Changes in the Mitochondrial Dynamics and Functions Together with the mRNA/miRNA Network in the Heart Tissue Contribute to Hypoxia Adaptation in Tibetan Sheep

Abstract

This study aimed to provide insights into molecular regulation and mitochondrial functionality under hypoxia by exploring the mechanism of adaptation to hypoxia, blood indexes, tissue morphology, mRNA/miRNA regulation, mitochondrial dynamics, and functional changes in Tibetan sheep raised at different altitudes. With regard to blood indexes and myocardial morphology, the HGB, HCT, CK, CK-MB, LDH, LDH1, SOD, GPX, LDL level, and myocardial capillary density were significantly increased in the sheep at higher altitudes (p < 0.05). The RNA-seq results suggested the DEmRNAs and DEmiRNAs are mainly associated with the PI3K-Akt, Wnt, and PPAR signaling pathways and with an upregulation of oncogenes (CCKBR, GSTT1, ARID5B) and tumor suppressor factors (TPT1, EXTL1, ITPRIP) to enhance the cellular metabolism and increased ATP production. Analyzing mRNA−miRNA coregulation indicated the mitochondrial dynamics and functions to be significantly enriched. By analyzing mitochondrial dynamics, mitochondrial fusion was shown to be significantly increased and fission significantly decreased in the heart with increasing altitude (p < 0.05). There was a significant increase in the density of the mitochondria, and a significant decrease in the average area, aspect ratio, number, and width of single mitochondrial cristae with increasing altitudes (p < 0.05). There was a significant increase in the NADH, NAD+ and ATP content, NADH/NAD+ ratio, and CO activity, while there was a significant decrease in SDH and CA activity in various tissues with increasing altitudes (p < 0.05). Accordingly, changes in the blood indexes and myocardial morphology of the Tibetan sheep were found to improve the efficiency of hemoglobin-carrying oxygen and reduce oxidative stress. The high expression of oncogenes and tumor suppressor factors might facilitate cell division and energy exchange, as was evident from enhanced mitochondrial fission and OXPHOS expression; however, it reduced the fusion and TCA cycle for the further rapid production of ATP in adaptation to hypoxia stress. This systematic study has for the first time delineated the mechanism of hypoxia adaptation in the heart of Tibetan sheep, which is significant for improving the ability of the mammals to adapt to hypoxia and for studying the dynamic regulation of mitochondria during hypoxia conditions.

Keywords: Qinghai–Tibet Plateau; RNA-seq; Tibetan sheep; dynamics; hypoxia; mitochondria; oxidative phosphorylation.

Figure 1

The morphological characteristics of the hearts in Tibetan sheep by H&E staining (40×). (a–c) Morphological characteristics of heart. (d) Capillary density per unit area. (e) Average diameter of single capillary per unit area. (TS25) 2500 m altitude Tibetan sheep. (TS35) 3500 m altitude Tibetan sheep. (TS45) 4500 m altitude Tibetan sheep. Data shown on graph are means ± SEM. TS25 as control, * p < 0.05, ** p < 0.01.

Figure 2

Results of mRNA-seq in heart tissue of Tibetan sheep at different altitudes. The Heatmap shows the relative expression pattern of DEmRNAs between groups. Each column represents a sample, and each row represents the expression of a single mRNA between different samples. The color transitions from blue (low expression) to red (high expression). (a) The heatmap between TS25 and TS35. (b) The heatmap between TS25 and TS45. (c) The heatmap between TS35 and TS45. (d) Statistical of DEmRNAs in three group. (TS25) 2500 m altitude Tibetan sheep. (TS35) 3500 m altitude Tibetan sheep. (TS45) 4500 m altitude Tibetan sheep.

Figure 3

Results of miRNA-Seq in heart tissue of Tibetan sheep at different altitudes. The scatter plot shows that DEmiRNASs were up-regulated and down-regulated between groups. The abscissa is the expression level of the control group, and the ordinate is the expression level of the experimental group. Red indicates up-regulated miRNA, green indicates down-regulated miRNA, and blue indicates co expressed miRNA. (a) Scatter plot of TS35 DEmiRNAs relative to TS25. (b) Scatter plot of TS45 DEmiRNAs relative to TS25. (c) Scatter plot of TS45 DEmiRNAs relative to TS35. (d) Statistical of DEmiRNAs in three group. (TS25) 2500 m altitude Tibetan sheep. (TS35) 3500 m altitude Tibetan sheep. (TS45) 4500 m altitude Tibetan sheep.

