Distinct Transcriptional Responses of Skeletal Muscle to Short-Term Cold Exposure in Tibetan Pigs and Bama Pigs

Abstract

Piglets are susceptible to cold, and piglet death caused by cold stress leads to economic losses in the pig industry in cold areas. Skeletal muscle plays a key role in adaptive thermogenesis in mammals, but the related mechanism in pigs is unclear. In this study, cold-tolerant Tibetan pigs and cold-sensitive Bama pigs were subjected to either a cold environment (4 °C) or a room temperature environment (25 °C) for 3 days. The biceps femoris (BF) and longissimus dorsi muscle (LDM) were collected for phenotypic analysis, and the BF was used for genome-wide transcriptional profiling. Our results showed that Tibetan pigs had a higher body temperature than Bama pigs upon cold stimulation. RNA-seq data indicated a stronger transcriptional response in the skeletal muscle of Tibetan pigs upon cold stimulation, as more differentially expressed genes (DEGs) were identified with the same criteria (p < 0.05 and fold change > 2). In addition, distinct pathway signaling patterns in skeletal muscle upon cold exposure were found between the breeds of pigs. Mitochondrial beta-oxidation-related genes and pathways were significantly upregulated in Tibetan pigs, indicating that Tibetan pigs may use fatty acids as the primary fuel source to protect against cold. However, the significant upregulation of inflammatory response- and glycolysis-related genes and pathways in the skeletal muscle of Bama pigs suggested that these pigs may use glucose as the primary fuel source in cold environments. Together, our study revealed the distinct transcriptional responses of skeletal muscle to cold stimulation in Tibetan pigs and Bama pigs and provided novel insights for future investigation of the cold adaptation mechanism in pigs.

Keywords: cold exposure; pig; skeletal muscle; transcriptome.

Figure 1

Effects of short-term cold exposure on the core body temperature, muscle fiber area distribution, and muscle fiber type composition in Tibetan pigs (TP) and Bama pigs (BP). (A) Core body temperature of TP and BP during short-term cold exposure. (B–F) H&E staining of tissue sections of the BF and LDM from TP and BP (B). Representative images are shown. Scale bar, 50 μm. The myofiber cross-sectional area distribution in the biceps femoris (BF) and longissimus dorsi muscles (LDM) of TP and BP was measured by Image J (n = 3 muscle samples) (C–F). (G–I) Immunofluorescence analysis of fiber type composition in the BF and LDM of TP and BP (G). The different myosin heavy chain isoforms are indicated by green (fast fibers) and red (slow fibers) fluorescence. Representative micrographs and the corresponding quantitative results (H,I) are shown (n = 3). Scale bar, 50 μm. (J,K) mRNA expression levels of fast and slow myofiber markers in the BF and LDM of TP and BP (n = 3). The results are presented as the mean ± standard error mean (SEM). * p < 0.05; ** p < 0.01.

Figure 2

RNA-seq analysis of differential expressed genes (DEGs) in the BF of TP and BP under room temperature (RT) or cold (CD) conditions. (A,B) PCA of samples from TP and BP. (C,D) Volcano plots showing significance on the y-axis (−log 10 p-value) plotted against the gene expression ratio (log₂ FC) on the x-axis; the p < 0.05 significance level is indicated by gray dashed horizontal lines. (E) The numbers of upregulated (black) and downregulated (striped) DEGs between Tibetan and Bama pigs. (F) Venn diagram based on the number of DEGs (FC > 2, p-value < 0.05).

Figure 3

Functional enrichment analysis and verification of the genes commonly regulated by cold exposure in TP and BP. (A)Venn diagram showing the number of commonly regulated DEGs between the two breeds. (B) Heatmap and detailed list of genes with the same expression trends. (C) All gene ontology (GO) terms enriched with the commonly regulated genes. (D) The protein-protein interaction (PPI) network of the commonly regulated genes. (E,F) qPCR validation of commonly regulated genes in the BF (E) and LDM (F); these genes are involved in glucose and lipid metabolism, as well as skeletal muscle cell differentiation. All values are presented as the mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 4

Short-term cold exposure uniquely regulates inflammatory response-related genes in BP. (A) Venn diagram showing the number of uniquely regulated DEGs in BP. (B) GO terms and KEGG pathway analyses of upregulated DEGs. (C) A PPI network of uniquely regulated genes in BP. The molecular complex detection (MCODE) algorithm was applied to identify densely connected network components. Each MCODE network was assigned a unique color. The three highest scoring terms with the highest by p-value were retained as the functional descriptions of the corresponding components, as shown in the table under the network diagram. (D) Heatmaps showing the relative changes in the expression of genes involved in the inflammatory response, the formation of collagen fibril organization, and glucose and lipid metabolism (n = 3). (E,F) qPCR validation of genes involved in the inflammatory response in the BF (E) and LDM (F). All values are presented as the mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 5

Short-term cold exposure uniquely regulates lipid catabolism-related genes in TP. (A) Venn diagram showing the number of uniquely regulated DEGs in TP. (B) GO and KEGG pathway analyses of upregulated DEGs. (C) Heatmaps showing the relative changes in the expression of genes involved in the response to oxidative stress and in glucose and lipid metabolism (n = 3). (D,E) qPCR validation of randomly selected genes that were uniquely upregulated in the BF (D) and LDM (E) of TP. (F,G) Expression of the fatty acid uptake- and lipolysis-related proteins CPT1A, HSL (LIPE), pS660HSL, PDK4, and UCP3 in the BF (F) and LDM (G) in TP and BP. All values are presented as the mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 6

Schematic diagram of the distinct transcriptomic responses to short-term cold exposure in the BF of Tibetan and Bama pigs; pathways and genes involved in fatty acid, glucose, and glycogen metabolism are shown in the diagram.