Pig Skeletal Muscle Research Articles

Integrative single-cell RNA-seq and ATAC-seq analysis of myogenic differentiation in pig

BackgroundSkeletal muscle development is a multistep process whose understanding is central in a broad range of fields and applications, from the potential medical value to human society, to its economic value associated with improvement of agricultural animals. Skeletal muscle initiates in the somites, with muscle precursor cells generated in the dermomyotome and dermomyotome-derived myotome before muscle differentiation ensues, a developmentally regulated process that is well characterized in model organisms. However, the regulation of skeletal muscle ontogeny during embryonic development remains poorly defined in farm animals, for instance in pig. Here, we profiled gene expression and chromatin accessibility in developing pig somites and myotomes at single-cell resolution.ResultsWe identified myogenic cells and other cell types and constructed a differentiation trajectory of pig skeletal muscle ontogeny. Along this trajectory, the dynamic changes in gene expression and chromatin accessibility coincided with the activities of distinct cell type-specific transcription factors. Some novel genes upregulated along the differentiation trajectory showed higher expression levels in muscular dystrophy mice than that in healthy mice, suggesting their involvement in myogenesis. Integrative analysis of chromatin accessibility, gene expression data, and in vitro experiments identified EGR1 and RHOB as critical regulators of pig embryonic myogenesis.ConclusionsCollectively, our results enhance our understanding of the molecular and cellular dynamics in pig embryonic myogenesis and offer a high-quality resource for the further study of pig skeletal muscle development and human muscle disease.

Characterization of pig skeletal muscle transcriptomes in response to low temperature

The response of mammals to cold environment is a complex physiological activity, and its underlying mechanism must be analyzed from multiple perspectives. Skeletal muscle is an important thermogenic tissue that maintains body temperature in mammals. We dissected the molecular mechanism of pig skeletal muscle response to a cold environment by performing comparative transcriptome analysis in the Enshi black pig. Three pigs were subjected to acute cold stress (3 days), three pigs were subjected to cold acclimation (58 days), and three pigs were used as controls. RNA-seq was used to screen the differentially expressed genes (DEGs) of skeletal muscle. Using RNA-seq methods, we identified 1241 DEGs within the acute cold stress group and 1886 DEGs within the cold acclimation group. Prolonged cold exposure induced more gene expression changes. A total of 540 key cold-responsive DEGs were found, and their trends were consistent within the acute cold stress group and cold acclimation group. Gene expression pattern analysis showed that there were significant differences between the low-temperature treatment groups and the control group, and there were also differences between individuals after long-term low-temperature treatment. Analysis of DEGs revealed that 134 pathways were significantly enriched in the cold adaptation group, 98 pathways were significantly enriched in the acute cold stress group, and 71 pathways were shared between the two groups. The 71 shared pathways were mainly related to lipid, amino acid, and carbohydrate metabolism; signal transduction; endocrine, immune, and nervous system; cardiovascular disease; infectious diseases caused by bacteria or viruses; and neurodegenerative disease. In conclusion, this study provides insights into the molecular mechanism of porcine skeletal muscle response under low-temperature environment. The data may assist further research on the mechanism of pig response to cold exposure.

Coordinated transcriptional and post-transcriptional epigenetic regulation during skeletal muscle development and growth in pigs

