muscle biopsy group, and with PRD of all athletes ( = 0.24, P = 0.010), indicating thatACTN3 XX genotype carriers exhibit a higher proportion of slow-twitch fibres and prefer to skate long-distance races. However, the majority of the association between muscle fibre type and PRD was independent of ACTN3 genotype. In conclusion, the ACTN3 R577X polymorphism is associated with preferred racing distance in speed skaters and muscle fibre type composition. Thus, it is probably partly via associations with fibre type that the R577X polymorphism contributes to a small but perhaps important component of the ability to perform at a high level in speed skating.

The aim of the present experiment was to determine the effect of sex and age on histochemical and morphometric characteristics of muscle fibres (myocytes) in lambs born by single, twin, triplet and quadruplet birth. Thirty lambs were slaughtered at 60 days of age; thirty were weaned at 60 days and fed until 120 days with flakes (60%) and food supplements, and then slaughtered. Muscle tissues were obtained from two muscles, namely m. semitendinosus and m. longissimus dorsi of all lambs. For each fibre type, area perimeter and diameter (maximum and minimum) were measured and slow-twitch oxidative fibres, fast-twitch glycolytic fibres, fast-twitch oxidative-glycolytic fibres were histochemically differentiated. The muscles were stained for myosin ATPase, and succinic dehydrogenase. At 60 days, females had fibres larger than males, whereas the opposite was observed at 120 days. Besides, at 60 days, the lambs born by single birth had fibres larger than those born by multiple birth, whereas the opposite was observed at 120 days. Single lambs were heavier than twin lambs and multiple lambs. Fast-twitch glycolytic fibres had the largest size, followed by slow-twitch oxidative and fast-twitch oxidative glycolytic fibres. The dimensions of fibre types in m. longissimus dorsi were larger than in m. semitendinosus (P &lt; 0.001).These muscle fibre characteristics are thought to be important factors influencing meat quality, which is often related to metabolic and contractile properties as determined by the muscle fibre type distribution.

Muscle fibers are the fundamental units of skeletal muscle. Based on contraction speed and metabolic properties, muscle fibers are categorized into fast-twitch and slow-twitch fibers. Further subdivision based on MyHC gene isoforms identifies them as type I, IIA, IIB, and IIX fibers. There is potential for interconversion among these muscle fiber types. The proportions of different muscle fibers determine muscle functional properties and affect muscle quality. Compared with muscles mainly harboring fast-twitch fibers, muscles predominantly composed of slow-twitch fibers are characterized by enhanced water-holding capacity, tenderness, and superior flavor. During the formation and transformation of animal skeletal muscle fibers, the expression of a series of muscle-specific genes is precisely regulated by cis-regulatory elements. These cis-regulatory elements achieve precise regulation of the target genes through interactions with transcription factors and other regulatory proteins, thereby ensuring the formation and transformation of muscle fibers. Based on introducing the types and characteristics of muscle fibers, we summarize and prospect the role of the transcription factors and cis-regulatory elements in the formation and transformation of fast-twitch and slow-twitch muscle fibers in livestock. The aim of this review is to deepen the understanding of the relationship between gene expression regulation and muscle fiber diversity, and to provide theoretical support for the improvement of meat quality in livestock.

Skeletal muscle adapts to the stress of endurance and sprint exercise and training. There are 2 main types of skeletal muscle fibre--slow twitch (ST) and fast twitch (FTa, FTb, FTc). Exercise may produce transitions between FT and ST fibres. Sprint training has decreased the proportion of ST fibres and significantly increased the proportion of FTa fibres, while endurance training may convert FTb to FTa fibres, and increase the proportion of ST fibres (i.e. FTb----FTa----FTc----ST). However, the high proportion of ST fibres documented for elite endurance athletes may be simply the result of natural selection. ST fibres function predominantly during submaximal exercise, whereas FT fibres are recruited as exercise intensity approaches VO2max and/or glycogen stores are depleted. Long distance runners have greater ST and FT fibre areas than untrained controls. However, doubt remains as to whether the ST or FT fibre area is greatest in endurance athletes. Increases in FT fibre area seem to occur during the first 2 months of training whereas ST fibre areas appear to increase after 2 to 6 months of training. Sprint training leads to the preferential use of FT fibres and male, but not female sprinters have larger FT fibres than untrained controls. Mitochondrial proteins and oxidative enzymes, as opposed to VO2max, are important determinants of the duration of endurance exercise. Endurance training increases intramuscular glycogen stores in both FT and ST fibres and produces a 'glycogen-sparing' effect which is characterised by an increased free fatty acid (FFA) metabolism. The activity of glycogen synthase is also increased by endurance training. Sprint training increases glycogen concentrations similarly in all fibre types, reduces the rate of glycogen utilisation at submaximal workloads and allows supramaximal workloads to be maintained for longer periods of time. During endurance exercise the pattern of glycogen depletion varies between muscle fibre types and between muscle groups. Glycogen stores in ST fibres are utilised initially, followed by stores in FTa then FTb fibres. Sprint activities are associated with a much greater rate of glycogen depletion. However, it is unlikely that glycogen depletion causes fatigue during sprinting. Sprint work is associated with a preferential depletion of glycogen from FTb then FTa and ST fibres. Endurance training appears to increase triglyceride stores adjacent to mitochondria and ST fibres have greater triglyceride stores than FT fibres. Endurance exercise is associated with a preferential use of triglycerides from ST fibres and endogenous triglycerides may account for over 50% of the total lipid oxidised during exercise.(ABSTRACT TRUNCATED AT 400 WORDS)

