Pig Skeletal Muscle Research Articles

Transcriptome analysis revealed chimeric RNAs, single nucleotide polymorphisms and allele-specific expression in porcine prenatal skeletal muscle

Prenatal skeletal muscle development genetically determines postnatal muscle characteristics such as growth and meat quality in pigs. However, the molecular mechanisms underlying prenatal skeletal muscle development remain unclear. Here, we performed the first genome-wide analysis of chimeric RNAs, single nuclear polymorphisms (SNPs) and allele-specific expression (ASE) in prenatal skeletal muscle in pigs. We identified 14,810 protein coding genes and 163 high-confidence chimeric RNAs expressed in prenatal skeletal muscle. More than 94.5% of the chimeric RNAs obeyed the canonical GT/AG splice rule and were trans-splicing events. Ten and two RNAs were aligned to human and mouse chimeric transcripts, respectively. We detected 106,457 high-quality SNPs (6,955 novel), which were mostly (89.09%) located within QTLs for production traits. The high proportion of non-exonic SNPs revealed the incomplete annotation status of the current swine reference genome. ASE analysis revealed that 11,300 heterozygous SNPs showed allelic imbalance, whereas 131 ASE variants were located in the chimeric RNAs. Moreover, 4 ASE variants were associated with various economically relevant traits of pigs. Taken together, our data provide a source for studies of chimeric RNAs and biomarkers for pig breeding, while illuminating the complex transcriptional events underlying prenatal skeletal muscle development in mammals.

Activation of ubiquitin‐proteasome system components in heat‐stressed pig skeletal muscle

Hyperthermia causes metabolic changes and leads to lower lean tissue accretion and increased adipose tissue deposition in animals. The direct effect of heat stress on muscle breakdown has been reported in poultry, rabbits and lactating cows. Muscle breakdown occurs by activating the ubiquitin‐proteasome system and the autophagy‐lysosome pathways. To study the mechanism by which heat stress induces muscle breakdown, the ubiquitin‐protease system in the pig skeletal muscle was examined. Pigs (35 ± 0.8 kg) were assigned to one of three treatments: thermal neutral fed ad libitum (TN, 21°C, n=8), heat stress fed ad libitum (HS, 35°C, n=8), or thermal neutral, pair‐fed to HS intake (PF, n=8) for 7 days. Longissimus dorsi muscle was biopsied on day 3 and 7 relative to start of treatments for real‐time PCR and immunoblotting assays. Heat stress increased activating transcription factor‐4 (ATF4), a stress‐response transcription factor, mRNA abundance by more than 1.5 fold regardless of days (P < 0.05). Heat stress increased growth arrest and DNA damage‐inducible 45a (Gadd45a) mRNA abundance by about 1.5 fold compared with PF muscle (P < 0.05). Similarly, the protein abundance of skeletal muscle Gadd45a was elevated in HS pigs by 50% as well as in PF pigs compared with TN pigs (P < 0.05). The phosphorylation of GSK3 β was similar between groups on day 3 and not different in HS versus PF and TN muscle. However, PF pigs had significantly higher phosphorylated GSK3β compared with TN pigs (P < 0.05). HS pigs exhibited intermediate atrogin‐1 mRNA abundance, which was not different from either TN or PF pigs. However, HS pigs had a significantly higher muscle ring finger‐1 (MuRF1) mRNA abundance (approximately 2 fold) compared with PF pigs (P < 0.05). In conclusion, heat stress activated components of muscle breakdown by inducing AFT4, which increased Gadd45a expression, and subsequent apparent activation of the ubiquitin‐protease system.Support or Funding InformationResults described herein were supported by Agriculture and Food Research Initiative Competitive Grant nos. 2011‐6700330007 and 2014‐67015‐21627 from the USDA National Institute of Food and Agriculture.