BackgroundN6-methyladenosine (m6A) and DNA 5-methylcytosine (5mC) methylation plays crucial roles in diverse biological processes, including skeletal muscle development and growth. Recent studies unveiled a potential link between these two systems, implicating the potential mechanism of coordinated transcriptional and post-transcriptional regulation in porcine prenatal myogenesis and postnatal skeletal muscle growth.MethodsImmunofluorescence and co-IP assays were carried out between the 5mC writers and m6A writers to investigate the molecular basis underneath. Large-scale in-house transcriptomic data were compiled for applying weighted correlation network analysis (WGCNA) to identify the co-expression patterns of m6A and 5mC regulators and their potential role in pig myogenesis. Whole-genome bisulfite sequencing (WGBS) and methylated RNA immunoprecipitation sequencing (MeRIP-seq) were performed on the skeletal muscle samples from Landrace pigs at four postnatal growth stages (days 30, 60, 120 and 180).ResultsSignificantly correlated expression between 5mC writers and m6A writers and co-occurrence of 5mC and m6A modification were revealed from public datasets of C2C12 myoblasts. The protein-protein interactions between the DNA methylase and the m6A methylase were observed in mouse myoblast cells. Further, by analyzing transcriptome data comprising 81 pig skeletal muscle samples across 27 developmental stages, we identified a 5mC/m6A epigenetic module eigengene and decoded its potential functions in pre- or post-transcriptional regulation in postnatal skeletal muscle development and growth of pigs. Following integrative multi-omics analyses on the WGBS methylome data and MeRIP-seq data for both m6A and gene expression profiles revealed a genome/transcriptome-wide correlated dynamics and co-occurrence of 5mC and m6A modifications as a consequence of 5mC/m6A crosstalk in the postnatal myogenesis progress of pigs. Last, we identified a group of myogenesis-related genes collaboratively regulated by both 5mC and m6A modifications in postnatal skeletal muscle growth in pigs.ConclusionsOur study discloses a potential epigenetic mechanism in skeletal muscle development and provides a novel direction for animal breeding and drug development of related human muscle-related diseases.

Free Amino Acid–Enriched Diets Containing Rapidly but Not Slowly Digested Carbohydrate Promote Amino Acid Absorption from Intestine and Net Fluxes across Skeletal Muscle of Pigs

The approach to matching appropriate carbohydrates alongside free amino acids to achieve optimal muscle growth remains unclear. We investigated whether the consumption of a diet containing rapidly digested carbohydrate and free amino acids can enhance intestinal absorption and muscular uptake of amino acids in pigs. Twelve barrows (28 kg; 11 wk old) with catheters installed in the portal vein, mesenteric vein, femoral artery, and femoral vein were randomly assigned to consume 1 of 2 free amino acid-enriched diets (3.34%) containing rapidly [waxy corn starch (WCS)] or slowly [pea starch (PS)] digested carbohydrate for 27 d. Blood was collected to determine the fluxes of plasma glucose and amino acids across the portal vein and the hindlimb muscle. Dietary in vitro carbohydrate digestive rates were also determined. Data were analyzed using repeated-measures (time × group) ANOVA. Carbohydrate in vitro cumulative digestibility at 30 and 240 min was 69.00% and 95.25% for WCS and 23.25% and 81.15% for PS, respectively. The animal experiment presented WCS increased individual amino acids (lysine, 0.67 compared with 0.53 mmol/min; threonine, 0.40 compared with 0.29 mmol/min; isoleucine, 0.33 compared with 0.22 mmol/min; glutamate, 0.51 compared with 0.35 mmol/min; and proline, 0.51 compared with 0.27 mmol/min), essential amino acid (EAA; 3.26 compared with 2.65 mmol/min), and branched-chain amino acid (BCAA; 0.86 compared with 0.65 mmol/min) fluxes across the portal vein during 8 h postprandial, as well as individual amino acids (isoleucine, 0.08 compared with 0.02 mmol/min; leucine, 0.06 compared with 0.02 mmol/min; and glutamine, 0.44 compared with 0.25 mmol/min), EAA (0.50 compared with 0.21 mmol/min), and BCAA (0.17 compared with 0.06 mmol/min) net fluxes across the hindlimb muscle during 8 h postprandial compared with PS (P < 0.05). A diet containing rapidly digested carbohydrate and free amino acids can promote intestinal absorption and net fluxes across hindlimb muscle of amino acids in pigs.

CircCSDE1 Regulates Proliferation and Differentiation of C2C12 Myoblasts by Sponging miR-21-3p.