Figure 4

Functional annotation analysis of DEmRNAs in heart tissue of Tibetan sheep at different altitudes. (a) Top 20 KEGG enrichment pathways between TS25 and TS35. (b) Top 20 KEGG enrichment pathways between TS25 and TS45. (c) Top 20 KEGG enrichment pathways between TS35 and TS45. The ordinate is the pathway, and the abscissa is the enrichment factor. Darker colors indicate smaller q-values. (d) Histogram of GO annotation results of DEmRNAs. The abscissa is the second level GO term, and the ordinate is the number of DEmRNAs in the term. (e) Venn diagram of mRNA interactions based on the overlapping mRNAs among the three groups. (TS25) 2500 m altitude Tibetan sheep. (TS35) 3500 m altitude Tibetan sheep. (TS45) 4500 m altitude Tibetan sheep.

Figure 5

Functional annotation of DEmiRNA target gene and co-expression network. (A) Histogram of GO annotation results of top 500 DEmRNAs in mRNA–miRNA correlation analysis results. The abscissa is the second level GO term, and the ordinate is the number of DEmRNAs in the term. (B) Gene co-expression network analyses of hypoxic DEmRNAs and DEmiRNAs. (a) Mitochondria dynamic related miRNA and target genes. (b) Mitochondria function related miRNA and target genes. (C) Top 20 KEGG enrichment pathways of top 500 mRNA in mRNA–miRNA correlation analysis results. The ordinate is the pathway, and the abscissa is the enrichment factor.

Figure 6

The expression of mitochondria fission (Drp1 (a), Fis1 (b), MFF (c)) and fusion (Mic60 (d), OPA1 (e)) genes in five tissues of Tibetan sheep. (1–5) Heart, liver, lung, brain, quadriceps femoris. (TS25) 2500 m altitude Tibetan sheep. (TS35) 3500 m altitude Tibetan sheep. (TS45) 4500 m altitude Tibetan sheep. Date shown on graph are means ± SEM. * p < 0.05, ** p < 0.01.

Figure 7

Immunostaining of mitochondria fission (Drp1 (A), Fis1 (B), MFF (C)) and fusion (Mic60 (D), OPA1 (E)) proteins in five tissues of Tibetan sheep (20×). (a–c) Heart. (d–f) Liver. (g–i) Lung. (j–l) Brain. (m–o) Quadriceps femoris. (1–5) Heart, liver, lung, brain, quadriceps femoris. (TS25) 2500 m altitude Tibetan sheep. (TS35) 3500 m altitude Tibetan sheep. (TS45) 4500 m altitude Tibetan sheep. Date shown on graph are means ± SEM. The IOD in heart of TS25 as control, * p < 0.05, ** p < 0.01.

Figure 8

Representative electron micrographs of mitochondria in five tissues of Tibetan sheep. (a–c) Heart. (d–f) Liver. (g–i) Lung. (j–l) Brain. (m–o) Quadriceps femoris. (a1–o1) 2000×. (a2–o2) 10,000×. (TS25) 2500 m altitude Tibetan sheep. (TS35) 3500 m altitude Tibetan sheep. (TS45) 4500 m altitude Tibetan sheep.

Figure 9

Analysis of five mitochondria dynamic-related indexes in five tissues of Tibetan sheep. (a) The structure of the mitochondrial cristae. (b) The number of mitochondrial cristae. (c) The width of mitochondria cristae. (d) Mitochondria density. (e) Mitochondria area. (f) Aspect ratio. (1–5) Heart, liver, lung, brain, quadriceps femoris. (TS25) 2500 m altitude Tibetan sheep. (TS35) 3500 m altitude Tibetan sheep. (TS45) 4500 m altitude Tibetan sheep. Date shown on graph are means ± SEM. With the indexes in heart of TS25 as control, * p < 0.05, ** p < 0.01.

Figure 10

Analysis of mitochondria function-related indexes in five tissues of Tibetan sheep. (a) Nicotinamide adenine dinucleotide (NADH (reduced state)). (b) Nicotinamide adenine dinucleotide (NAD+ (oxidation state)). (c) The ratio of NADH/NAD+. (d) Citric acid (CA). (e) Succinate dehydrogenase (SDH). (f) Cytochrome c oxidase (CO). (g) Adenosine triphosphate (ATP). (1–5) Heart, liver, lung, brain, quadriceps femoris. (TS25) 2500 m altitude Tibetan sheep. (TS35) 3500 m altitude Tibetan sheep. (TS45) 4500 m altitude Tibetan sheep. Date shown on graph are means ± SEM. * p < 0.05, ** p < 0.01.