Dysregulation of sirtuins and key metabolic genes in skeletal muscle of pigs with spontaneous intrauterine growth restriction is associated with alterations of circulating IGF-1

Prenatal and early postnatal life determines future health, and intrauterine growth restriction (IUGR) – associated low birth weight predisposes to metabolic syndrome in adulthood. We hypothesize here that IUGR might induce hormonal and gene expression alterations predisposing to metabolic disease.Using a porcine model of spontaneous IUGR, we determined in utero (71, 112days post-conception) and early-postnatal (2days post-birth) IGF-1, insulin and leptin levels, and in parallel we investigated, in skeletal muscle, the developmental expression patterns of sirtuins and metabolic and signaling genes IRS1, GLUT4, HK2 and GAPDH.IUGR was associated with impaired IGF-1 plasmatic levels. Gene expression of sirtuin 1, 5, 6, 7, GLUT4 and HK2 exhibited significant correlations with gestational age or body weight. SIRT1 and HK2 expression displayed an age- and weight-dependent downregulation in controls, which was lost in IUGR pigs. Conversely, SIRT2 and GLUT4 were upregulated in IUGR pigs. Within the set of genes studied, we found a significant correlation between IGF-1 levels and gene expression in control, but not IUGR samples, indicating that lower IGF-1 may be a limiting factor in IUGR. IUGR-dependent gene alterations were partly linked to epigenetic changes on histone H3 acetylation and methylation.Overall, our data indicate that several sirtuins and metabolic genes display specific gene expression trajectories during fetal and early postnatal life. Gene expression alterations observed in IUGR are correlated to IGF-1 dysregulation. Given the importance of the genes studied in metabolic control, their perinatal alterations might contribute to the predisposition to metabolic disease of adulthood.

Molecular characterization and tissue expression profile of porcine Ephrin-B2.

Ephrin-B2 (EFNB2) is a signaling molecule that plays an important role in cell adhesion, proliferation, and migration in humans. However, little is known about this molecule in pigs. In order to investigate whether EFNB2 is associated with the skeletal muscle in pigs, we cloned the full-length cDNA of EFNB2 (GenBank accession No. KF500033) from the longissimus dorsi muscle of Yorkshire pigs by rapid amplification of cDNA ends. The results indicated that its full-length cDNA comprises 1991 bp, with an open reading frame of 1002 bp, a 5' end of 88 bp, and a 3' end of 901 bp. We analyzed the homology of porcine EFNB2 with sequences from other species, and the phylogenetic tree showed that pig EFNB2 was most closely related to that from sheep, followed by domestic cats and wolf, with mackerel being the most distantly related. Porcine EFNB2 is a water-soluble protein with a theoretical molecular weight of 36,928.1 Da, an isoelectric point of 8.98, and a hydrophilic transmembrane-spanning region. It contains 19 glycosylation sites and eight phosphorylation sites. The tertiary structure of the EFNB2 protein showed a forniciform helix structure. The porcine EFNB2 gene was expressed in ten different tissues from 25-day-old Shaziling and Yorkshire piglets, with the highest expression observed in the longissimus dorsi. These results lay the foundation for further study on the EFNB2 gene in pigs.

Development of a decellularized scaffold of pig skeletal muscle tissue

Development of a decellularized scaffold of pig skeletal muscle tissue

Signaling Pathways Related to Protein Synthesis and Amino Acid Concentration in Pig Skeletal Muscles Depend on the Dietary Protein Level, Genotype and Developmental Stages.

Muscle growth is regulated by the homeostatic balance of the biosynthesis and degradation of muscle proteins. To elucidate the molecular interactions among diet, pig genotype, and physiological stage, we examined the effect of dietary protein concentration, pig genotype, and physiological stages on amino acid (AA) pools, protein deposition, and related signaling pathways in different types of skeletal muscles. The study used 48 Landrace pigs and 48 pure-bred Bama mini-pigs assigned to each of 2 dietary treatments: lower/GB (Chinese conventional diet)- or higher/NRC (National Research Council)-protein diet. Diets were fed from 5 weeks of age to respective market weights of each genotype. Samples of biceps femoris muscle (BFM, type I) and longissimus dorsi muscle (LDM, type II) were collected at nursery, growing, and finishing phases according to the physiological stage of each genotype, to determine the AA concentrations, mRNA levels for growth-related genes in muscles, and protein abundances of mechanistic target of rapamycin (mTOR) signaling pathway. Our data showed that the concentrations of most AAs in LDM and BFM of pigs increased (P<0.05) gradually with increasing age. Bama mini-pigs had generally higher (P<0.05) muscle concentrations of flavor-related AA, including Met, Phe, Tyr, Pro, and Ser, compared with Landrace pigs. The mRNA levels for myogenic determining factor, myogenin, myocyte-specific enhancer binding factor 2 A, and myostatin of Bama mini-pigs were higher (P<0.05) than those of Landrace pigs, while total and phosphorylated protein levels for protein kinase B, mTOR, and p70 ribosomal protein S6 kinases (p70S6K), and ratios of p-mTOR/mTOR, p-AKT/AKT, and p-p70S6K/p70S6K were lower (P<0.05). There was a significant pig genotype-dependent effect of dietary protein on the levels for mTOR and p70S6K. When compared with the higher protein-NRC diet, the lower protein-GB diet increased (P<0.05) the levels for mTOR and p70S6K in Bama mini-pigs, but repressed (P<0.05) the level for p70S6K in Landrace pigs. The higher protein-NRC diet increased ratio of p-mTOR/mTOR in Landrace pigs. These findings indicated that the dynamic consequences of AA profile and protein deposition in muscle tissues are the concerted effort of distinctive genotype, nutrient status, age, and muscle type. Our results provide valuable information for animal feeding strategy.