The growth and development of skeletal muscle is regulated by many factors, and recent studies have shown that circular RNAs (circRNAs) can participate in this process. The model of porcine skeletal muscle injury was constructed to search for circRNAs that can regulate the growth and development of skeletal muscle in pigs. Using whole-transcriptome sequencing and bioinformatics analysis, a novel circRNA (circCSDE1) was screened out, which is highly expressed in skeletal muscle. Functional studies in C2C12 cells demonstrated that circCSDE1 could promote proliferation and inhibit myoblast differentiation, while opposing changes were observed by circCSDE1 knockdown. A dual-luciferase reporter assay revealed that circCSDE1 directly targeted miR-21-3p to regulate the expression of the downstream target gene (Cyclin-dependent kinase 16, CDK16). Moreover, miR-21-3p could inhibit proliferation and promote myoblast differentiation in C2C12 cells, opposite with the effects of circCSDE1. Additionally, the rescue experiments offered further evidence that circCSDE1 and its target, miR-21-3p, work together to regulate myoblast proliferation and differentiation. This study provides a theoretical basis for further understanding the regulatory mechanisms of circRNAs.

Min pig skeletal muscle response to cold stress.

The increased sensitivity of pigs to ambient temperature is due to today’s intensive farming. Frequent climate disasters increase the pressure on healthy pig farming. Min pigs are an indigenous pig breed in China with desirable cold resistance characteristics, and hence are ideal for obtaining cold-resistant pig breeds. Therefore, it is important to discover the molecular mechanisms that are activated in response to cold stress in the Min pig. Here, we conducted a transcriptomic analysis of the skeletal muscle of Min pigs under chronic low-temperature acclimation (group A) and acute short cold stress (group B). Cold exposure caused more genes to be upregulated. Totals of 125 and 96 differentially expressed genes (DEGs) were generated from groups A and B. Sixteen common upregulated DEGs were screened; these were concentrated in oxidative stress (SRXN1, MAFF), immune and inflammatory responses (ITPKC, AREG, MMP25, FOSL1), the nervous system (RETREG1, GADD45A, RCAN1), lipid metabolism (LRP11, LIPG, ITGA5, AMPD2), solute transport (SLC19A2, SLC28A1, SLCO4A1), and fertility (HBEGF). There were 102 and 73 genes that were specifically differentially expressed in groups A and B, respectively. The altered mRNAs were enriched in immune, endocrine, and cancer pathways. There were 186 and 91 differentially expressed lncRNAs generated from groups A and B. Analysis of the target genes suggested that they may be involved in regulating the MAPK signaling pathway for resistance to cold. The results of this study provide a comprehensive overview of cold exposure–induced transcriptional patterns in skeletal muscle of the Min pig. These results can guide future molecular studies of cold stress response in pigs for improving cold tolerance as a goal in breeding programs.

Identification of a missense variant in the porcine AGPAT gene family associated with intramuscular fat content through whole-genome sequencing.

The 1-acylglycerol-3-phosphate O-acyltransferases (AGPATs) are enzymes that catalyze the conversion of lysophosphatidic acid to phosphatidic acid, which is a precursor of triacylglycerol, the main fat reservoir in mammals. We used whole-genome sequencing of 205 pigs to identify 6639 genetic variants in the porcine AGPAT gene family. Of these, 166 common variants in the AGPAT5 gene had significant associations with fat content and composition traits. We preselected a missense single nucleotide polymorphism in exon 6 of AGPAT5 (rs196952262, A>G) for validation of its associations in 1034 pigs from the same Duroc line. The A allele showed a positive additive effect for intramuscular fat content (+1.12% ± 0.21, p < 0.001, for gluteus medius and +0.89% ± 0.33, p < 0.01, for longissimus). We also observed significant associations with fatty acid composition that were, at least in part, independent of the increased intramuscular fat. The A allele resulted in more monounsaturated fatty acids (+0.34% ± 0.15, p < 0.05, for longissimus) and a greater monounsaturated/polyunsaturated fatty acids ratio (+0.11 ± 0.04, p < 0.01, for gluteus medius and +0.13 ± 0.05, p < 0.05, for longissimus). The effect of the AGPAT5 variant on intramuscular fat was more noticeable in fatter pigs, and AGPAT5 interacts with other genes that affect overall fatness such as LEPR. AGPAT5 was the most expressed gene of the AGPAT family in pig skeletal muscle. This variant can be used as a marker in assisted selection for modulating pig fat deposition and fatty acid content.