Muscle fibre composition and capillarity were evaluated in frozen sections stained for myosin ATPase of the soleus and the white area of the medial head of the gastrocnemius of rats made hyperthyroid by injections of triiodothyronine (300-400 micrograms/kg body weight, every other day) for 2, 3 and 4 weeks. O2 consumption of homogenates of these muscles in the presence of excess inorganic phosphate (Pi) and ADP with pyruvate and malate as substrates was also measured. Increased oxidative capacity was observed in the soleus homogenates of hyperthyroid animals after 2 weeks of treatment while no changes were observed in the oxidative capacity of the homogenates of the white area of the medial head of the gastrocnemius, even after 4 weeks of treatment. Hyperthyroid animals showed a greater capillarity than controls in both muscles. In the soleus this was evident after 2 weeks of treatment while in the white area of the medial head of the gastrocnemius, it was evident only after 4 weeks of treatment. Fibre composition was affected in the soleus after 4 weeks of treatment. In control animals two fibre types were present in the soleus: slow-twitch oxidative fibres (s.o. or type I fibres) with a high ATPase activity after acid pre-incubation and fast-twitch glycolytic oxidative fibres (f.o.g. or type IIa fibres) with a low ATPase activity after acid pre-incubation. In the soleus of the hyperthyroid animals, a third fibre type with intermediate ATPase activity after acid pre-incubation was also present. This most probably represents a change in the type of myosin being synthesized by some fibres. No changes in fibre composition were observed in the white area of medial head of the gastrocnemius which was made up of only fast-twitch glycolytic fibres (f.g. or type IIb fibres). The changes in oxidative capacity and capillarity in the soleus preceded and did not seem to be related to the changes in the type of myosin being synthesized. The increased capillarity found in the white area of the medial head of the gastrocnemius of the hyperthyroid animals, in the absence of an increase in the oxidative capacity, indicates that the latter is not the only factor that determines capillarity in skeletal muscle.

The expression of myosin heavy chain (MHC) genes in human skeletal muscle is related to metabolic characteristics involved in the pathogenesis of type 2 diabetes

The succinate dehydrogenase (SDH) activity and cross-sectional area of individual muscle fibers in the cat diaphragm were quantified using a computerized image-processing system. The population distributions of fiber-SDH activities and cross-sectional areas showed considerable range within each diaphragm. Despite an overlap in the distribution of SDH activities between fast-twitch (i.e., those staining darkly for myosin ATPase) and slow-twitch (i.e., those staining lightly for myosin ATPase) fibers, differences between the two populations of fibers were observed. Fast-twitch fibers generally had lower SDH activities and greater cross-sectional areas than slow-twitch fibers. However, the range of SDH activities and cross-sectional areas of fast-twitch fibers was much greater than in slow-twitch fibers. The population distributions of SDH activities and cross-sectional areas of both fast- and slow-twitch fibers were unimodal. The unimodal distribution of SDH activities in fast-twitch fibers suggested that these fibers could not be clearly subdivided into two groups based on differences in their oxidative capacity (i.e., high- and low-oxidative fibers). These results were discussed in the context of the classification of fast-twitch glycolytic and fast-twitch oxidative-glycolytic fibers using qualitative histochemistry. In addition, the functional significance of the unimodal distribution of oxidative capacities in fast-twitch fibers was discussed in relation to the distribution of fatigue properties in fast-twitch motor units. A significant negative correlation between cross-sectional area and SDH activity in both fast- and slow-twitch fibers was also observed. The importance of this negative correlation in relationship to the diffusion of energy substrates for oxidative metabolism was discussed.