Analysis of MicroRNA Expression Profiles in Weaned Pig Skeletal Muscle after Lipopolysaccharide Challenge.

MicroRNAs (miRNAs) constitute a class of non-coding RNAs that play a crucial regulatory role in skeletal muscle development and disease. Several acute inflammation conditions including sepsis and cancer are characterized by a loss of skeletal muscle due primarily to excessive muscle catabolism. As a well-known inducer of acute inflammation, a lipopolysaccharide (LPS) challenge can cause serious skeletal muscle wasting. However, knowledge of the role of miRNAs in the course of inflammatory muscle catabolism is still very limited. In this study, RNA extracted from the skeletal muscle of pigs injected with LPS or saline was subjected to small RNA deep sequencing. We identified 304 conserved and 114 novel candidate miRNAs in the pig. Of these, four were significantly increased in the LPS-challenged samples and five were decreased. The expression of five miRNAs (ssc-miR-146a-5p, ssc-miR-221-5p, ssc-miR-148b-3p, ssc-miR-215 and ssc-miR-192) were selected for validation by quantitative polymerase chain reaction (qPCR), which found that ssc-miR-146a-5p and ssc-miR-221-5p were significantly upregulated in LPS-challenged pig skeletal muscle. Moreover, we treated mouse C2C12 myotubes with 1000 ng/mL LPS as an acute inflammation cell model. Expression of TNF-α, IL-6, muscle atrophy F-box (MAFbx) and muscle RING finger 1 (MuRF1) mRNA was strongly induced by LPS. Importantly, miR-146a-5p and miR-221-5p also showed markedly increased expression in LPS-treated C2C12 myotubes, suggesting the two miRNAs may be involved in muscle catabolism systems in response to acute inflammation caused by a LPS challenge. To our knowledge, this study is the first to examine miRNA expression profiles in weaned pig skeletal muscle challenged with LPS, and furthers our understanding of miRNA function in the regulation of inflammatory muscle catabolism.

Low dose of IGF-I increases cell size of skeletal muscle satellite cells via Akt/S6K signaling pathway.

The objective of this study was to investigate the effect of insulin growth factor-I (IGF-I) on the size of pig skeletal muscle satellite cells (SCs). Using microarray, real-time RT-PCR, radioimmunoassay analysis and western blot, we first showed that supplementation of low-dose of IGF-I in culture medium resulted in enlarged cell size of Lantang SCs, only Akt and S6K were up-regulated at both the mRNA and protein levels among almost all of the mTOR pathway key genes, but had no effect on cell number. To elucidate the signaling mechanisms responsible for regulating cell size under low-dose of IGF-I treatment, we blocked Akt and S6K activity with the specific inhibitors MK2206 and PF4708671, respectively. Both inhibitors caused a decrease in cell size. In addition, MK2206 lowered the protein level of p-Akt (Ser473), p-S6K (Thr389), and p-rpS6 (Ser235/236), whereas PF4708671 lowered the protein level of p-S6K (Thr389) and p-rpS6 (Ser235/236). However, low dose of IGF-I didn't affect the protein level of p-mTOR (Ser2448) and p-mTOR (Ser2481). When both inhibitors were applied simultaneously, the effect was the same as that of the Akt inhibition alone. Taken together, we report for the first time that low-dose of IGF-I treatment increases cell size via Akt/S6K signaling pathway.