A Combined Differential Proteome and Transcriptome Profiling of Fast- and Slow-Twitch Skeletal Muscle in Pigs.

Skeletal muscle fiber types can contribute in part to affecting pork quality parameters. Biceps femoris (Bf) (fast muscle or white muscle) and Soleus (Sol) (slow muscle or red muscle) are two typical skeletal muscles characterized by obvious muscle fiber type differences in pigs. However, the critical proteins and potential regulatory mechanisms regulating porcine skeletal muscle fibers have yet to be clearly defined. In this study, the isobaric Tag for Relative and Absolute Quantification (iTRAQ)-based proteome was used to identify the key proteins affecting the skeletal muscle fiber types with Bf and Sol, by integrating the previous transcriptome data, while function enrichment analysis and a protein–protein interaction (PPI) network were utilized to explore the potential regulatory mechanisms of skeletal muscle fibers. A total of 126 differentially abundant proteins (DAPs) between the Bf and Sol were identified, and 12 genes were found to be overlapping between differentially expressed genes (DEGs) and DAPs, which are the critical proteins regulating the formation of skeletal muscle fibers. Functional enrichment and PPI analysis showed that the DAPs were mainly involved in the skeletal-muscle-associated structural proteins, mitochondria and energy metabolism, tricarboxylic acid cycle, fatty acid metabolism, and kinase activity, suggesting that PPI networks including DAPs are the main regulatory network affecting muscle fiber formation. Overall, these data provide valuable information for understanding the molecular mechanism underlying the formation and conversion of muscle fiber types, and provide potential markers for the evaluation of meat quality.

Identification and Functional Prediction of Circular RNAs Related to Growth Traits and Skeletal Muscle Development in Duroc pigs.

Porcine skeletal muscle is a highly heterogeneous tissue type, and the Longissimus Dorsi muscle (LDM), as the most economical and physiologically metabolized skeletal muscle in pigs, has always been the focus of research and improvement in pig molecular breeding. Circular RNA, as an important new member of regulatory non-coding RNA after microRNA, has become a frontier hot spot in life science research. This study aims to explore candidate circRNAs related to growth, meat quality, and skeletal muscle development among Duroc pigs with different average daily gain (ADG). Eight pigs were selected and divided into two groups: H group (high-ADG) and L group (low-ADG), followed by RNA-Seq analysis to identify circRNAs. The results showed that backfat at 6-7 rib (BF) and Intramuscular fat (IMF) content in the H group was significantly lower than L group, but ribeye area (REA) in the H group was higher than in the L group. In RNA-seq, 296 Differentially expressed (DE) circRNAs (157 upregulated and 139 downregulated) were identified and exons flanking long introns are easier to circularize to produce circRNAs. Most of the DE circRNAs were enriched in Quantitative trait locus (QTL) regions related to meat quality and growth traits. In addition, a gene can produce one or more circRNA transcripts. It was also found that the source genes of DE circRNAs were enriched in MAPK, FoXO, mTOR, PI3K-Akt, and Wnt signaling pathways. The results showed that different ADG, carcass, and meat quality traits among half-sibling Duroc pigs with the same diet may be due to the DE circRNAs related to skeletal muscle growth and development.

The impact of Zearalenone on heat-stressed skeletal muscle in pigs.

