Comprehensive RNA-Seq Profiling Reveals Temporal and Tissue-Specific Changes in Gene Expression in Sprague-Dawley Rats as Response to Heat Stress Challenges

Abstract

Understanding heat stress physiology and identifying reliable biomarkers are paramount for developing effective management and mitigation strategies. However, little is known about the molecular mechanisms underlying thermal tolerance in animals. In an experimental model of Sprague-Dawley rats subjected to temperatures of 22 ± 1°C (control group; CT) and 42°C for 30 min (H30), 60 min (H60), and 120 min (H120), RNA-sequencing (RNA-Seq) assays were performed for blood (CT and H120), liver (CT, H30, H60, and H120), and adrenal glands (CT, H30, H60, and H120). A total of 53, 1,310, and 1,501 differentially expressed genes (DEGs) were significantly identified in the blood (P < 0.05 and |fold change (FC)| >2), liver (P < 0.01, false discovery rate (FDR)-adjusted P = 0.05 and |FC| >2) and adrenal glands (P < 0.01, FDR-adjusted P = 0.05 and |FC| >2), respectively. Of these, four DEGs, namely Junb, P4ha1, Chordc1, and RT1-Bb, were shared among the three tissues in CT vs. H120 comparison. Functional enrichment analyses of the DEGs identified in the blood (CT vs. H120) revealed 12 biological processes (BPs) and 25 metabolic pathways significantly enriched (FDR = 0.05). In the liver, 133 BPs and three metabolic pathways were significantly detected by comparing CT vs. H30, H60, and H120. Furthermore, 237 BPs were significantly (FDR = 0.05) enriched in the adrenal glands, and no shared metabolic pathways were detected among the different heat-stressed groups of rats. Five and four expression patterns (P < 0.05) were uncovered by 73 and 91 shared DEGs in the liver and adrenal glands, respectively, over the different comparisons. Among these, 69 and 73 genes, respectively, were proposed as candidates for regulating heat stress response in rats. Finally, together with genome-wide association study (GWAS) results in cattle and phenome-wide association studies (PheWAS) analysis in humans, five genes (Slco1b2, Clu, Arntl, Fads1, and Npas2) were considered as being associated with heat stress response across mammal species. The datasets and findings of this study will contribute to a better understanding of heat stress response in mammals and to the development of effective approaches to mitigate heat stress response in livestock through breeding.

Keywords: RNA-sequencing; heat stress response; mammals; rats' transcriptome; thermal tolerance.

Figure 1

Number of lowly (RPKM ≤ 50), medium (50 < RPKM < 500), and highly (RPKM ≥ 500) expressed genes in each library. (A–C) refer to the numbers of lowly, medium and highly expressed genes identified in blood, liver and adrenal glands, respectively.

Figure 2

Significantly (FDR = 0.05) enriched biological processes (BPs) for differentially expressed genes (DEGs) in the blood, liver, and adrenal glands. CT means rats were kept at 22 ± 1°C and relative humidity 50%; H30, H60, and H120 mean rats were kept at 42°C for 30, 60, and 120 min, and the relative humidity 50% conditions. (A) All of the 12 significantly enriched BPs for DEGs in blood for CT vs. H120. (B–D) The top 20 significantly enriched BPs for DEGs in liver for CT vs. H30, CT vs. H60, and CT vs. H120, respectively. (E–G) The top 20 significantly enriched BPs for DEGs in adrenal glands for CT vs. H30, CT vs. H60, and CT vs. H120, respectively. The red words mean these BPs are significantly enriched in three comparisons of the same tissue; the blue words mean these BPs are significantly enriched in only two comparisons of the same tissue; the black words mean these BPs are specifically enriched in each comparison of the tissue. The red boxes mean these BPs are shared among different tissues.

Figure 3

Analysis of shared and specific genes across treatments and tissues. (A) Venn diagram of DEGs in blood, liver, and adrenal gland tissues under the same heat stress conditions. CT means rats were kept at 22 ± 1°C and relative humidity 50%; H30, H60, and H120 mean rats were kept at 42°C for 30, 60, and 120 min, and the relative humidity 50% conditions. (B) Venn diagram of DEGs in comparisons of CT vs. H30, CT vs. H60, and CT vs. H120 in the liver and adrenal gland tissues, respectively.

Figure 4

Temperature-dependent gene expression patterns and their functions. CT means rats were kept at 22 ± 1°C and relative humidity 50%; H30, H60, and H120 mean rats were kept at 42°C for 30, 60, and 120 min, and the relative humidity 50% conditions. (A) The expression pattern of 91 genes of liver identified in comparisons of CT vs. H30, CT vs. H60, and CT vs. H120. (B) Pie plot of biological processes (BPs) enriched by genes of the liver, which were significantly (P < 0.05) enriched in patterns. (C) Significantly enriched pathways for genes in liver that were significantly (P < 0.05) enriched in patterns. (D) The expression pattern of 73 genes of the adrenal glands identified in comparisons of CT vs. H30, CT vs. H60, and CT vs. H120. (E) Pie plot of biological processes (BPs) enriched by genes of adrenal glands, which were significantly (P < 0.05) enriched in patterns. (F) Significantly enriched pathways for genes in the adrenal glands that were significantly (P < 0.05) enriched in patterns.

Figure 5

Phenome-wide association studies (PheWAS) results for five genes in humans. (A) PheWAS results of three genes, which had the most significant SNPs for rectal temperature (RT), respiration rate score (RR), and drooling score (DS) traits. (B) PheWAS results of two genes with SNPs identified for at least two SNP traits. The human complex traits were summarized mainly from six aspects, including cardiovascular, endocrinal, environmental, immunological, metabolic, and others (e.g., neurological, psychiatric, activities, respiratory). Only the name of the most significant trait in each aspect has been reported.