Dietary supplementation with monosodium l-glutamate modifies lipid composition and gene expression related to lipid metabolism in growing pigs fed a normal- or high-fat diet

Monosodium glutamate (MSG) is used worldwide as a flavor enhancer. Its effects on body weight and fat content are debated. Using the pig model in which animals were fed normal- or high-fat isocaloric diets, the present study tested whether dietary MSG modified body weight, relative amount of carcass fat, expression of genes related to fatty acid metabolism, and carcass lipid composition. A total of 32 growing pigs were randomly assigned to four dietary treatment groups: normal-fat diet, normal-fat diet+30g/kg MSG, high-fat diet, or high-fat diet+30g/kg MSG. There were 8 animals per treatment group. After a 30-day feeding period, body weight and carcass composition were measured. The expression of genes related to lipid metabolism in longissimus dorsi muscle and white adipose tissue was also assessed. Our results showed that MSG supplementation given with either normal- or high-fat diet did not affect body weight and the relative amount of total carcass fat. However, MSG supplementation in pigs fed the normal- or high fat diet modified the lipid content and fatty acid profiles in skeletal muscles. MSG modified differently the expression of genes related to lipid metabolism in the muscles and white adipose tissue of animals depending on the normal- or high-fat diet used. In addition, the expression of myosin heavy chains in skeletal muscle fibers was modified by MSG supplementation in both normal- and high-fat groups of animals. Collectively, our results indicate that MSG supplementation is not obesogenic but differentially regulates gene expression related to lipid metabolism, lipid composition and muscle fiber composition in the skeletal muscle of pigs.

Conjugated linoleic acid supplementation during late gestation and lactation of sows affects myofiber type in their litters

The purpose of this study is to determine the effect of dietary conjugated linoleic acid (CLA) supplementation of pregnant and lactating sows on muscle growth and myofiber type of their piglets. There are thirty healthy, pregnant Rongchang sows with similar body condition and closed expected day of parturition for the experiment in vivo. Three experiment groups carry out with/without CLA (1%, 2% and 0%). The experiment began on 85th day of gestation and ended at 28th day after piglet birth. The results show that (1) piglets from CLA-fed sows had heavier longissimus dorsi and higher percentages of slow myofibers than control piglets (P<0.05); (2) moreover, real-time PCR showed that CLA supplementation induce increasing expression of MyHC 1, MyOG, and MyOD genes while decreasing expression of MyHC 2a, MyHC 2x, and MSTN genes in piglets. We also check the expression of genes which appear in 2 in vitro experiment. c9, t11-CLA acted to induce the differentiation of cultured pig skeletal muscle cells and up-regulates the expression levels of MyHC 1 and MyOG, but t10, c12-CLA strongly inhibited cellular differentiation and down-regulates expression of MyOG. These findings suggest that dietary CLA during early life may affect muscle development and myofiber type in pigs.

Evaluation of housekeeping genes for normalizing real-time quantitative PCR assays in pig skeletal muscle at multiple developmental stages

Quantitative PCR (qPCR) requires a constantly expressed housekeeping gene as an internal control, the expression of which is similar in different biological samples. In the present study, we evaluated the applicability and compared the consistency of the gene expression of 16 reference genes, i.e., 10 common and 6 candidate genes, through qPCR assays in pig skeletal muscles at multiple developmental stages. The stability of these 16 potential reference genes was investigated using the geNorm and NormFinder methods. Our results indicated that DRAP1 and RNF7 were the most appropriate combination to normalize gene expression in the Yorkshire samples, the combination of DRAP1 and WSB2 were appropriate in the Tongcheng samples, H3F3A and DRAP1 in prenatal periods, DRAP1 and RNF7 in postnatal periods, and the combination of DRAP1 and WSB2 was most suitable for accurate normalization in whole samples. This result provides a strong basis for similar studies in pigs that explore the molecular mechanisms underlying skeletal muscle development.