Heat stress (HS) and Zearalenone (ZEN) exposure affect growth, production efficiency, and animal welfare; and, under extreme situations, both can be lethal. Given that both HS and ZEN independently cause oxidative stress, we hypothesized that simultaneous exposure to HS and ZEN would cause greater oxidative stress in porcine skeletal muscle than either condition, alone. To address this hypothesis, crossbred, prepubertal gilts were treated with either vehicle control (cookie dough) or ZEN (40 μg/kg) and exposed to either thermoneutral (TN; 21.0 °C) or 12-h diurnal HS conditions (night: 32.2 °C; day: 35.0 °C) for 7 d. Pigs were euthanized immediately following the environmental challenge and the glycolytic (STW) and oxidative (STR) portions of the semitendinosus muscle were collected for analysis. In STR, malondialdehyde (MDA) concentration, a marker of oxidative stress, tended to increase following ZEN exposure (P = 0.08). HS increased CAT (P = 0.019) and SOD1 (P = 0.049) protein abundance, while ZEN decreased GPX1 protein abundance (P = 0.064) and activity (P = 0.036). In STR, HS did not alter protein expression of HSP27, HSP70, or HSP90. Conversely, in STW, MDA-modified proteins remained similar between all groups. Consistent with STR, ZEN decreased GPX1 (P = 0.046) protein abundance in STW. In STW, ZEN decreased protein abundance of HSP27 (P = 0.032) and pHSP27 (P = 0.0068), while HS increased protein expression of HSP70 (P = 0.04) and HSP90 (P = 0.041). These data suggest a muscle fiber type-specific response to HS or ZEN exposure, potentially rendering STR more susceptible to HS- and/or ZEN-induced oxidative stress, however, the combination of HS and ZEN did not augment oxidative stress.

Modeling microRNA-driven post-transcriptional regulation using exon-intron split analysis in pigs.

The contribution of microRNAs (miRNAs) to mRNA post-transcriptional regulation has often been explored by the post hoc selection of downregulated genes and determining whether they harbor binding sites for miRNAs of interest. This approach, however, does not discriminate whether these mRNAs are also downregulated at the transcriptional level. Here, we have characterized the transcriptional and post-transcriptional changes in mRNA expression in two porcine tissues: gluteus medius muscle of fasted and fed Duroc gilts and adipose tissue of lean and obese Duroc-Göttingen minipigs. Exon-intron split analysis of RNA-seq data allowed us to identify downregulated mRNAs with high post-transcriptional signals in fed or obese states, and we assessed whether they harbor binding sites for upregulated miRNAs in any of these two physiological states. We found 26 downregulated mRNAs with high post-transcriptional signals in the muscle of fed gilts and 21 of these were predicted targets of miRNAs upregulated in fed pigs. For adipose tissue, 44 downregulated mRNAs in obese minipigs displayed high post-transcriptional signals, and 25 of these were predicted targets of miRNAs upregulated in the obese state. These results suggest that the contribution of miRNAs to mRNA repression is more prominent in the skeletal muscle system. Finally, we identified several genes that may play relevant roles in the energy homeostasis of the pig skeletal muscle (DKK2 and PDK4) and adipose (SESN3 and ESRRG) tissues. By differentiating transcriptional from post-transcriptional changes in mRNA expression, exon-intron split analysis provides a valuable view of the regulation of gene expression, complementary to canonical differential expression analyses.

Are n-3 PUFAs from Microalgae Incorporated into Membrane and Storage Lipids in Pig Muscle Tissues?-A Lipidomic Approach.

For the study of molecular mechanisms of to lipid transport and storage in relation to dietary effects, lipidomics has been rarely used in farm animal research. A feeding study with pigs (German Landrace sows) and supplementation of microalgae (Schizochytrium sp.) was conducted. The animals were allocated to the control group (n = 15) and the microalgae group (n = 16). Shotgun lipidomics was applied. This study enabled us to identify and quantify 336 lipid species from 15 different lipid classes in pig skeletal muscle tissues. The distribution of the lipid classes was significantly altered by microalgae supplementation, and ether lipids of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidic acid (PA) were significantly decreased. The total concentration of triacylglycerides (TAGs) was not affected. TAGs with high degree of unsaturation (TAG 56:7, TAG 56:6, TAG 54:6) were increased in the microalgae group, and major abundant species like TAG 52:2 and TAG 52:1 were not affected by the diet. Our results confirmed that dietary DHA and EPA are incorporated into storage and membrane lipids of pig muscles, which further led to systemic changes in the lipidome composition.

Quantitative Estimation of Mechanical Anisotropy Using Acoustic Radiation Force (ARF)-Induced Peak Displacements (PD): In Silico and Experimental Demonstration.

