Adaptation Mechanism Of Hypoxia Based On MRNA/miRNA Regulation Network,Mitochondrial Dynamics And Functions On Qinghai-Tibet Plateau In Tibetan Sheep

Long-term exposure of mammals to high-altitude hypoxia,not only affects their production performance,but also easily induces a variety of high-altitude diseases and even death.Mitochondria is the main place for aerobic respiration of cells,provide sufficient energy for cell proliferation,migration and survival,and play an important role in regulating biological processes such as apoptosis,signal transmission and calcium homeostasis.The function of mitochondria is mainly regulated by the fusion and fission of mitochondrial dynamics,but so far,the regulation mechanism of mitochondrial dynamics and functions under hypoxia is still unclear.Therefore,elucidating the regulatory mechanism of mitochondria in the process of hypoxia adaptation is of great significance for improving mammalian hypoxia adaptation capacity,increasing animal products,and the development of plateau medicine.Tibetan sheep are mainly distributed in the Qinghai-Tibet Plateau and its adjacent areas with an altitude of 2500～4500 m,which one of the livestock species to study hypoxia adaptation.In this study,Tibetan sheep at an altitudes of 2500 m（TS25）,3500 m（TS35）,and4500 m（TS45）were used as the research object,phenotyping,high-throughput sequencing,transmission electron microscopy,and other methods were used to study the phenotypic differences,regulation of heart-related mRNAs and miRNAs,and then analysis the adaptive changes of mitochondrial dynamics and functions in heart,liver,lung,brain and quadriceps femoris.The main results can be listed as follows:（1）The main physiological and biochemical indexes of blood and the morphology of myocardial in Tibetan sheep were significantly correlated with the changes of altitude.The HGB,HCT,CK,CK-MB,LDH,LDH1,SOD,GPX,TG,Ca²⁺,CHO and LDL level were significantly higher in TS45 than that of TS25（P<0.05）;The PO₂,SO₂ and PCO₂ level in the TS45 were significantly lower than that of TS35 and TS25 Tibetan sheep（P<0.05）.These changes might reduce oxidative stress of blood,increase blood flow and fluidity to improve the efficiency of hemoglobin-carrying oxygen（2）The results of mRNA-seq and miRNA-seq showed that a total of 18140 mRNAs and1046 miRNAs were found in heart of Tibetan sheep at different altitudes.After filtering,544DEmRNAs and 40 DEmiRNAs were found in TS25-vs-TS35;1349 DEmRNAs and 69DEmiRNAs were found in TS25-vs-TS45;1418 DEmRNAsand 92 DEmiRNAs were found in TS35-vs-TS45.The function analysis showed that DEmRNAs and DEmiRNAs are mainly associated with the PI3K-Akt,Wnt,and PPAR signaling pathways and with an up-regulation of oncogenes（CCKBR,GSTT1,ARID5B）and tumor suppressor factors（TPT1,EXTL1,ITPRIP）to enhance the cellular metabolism and increased ATP production to resist hypoxia stress.Analyzing mRNA-miRNA coregulation indicate that the regulation of mitochondrial dynamics and functions were play an important role in the process of hypoxia adaptation.（3）The mitochondrial fission and fusion rates in the five tissues of Tibetan sheep changed regularly with the increase of altitude.Taking heart tissues as an example,the expression of Drp1,Fis1 and MFF were significantly higher in the TS45 than that of TS25Tibetan sheep（P<0.05）.The Mic60 and OPA1 expression was significantly lower in the TS45 and TS35 Tibetan sheep than that of TS35（P<0.05）,and the expression level in TS45was significantly lower than those in TS35（P<0.05）.Therefore,mitochondria fusion rate was decreased and fission rate was increased in heart with the increase of altitude.Taking heart tissues as an example,the ultrastructural observation of mitochondria showed that mitochondria average density was significantly higher in the TS45 than that of TS35 and TS25 Tibetan sheep（P<0.05）.The average of area and aspect ratio were significantly lower in the TS45 than that of TS35 and TS25 Tibetan sheep（P<0.05）.In addition,the number and width of single mitochondrial cristae was decrease with the increase of altitude,while the number of cristae was significantly lower in TS45 than that of TS25Tibetan sheep（P<0.05）.Therefore,there are more and smaller mitochondria in the heart tissue of TS45 Tibetan sheep with the changes of fission and fusion rates.（4）Changes in mitochondria function maintain cellular homeostasis and promote ATP production resist hypoxia stress.Divergent HSPs showed different patterns with the increase of altitude,HSP27 expression in five tissues was significantly increased with the increase of altitude（P<0.05）.HSP60 expression was significantly increased in all of the tissues of TS35than of TS25,but was significantly decreased at TS45 than that of TS35（P<0.05）.By measuring the expression and content/activity of glycolysis-related gene and enzymes,it was found that HIF1A and PDK4 expression,LDH activity,LA and PA content and LA/PA ratio in five tissues were significantly increased with the increase of altitude（P<0.05）,which indicate that glycolysis was significantly up-regulated with the increase of altitude.The tricarboxylic acid cycle was down-regulated and oxidative phosphorylation was up-regulated with the increase of altitude.By measuring the content/activity of tricarboxylic acid cycle and oxidative phosphorylation-related enzymes,it was found that CA content in five tissues was significantly decreased with the increase of altitude（P<0.05）.NADH and NAD+content,NDH/NAD+ratio and CO activity were significantly higher in TS45 than that of TS25 Tibetan sheep（P<0.05）.The SDH activity in five tissues was decrease with the increase of altitude,while was significantly lower in heart of TS45 and TS35 than that of TS25 Tibetan sheep（P<0.05）.The ATP content in all of tissues was significantly increased with the increase of altitude（P<0.01）.The obvious changes in blood indexes and myocardial tissue morphology of Tibetan sheep were found to adapt to the hypoxic environment with the increase of altitude.The high expression of oncogenes-related genes and tumor suppressor factors might enhance cellular metabolism,so that enhanced mitochondria fission rate and reduced fusion rate of heart tissue,and then up-regulate glycolysis and oxidative phosphorylation and down-regulate the tricarboxylic acid cycle,so as to reduce oxygen consumption and greatly production of ATP to adapt to hypoxic environment.