Effect of Heat Stress on Pig Skeletal Muscle Metabolism

Hyperthermic pigs differ metabolically, including altered insulin profiles, reduced lipolysis, and compromised intestinal integrity, leading to lower lean tissue and elevated adipose tissue mass. This is distinct from thermal neutral pigs on a similar plane of nutrition. To delineate between direct and indirect (via feed intake changes) HS effects, skeletal muscle substrate metabolism was assessed. Pigs (35 ± 0.8 kg) were assigned to three treatments: thermal neutral fed ad libitum (TN, 21°C, n=8), heat stress fed ad libitum (HS, 35°C, n=8), and thermal neutral, pair-fed to HS intake (PF, n=8) for 7 days. Body temperature (TB) and feed intake (FI) were recorded daily. Longissimus dorsi muscle was biopsied on day -2, 3, and 7 relative to start of treatments for metabolic assays. Heat stress increased TB and decreased FI (P < 0.05). Heat stress tended to decrease acid soluble metabolites (P = 0.07), and did not alter glucose oxidation. Metabolic flexibility decreased in HS versus TN and PF (P < 0.05). Total fatty acid oxidation and pyruvate dehydrogenase (PDH) activity increased in PF (P < 0.05), however, TN and HS did not differ. Heat stress inhibited citrate synthase and beta hydroxyacyl-CoA dehydrogenase (BHAD) activities (P < 0.05). Heat stress did not alter PDH phosphorylation or carnitine palmitoyltransferase 1 abundances, but reduced acetyl-CoA carboxylase (ACC) protein abundance (P < 0.05). In conclusion, HS altered skeletal muscle fatty acid oxidation and metabolic flexibility, likely involving BHAD and ACC regulation.

FRET-Based RyR-Selective Detection Reveals No Significant Competition between CaM and S100A1 Binding to RyRs

Using fluorescence resonance energy transfer (FRET), we directly tested the hypothesis that S100A1 competes with calmodulin (CaM) for binding to intact, functional ryanodine receptors type I (RyR1) and II (RyR2) from skeletal and cardiac muscle, respectively. This hypothesis is largely based on competition assays using isolated sarcoplasmic reticulum (SR) membranes from skeletal muscle and evidence that S100A1 binds to peptides corresponding to one of the proposed CaM binding domains on RyR1. However, competition between S100A1 and CaM for RyR binding has not been directly detected. We targeted a donor-labeled FKBP12.6 (D-FKBP) to the cytoplasmic domain of RyR1 or RyR2 in SR vesicles isolated from pig skeletal or cardiac muscle. FRET was detected as a decrease of D-FKBP fluorescence in the presence of 100nM acceptor-labeled CaM (A-CaM) and used to index CaM binding to RyR. Upon pre-incubating SR with [S100A1] ranging from 0.01 to >100 μM, we found partial inhibition of FRET, with μM KI, for both skeletal and cardiac SR. By comparison, FRET was completely inhibited by unlabeled WT-CaM (KI ∼100nM), indicating that A-CaM and WT-CaM bind RyR with similar affinities and at the same site. Similar results were obtained using co-sedimentation assays conducted under similar experimental conditions, to detect competition between S100A1 and CaM binding to SR membranes. Taken together, these results indicate that CaM-RyR binding may not be significantly competed by S100A1 under normal physiologic conditions. Furthermore, structural analysis of FRET data suggests that S100A1 allosterically interacts with RyR-CaM binding. Initial results from a complementary FRET approach, using acceptor-labeled S100A1, further support the conclusion that S100A1 does not significantly compete with CaM-RyR binding in skeletal or cardiac muscle.

Interactome mapping reveals important pathways in skeletal muscle development of pigs.

The regulatory relationship and connectivity among genes involved in myogenesis and hypertrophy of skeletal muscle in pigs still remain large challenges. Presentation of gene interactions is a potential way to understand the mechanisms of developmental events in skeletal muscle. In this study, genome-wide transcripts and miRNA profiling was determined for Landrace pigs at four time points using microarray chips. A comprehensive method integrating gene ontology annotation and interactome network mapping was conducted to analyze the biological patterns and interaction modules of muscle development events based on differentially expressed genes and miRNAs. Our results showed that in total 484 genes and 34 miRNAs were detected for the duration from embryonic stage to adult in pigs, which composed two linear expression patterns with consensus changes. Moreover, the gene ontology analysis also disclosed that there were three typical biological events i.e., microstructure assembly of sarcomere at early embryonic stage, myofibril formation at later embryonic stage and function establishments of myoblast cells at postnatal stage. The interactome mappings of different time points also found the down-regulated trend of gene expression existed across the whole duration, which brought a possibility to introduce the myogenesis related miRNAs into the interactome regulatory networks of skeletal muscle in pigs.