Elastic degree of anisotropy (DoA) is a diagnostically relevant biomarker in muscle, kidney, breast, and other organs. Previously, elastic DoA was qualitatively assessed as the ratio of peak displacements (PD) achieved with the long-axis of a spatially asymmetric Acoustic Radiation Force Impulse (ARFI) excitation point spread function (PSF) aligned along versus across the axis of symmetry (AoS) in transversely isotropic materials. However, to better enable longitudinal and cross-sectional analyses, a quantitative measure of elastic DoA is desirable. In this study, qualitative ARFI PD ratios are converted to quantitative DoA, measured as the ratio of longitudinal over transverse shear elastic moduli, using a model empirically derived from Field II and finite element method (FEM) simulations. In silico, the median absolute percent error (MAPE) in ARFI-derived shear moduli ratio (SMR) was 1.75%, and predicted SMRs were robust to variations in transverse shear modulus, Young's moduli ratio, speed of sound, attenuation, density, and ARFI excitation PSF dimension. Further, ARFI-derived SMRs distinguished two materials when the true SMRs of the compared materials differed by as little as 10%. Experimentally, ARFI-derived SMRs linearly correlated with the corresponding ratios measured by Shear Wave Elasticity Imaging (SWEI) in excised pig skeletal muscle ( [Formula: see text], MAPE = 13%) and in pig kidney, in vivo ( [Formula: see text], MAPE = 5.3%). These results demonstrate the feasibility of using the ARFI PD to quantify elastic DoA in biological tissues.

PSV-17 The Interaction Between Heat Stress and Zearalenone Toxicity in Porcine Skeletal Muscle

Abstract Heat stress (HS) and Zearalenone (Zen) exposure negatively impact growth, production efficiency, animal welfare, and both can be lethal. Zearalenone, a mycotoxin, can contaminate a variety of feedstuffs. As both HS and Zen independently can cause oxidative stress, we hypothesized that concomitant exposure to HS and Zen would cause oxidative stress in pig skeletal muscle more than either agent alone. Prepubertal gilts (n = 26) were fed placebo or Zen (40 ug/kg) twice daily and exposed to thermoneutral conditions (TN; 21.0 °C) or a 12 h diurnal HS challenge (simulated night - 32.2 °C; simulated day - 35.0 °C) for 7 d. Immediately following the environmental challenge, pigs were euthanized and the oxidative (STR) portion of the semitendinosus muscle was collected. Heat stress increased rectal temperature following the simulated nighttime (TN: 38.9 ± 0.04 °C; HS: 39.3 ± 0.04; P &amp;lt; 0.0001) and daytime (TN: 39.2 ± 0.09 °C; HS: 39.9 ± 0.08 °C; P &amp;lt; 0.0001) conditions. In STR, malondialdehyde-modified protein abundance (P = 0.1) and concentration (P &amp;lt; 0.1), markers of oxidative stress, tended to increase following Zen exposure, largely driven by the HS-Zen group. Heat stress increased protein expression of antioxidant enzymes catalase (p&amp;lt; 0.05) and superoxide dismutase 1 (P &amp;lt; 0.05), while Zen decreased glutathione peroxidase 1 (P &amp;lt; 0.05) transcript (P &amp;lt; 0.05) and protein (P &amp;lt; 0.05) abundance and enzymatic activity (P &amp;lt; 0.05). Although a mild HS paradigm and low Zen exposure were employed, oxidative stress markers were altered suggesting oxidative skeletal muscle is susceptible to HS- and Zen-mediated changes in redox balance. In total, these data raise the possibility that more severe HS exposures and greater Zen doses, similar to those sometimes encountered in production settings, may compromise muscle health.

Application of a Photoacoustic Tomography System: A Case Study on the Monitoring of Pig Tissue Decomposition

Pig carcasses have been used in forensic research because they share several similarities to human cadaver, including decomposition. In several studies, the decomposition of pig’s cadaver for a certain time can be used as a model to determine the time of death of a human. This study was conducted to determine the relationship between the days of spoilage of pig tissue and the level of average sound intensity produced by the sample. Then, in this study, pig skeletal muscles were allowed to decay with a variation of 1–5 days. Afterward, these muscles were imaged using diode laser-based photoacoustic tomography. Results of the experiment show that the average acoustic intensity level from the first day until the fourth day has increased (78–92 a.u for young pig and 76–86 a.u for old pig) but decreased on the fifth day (88 a.u for young and 84 a.u for old pig). These results can improve forensic imaging because such results can be applied to determine the time of death of human by plotting the ratio of the average sound intensity level of the sample to the number of days of decomposition.