The distinct transcriptomes of fast-twitch and slow-twitch muscles in Mongolian horses

Skeletal muscles of teleost are mainly composed of slow-twitch muscles (SM) and fast-twitch muscles (FM) differed in contractile properties, metabolic capacities, and regeneration rate. The transcriptional regulatory mechanisms that control different muscle types have been elucidated in teleost according to transcriptome between SM and FM. However, the differences between SM and FM were affected not only by genotype but also by complicated epigenetic effects, including DNA methylation, which usually regulates genes in transcription level. To determine the essential role of DNA methylation in the regulation of different muscle types, we analyzed whole-genome methylation profiles of pelagic migratory fish Pseudocaranx dentex with abundant and well-separated SM and integrated DNA methylation profiles with the previously obtained transcriptome data. A total of 4,217 differentially methylated genes (DMGs) were identified, of which 3,582 were located in the gene body and 635 in the promoter. These DMGs mainly participated in muscle metabolite and cell junction. Enriched cell junction pathway reflected different capillary distribution between SM and FM. Through comprehensive analysis of methylome and transcriptome, 84 differentially expressed genes (DEGs) showed significant methylation variation in promoters between SM and FM, indicating that their expression was regulated by DNA methylation. Hypomethylated and highly expressed oxygen storage protein Myoglobin (myg) in SM indicated demethylation of myg promoter could upregulate its expression, thus increasing O2 supplying and meeting oxygen demands of SM. Hypermethylated and lowly expressed tnn (Troponin) and rlc (myosin regulatory light chain) in SM may be associated with low mobility of myosin cross bridges, which lead to slower and less frequent muscle contraction in SM than in FM. In addition, hypomethylated and highly expressed lbx1 (Ladybird homeobox protein homolog 1) and epo (erythropoietin) may be related to increased satellite cell numbers, and Semaphorin/Plexin genes may be related to higher rate of neuromuscular connection reconstruction, which further promote high muscle regeneration efficiency in SM. Our study elucidated the potential DNA methylation mechanisms that regulate physiological characteristics differences between SM and FM, which could facilitate our understanding of skeletal muscle adaptation in pelagic migratory fishes and further enrich the theoretical basis for the study of physiological characteristics and adaptive evolution in teleost fishes.

Nitrogen is one of the most important mineral elements for plant growth and development. Excessive nitrogen application not only pollutes the environment, but also reduces the quality of crops. However, are few studies on the mechanism of barley tolerance to low nitrogen at both the transcriptome and metabolomics levels. In this study, the nitrogen-efficient genotype (W26) and the nitrogen-sensitive genotype (W20) of barley were treated with low nitrogen (LN) for 3 days and 18 days, then treated with resupplied nitrogen (RN) from 18 to 21 days. Later, the biomass and the nitrogen content were measured, and RNA-seq and metabolites were analyzed. The nitrogen use efficiency (NUE) of W26 and W20 treated with LN for 21 days was estimated by nitrogen content and dry weight, and the values were 87.54% and 61.74%, respectively. It turned out to have a significant difference in the two genotypes under the LN condition. According to the transcriptome analysis, 7926 differentially expressed genes (DEGs) and 7537 DEGs were identified in the leaves of W26 and W20, respectively, and 6579 DEGs and 7128 DEGs were found in the roots of W26 and W20, respectively. After analysis of the metabolites, 458 differentially expressed metabolites (DAMs) and 425 DAMs were found in the leaves of W26 and W20, respectively, and 486 DAMs and 368 DAMs were found in the roots of W26 and W20, respectively. According to the KEGG joint analysis of DEGs and DAMs, it was discovered that glutathione (GSH) metabolism was the pathway of significant enrichment in the leaves of both W26 and W20. In this study, the metabolic pathways of nitrogen metabolism and GSH metabolism of barley under nitrogen were constructed based on the related DAMs and DEGs. In leaves, GSH, amino acids, and amides were the main identified DAMs, while in roots, GSH, amino acids, and phenylpropanes were mainly found DAMs. Finally, some nitrogen-efficient candidate genes and metabolites were selected based on the results of this study. The responses of W26 and W20 to low nitrogen stress were significantly different at the transcriptional and metabolic levels. The candidate genes that have been screened will be verified in future. These data not only provide new insights into how barley responds to LN, but also provide new directions for studying the molecular mechanisms of barley under abiotic stress.