A splice mutation in the PHKG1 gene causes high glycogen content and low meat quality in pig skeletal muscle.

Glycolytic potential (GP) in skeletal muscle is economically important in the pig industry because of its effect on pork processing yield. We have previously mapped a major quantitative trait loci (QTL) for GP on chromosome 3 in a White Duroc × Erhualian F2 intercross. We herein performed a systems genetic analysis to identify the causal variant underlying the phenotype QTL (pQTL). We first conducted genome-wide association analyses in the F2 intercross and an F19 Sutai pig population. The QTL was then refined to an 180-kb interval based on the 2-LOD drop method. We then performed expression QTL (eQTL) mapping using muscle transcriptome data from 497 F2 animals. Within the QTL interval, only one gene (PHKG1) has a cis-eQTL that was colocolizated with pQTL peaked at the same SNP. The PHKG1 gene encodes a catalytic subunit of the phosphorylase kinase (PhK), which functions in the cascade activation of glycogen breakdown. Deep sequencing of PHKG1 revealed a point mutation (C>A) in a splice acceptor site of intron 9, resulting in a 32-bp deletion in the open reading frame and generating a premature stop codon. The aberrant transcript induces nonsense-mediated decay, leading to lower protein level and weaker enzymatic activity in affected animals. The mutation causes an increase of 43% in GP and a decrease of>20% in water-holding capacity of pork. These effects were consistent across the F2 and Sutai populations, as well as Duroc × (Landrace × Yorkshire) hybrid pigs. The unfavorable allele exists predominantly in Duroc-derived pigs. The findings provide new insights into understanding risk factors affecting glucose metabolism, and would greatly contribute to the genetic improvement of meat quality in Duroc related pigs.

Genome-wide DNA methylation changes in skeletal muscle between young and middle-aged pigs

BackgroundAge-related physiological, biochemical and functional changes in mammalian skeletal muscle have been shown to begin at the mid-point of the lifespan. However, the underlying changes in DNA methylation that occur during this turning point of the muscle aging process have not been clarified. To explore age-related genomic methylation changes in skeletal muscle, we employed young (0.5 years old) and middle-aged (7 years old) pigs as models to survey genome-wide DNA methylation in the longissimus dorsi muscle using a methylated DNA immunoprecipitation sequencing approach.ResultsWe observed a tendency toward a global loss of DNA methylation in the gene-body region of the skeletal muscle of the middle-aged pigs compared with the young group. We determined the genome-wide gene expression pattern in the longissimus dorsi muscle using microarray analysis and performed a correlation analysis using DMR (differentially methylated region)-mRNA pairs, and we found a significant negative correlation between the changes in methylation levels within gene bodies and gene expression. Furthermore, we identified numerous genes that show age-related methylation changes that are potentially involved in the aging process. The methylation status of these genes was confirmed using bisulfite sequencing PCR. The genes that exhibited a hypomethylated gene body in middle-aged pigs were over-represented in various proteolysis and protein catabolic processes, suggesting an important role for these genes in age-related muscle atrophy. In addition, genes associated with tumorigenesis exhibited aged-related differences in methylation and expression levels, suggesting an increased risk of disease associated with increased age.ConclusionsThis study provides a comprehensive analysis of genome-wide DNA methylation patterns in aging pig skeletal muscle. Our findings will serve as a valuable resource in aging studies, promoting the pig as a model organism for human aging research and accelerating the development of comparative animal models in aging research.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-653) contains supplementary material, which is available to authorized users.

Dynamic changes in genes related to glucose uptake and utilization during pig skeletal and cardiac muscle development.