A molecular insight into the lipid changes of pig Longissimus thoracis muscle following dietary supplementation with functional ingredients.

In this work, the Longissimus thoracis pig skeletal muscle was used as a model to investigate the impact of two different diets, supplemented with n-3 polyunsaturated fatty acids from extruded linseed (L) and polyphenols from grape skin and oregano extracts (L+P), on the lipidomic profile of meat. A standard diet for growing-finishing pigs (CTRL) was used as a control. Changes in lipids profile were investigated through an untargeted lipidomics and transcriptomics combined investigation. The lipidomics identified 1507 compounds, with 195 compounds fitting with the MS/MS spectra of LipidBlast database. When compared with the CTRL group, the L+P diet significantly increased 15 glycerophospholipids and 8 sphingolipids, while the L diet determined a marked up-accumulation of glycerolipids. According to the correlations outlined between discriminant lipids and genes, the L diet may act preventing adipogenesis and the related inflammation processes, while the L+P diet promoted the expression of genes involved in lipids' biosynthesis and adipogenic extracellular matrix formation and functioning.

UCP1 Knockin Induces Lipid Dynamics and Transcriptional Programs in the Skeletal Muscles of Pigs.

Uncoupling protein 1 (UCP1), the hallmark protein responsible for nonshivering thermogenesis in adipose tissue (especially brown adipose tissue) has regained researchers’ attention in the context of metabolic disorders following the realization that UCP1 can be activated in adult humans and reconstituted in pigs. Both skeletal muscle and adipose tissue are highly dynamic tissues that interact at the metabolic and hormonal level in response to internal and external stress, and they coordinate in maintaining whole-body metabolic homeostasis. Here, we utilized lipidomics and transcriptomics to identify the altered lipid profiles and regulatory pathways in skeletal muscles from adipocyte-specific UCP1 knock-in (KI) pigs. UCP1 KI changed the contents of glycerophospholipids and acyl carnitines of skeletal muscles. Several metabolic regulatory pathways were more enriched in the UCP1 KI skeletal muscle. Comparison of the transcriptomes of adipose and skeletal muscle suggested that nervous system or chemokine signaling might account for the crosstalk between these two tissues in UCP1 KI pigs. Comparison of the lipid biomarkers from UCP1 KI pigs and other mammals suggested associations between UCP1 KI-induced metabolic alternations and metabolic and muscle dysfunction. Our study reveals the lipid dynamics and transcriptional programs in the skeletal muscle of UCP1 KI pigs and suggests that a network regulates metabolic homeostasis between skeletal muscle and adipose tissue.

ACSL4 Directs Intramuscular Adipogenesis and Fatty Acid Composition in Pigs.

Simple SummaryIn the livestock industry, intramuscular fat content is an important indicator of the meat quality of domestic animals. The variations of the Acyl-CoA Synthetase Long-Chain Family Member 4 (ACSL4) gene locus are associated with intramuscular fat content in different pig populations, but the detailed molecular function of ACSL4 in pig intramuscular adipogenesis remains obscure. Our study reveals the function of ACSL4 in pig intramuscular adipogenesis and provides new clues for improving the palatability of meat and enhancing the nutritional value of pork for human health.The intramuscular fat is a major quality trait of meat, affecting sensory attributes such as flavor and texture. Several previous GWAS studies identified Acyl-CoA Synthetase Long Chain Family Member 4 (ACSL4) gene as the candidate gene to regulate intramuscular fat content in different pig populations, but the underlying molecular function of ACSL4 in adipogenesis within pig skeletal muscle is not fully investigated. In this study, we isolated porcine endogenous intramuscular adipocyte progenitors and performed ACSL4 loss- and gain-of-function experiments during adipogenic differentiation. Our data showed that ACSL4 is a positive regulator of adipogenesis in intramuscular fat cells isolated from pigs. More interestingly, the enhanced expression of ACSL4 in pig intramuscular adipocytes could increase the cellular content of monounsaturated and polyunsaturated fatty acids, such as gamma-L eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA). The above results not only confirmed the function of ACSL4 in pig intramuscular adipogenesis and meat quality attributes, but also provided new clues for the improvement of the nutritional value of pork for human health.

Targeted myostatin loss-of-function mutation increases type II muscle fibers in Meishan pigs

Myostatin (MSTN) is a negative regulator of skeletal muscle growth and development. The skeletal muscle in MSTN−/– mice is significantly hypertrophied, with muscle fiber type II increasing significantly while muscle fiber type I decreasing. However, it is still not clear how the skeletal muscle types change in MSTN−/– pigs, and how the mechanism for MSTN regulates fiber types, especially in large animals like pigs. This study conducted a comprehensive analysis of the composition of skeletal muscle fibers in MSTN−/– pigs produced in our laboratory. It was observed that, compared with wild-type (WT) pigs, both the total mass of skeletal muscle and type IIb muscle fibers increased significantly (P<0.01), while the type I and type IIa muscle fibers decreased significantly (P<0.01), in MSTN−/– Meishan pigs. In addition, to explore the influence of MSTN on muscle fiber type and its regulation mechanism in the embryonic stage, this study selected a few genes (Myf5, Mef2d, MyoD and Six1) associated with muscle fiber type and validated their expression by quantitative RT-PCR. Herein, it was found that Myh7, Myh2, Myh4 and Myh1 can be detected in the skeletal muscle of pigs at 65 days of gestation (dg). Compared with WT pigs, in MSTN−/– Meishan pigs, Myh7 decreased significantly (P<0.01), while Myh4 (P<0.001) and Myh1 (P<0.05) increased significantly. Meanwhile, the increased expression of Myf5 (P<0.05), Mef2d (P<0.01) and Six1 (P<0.05) in MSTN−/– Meishan pigs suggested that MSTN should regulate the directional development of muscle fiber types in the early stage of embryonic development. Thus, at the embryonic stage, the type II muscle fibers began to increase in MSTN−/– pigs. These results can provide valuable information not only for pig meat quality improvement, but also for the study of human skeletal muscle development and disease treatment.

A Dystrophin Exon-52 Deleted Miniature Pig Model of Duchenne Muscular Dystrophy and Evaluation of Exon Skipping.

Duchenne muscular dystrophy (DMD) is a lethal X-linked recessive disorder caused by mutations in the DMD gene and the subsequent lack of dystrophin protein. Recently, phosphorodiamidate morpholino oligomer (PMO)-antisense oligonucleotides (ASOs) targeting exon 51 or 53 to reestablish the DMD reading frame have received regulatory approval as commercially available drugs. However, their applicability and efficacy remain limited to particular patients. Large animal models and exon skipping evaluation are essential to facilitate ASO development together with a deeper understanding of dystrophinopathies. Using recombinant adeno-associated virus-mediated gene targeting and somatic cell nuclear transfer, we generated a Yucatan miniature pig model of DMD with an exon 52 deletion mutation equivalent to one of the most common mutations seen in patients. Exon 52-deleted mRNA expression and dystrophin deficiency were confirmed in the skeletal and cardiac muscles of DMD pigs. Accordingly, dystrophin-associated proteins failed to be recruited to the sarcolemma. The DMD pigs manifested early disease onset with severe bodywide skeletal muscle degeneration and with poor growth accompanied by a physical abnormality, but with no obvious cardiac phenotype. We also demonstrated that in primary DMD pig skeletal muscle cells, the genetically engineered exon-52 deleted pig DMD gene enables the evaluation of exon 51 or 53 skipping with PMO and its advanced technology, peptide-conjugated PMO. The results show that the DMD pigs developed here can be an appropriate large animal model for evaluating in vivo exon skipping efficacy.