Skeletal and cardiac muscle have important roles in glucose uptake and utilization. However, changes in expression of protein coding genes and miRNAs that participate in glucose metabolism during development are not fully understood. In this study, we investigated the expression of genes related to glucose metabolism during muscle development. We found an age-dependent increase in gene expression in cardiac muscle, with enrichment in heart development- and energy-related metabolic processes. A subset of genes that were up-regulated until 30 or 180 days postnatally, and then down-regulated in psoas major muscle was significantly enriched in mitochondrial oxidative-related processes, while genes that up-regulated in longissimus doris muscle was significantly enriched in glycolysis-related processes. Meanwhile, expression of energy-related microRNAs decreased with increasing age. In addition, we investigated the correlation between microRNAs and mRNAs in three muscle types across different stages of development and found many potential microRNA-mRNA pairs involved in regulating glucose metabolism.

Development of rabbit monoclonal antibodies for detection of alpha-dystroglycan in normal and dystrophic tissue.

Alpha-dystroglycan requires a rare O-mannose glycan modification to form its binding epitope for extracellular matrix proteins such as laminin. This functional glycan is disrupted in a cohort of muscular dystrophies, the secondary dystroglycanopathies, and is abnormal in some metastatic cancers. The most commonly used reagent for detection of alpha-dystroglycan is mouse monoclonal antibody IIH6, but it requires the functional O-mannose structure for recognition. Therefore, the ability to detect alpha-dystroglycan protein in disease states where it lacks the full O-mannose glycan has been limited. To overcome this hurdle, rabbit monoclonal antibodies against the alpha-dystroglycan C-terminus were generated. The new antibodies, named 5–2, 29–5, and 45–3, detect alpha-dystroglycan from mouse, rat and pig skeletal muscle by Western blot and immunofluorescence. In a mouse model of fukutin-deficient dystroglycanopathy, all antibodies detected low molecular weight alpha-dystroglycan in disease samples demonstrating a loss of functional glycosylation. Alternately, in a porcine model of Becker muscular dystrophy, relative abundance of alpha-dystroglycan was decreased, consistent with a reduction in expression of the dystrophin-glycoprotein complex in affected muscle. Therefore, these new rabbit monoclonal antibodies are suitable reagents for alpha-dystroglycan core protein detection and will enhance dystroglycan-related studies.

Molecular characterization and expression patterns of the actinin-associated LIM protein (ALP) subfamily genes in porcine skeletal muscle

The actinin-associated LIM protein (ALP) subfamily has important functions in cell signal transduction, cell proliferation, and integration of cytoskeletal architecture. To detect their functions in pig skeletal muscle, we cloned and characterized the pig ALP subfamily genes, drew their genomic structure maps, and detected their tissue expression patterns. We identified a new spliced variant of PDLIM3 in pig skeletal muscle and named it as PDLIM3-4, which was only expressed in the heart and skeletal muscle. Our results showed that PDLIM3-4 was expressed in adult pig skeletal muscle with the highest expression level, and both PDLIM3-4 isoform and PDLIM4 had different expression profiles during the prenatal and postnatal stages of skeletal muscle development among the three pig breeds. These studies provide useful information for further research on the functions of pig ALP subfamily genes in skeletal muscle development.

Dystrophin insufficiency causes selective muscle histopathology and loss of dystrophin‐glycoprotein complex assembly in pig skeletal muscle

The purpose of this investigation was to determine the extent to which dystrophin insufficiency caused histomorphological changes in a novel pig model of Becker muscular dystrophy. In our procedures, we used a combination of biochemical approaches, including quantitative PCR and Western blots, along with a histological analysis using standard and immunohistological measures. We found that 8-wk-old male affected pigs had a 70% reduction in dystrophin protein abundance in the diaphragm, psoas major, and longissimus lumborum and a 5-fold increase in serum creatine kinase activity compared with healthy male littermates. Dystrophin insufficiency in the diaphragm and the longissimus resulted in muscle histopathology with disorganized fibrosis that often colocalized with fatty infiltration but not the psoas. Affected animals also had an 80-85% reduction in α-sarcoglycan localization in these muscles, indicating compromised assembly of the dystrophin glycoprotein complex. Controls used in this study were 4 healthy male littermates, as they are most closely related to the affected animals. We concluded that pigs with insufficient dystrophin protein expression have a phenotype consistent with human dystrophinopathy patients. Given that and their similarity in body size and physiology to humans, we further conclude that this pig line is an appropriate translational model for dystrophinopathies.