Mitophagy And Its Regulation In Skeletal Muscle Of Gansu Zokor Under Hypoxia

Eospalax cansus(E.cansus)is mainly distributed in northwest China,and it is a unique underground rat in China.In order to adapt to the living environment of underground low oxygen and high carbon dioxide,E.cansus has formed a unique physiological and biochemical mechanism in the evolution process and is an excellent model animal for studying hypoxia tolerance.Mitochondria is the main source of energy.It is very sensitive to changes in oxygen concentration.Hypoxia can easily damage mitochondrial structure and pose a threat to cell survival.E.cansus live in underground hypoxic tunnels all their lives.It needs to consume a lot of energy to dig tunnels and forage.What about the mitochondria of E.cansus in hypoxic environment?Does mitochondrial autophagy occur in skeletal muscle?Does the change in oxygen concentration affect the strength of mitochondrial autophagy?If mitochondrial autophagy occurs,how is it regulated?In order to answer the above questions,this study took E.cansus as the research object,and SD rats as controls.First,Observe the ultrastructure of skeletal muscle and mitochondria under different oxygen concentrations(normal oxygen:21%oxygen concentration;chronic hypoxia:10.5%oxygen concentration 44h;acute hypoxia:6.5%oxygen concentration 6h),analyze whether autophagy occurs.Simultaneously,the mitochondrial quantitative enzyme citrate synthase(C.S)content was measured,and the mitochondrial quantity change was analyzed.The autophagy marker protein LC3 was then detected.Finally,the mRNA and protein levels of HIF-1?,BNIP3,BNIP3L,and BECLIN1 in skeletal muscle of E.cansus and SD rats under different oxygen concentrations were measured by real-time quantification and Western blotting.To explore mitochondrial autophagy and its regulation in skeletal muscle of E.cansus.The main results are as follows:1.Ultrastructure of skeletal muscle and mitochondria of E.cansus and SD rats under hypoxiaWith the decrease of oxygen concentration,the muscle fibers of E.cansus skeletal muscle showed no damage and the Z-line was clear.With the decrease of oxygen concentration in skeletal muscle of SD rats,myofilaments swelled and muscle fibers were damaged.Under three oxygen concentrations,the mitochondrial distribution of E.cansus did not change,the mitochondrial structure was complete,the mitochondrial cristae were dense and clear,and autophagosomes appeared at different stages;With the decrease of oxygen concentration,the mitochondrial distribution of SD rats changed,mitochondria swelled and the vacuolation was severe.Autophagosomes were observed at 6.5%oxygen concentration.The content of mitochondrial quantitative enzyme,citrate synthase(CS),didn't show significant differences at three oxygen concentrations in E.cansus,but all were higher than SD rats;Compared with 21%oxygen concentration,the citrate synthase content of SD rats increased at 10.5%oxygen concentration,but there was no significant difference.But the citrate synthase content decreased significantly at 6.5%oxygen concentration.2.Hypoxia expression of LC3-?/LC3-? in skeletal muscle of E.cansus and SD ratsCompared with 21%oxygen concentration,the ratio of LC3-?/LC3-? in E.cansus showed no significant change under 10.5%oxygen concentration,and it increased extremely significantly under 6.5%oxygen concentration;The SD rats increased extremely significantly at 10.5%oxygen concentration and 6.5%oxygen concentration.The ratio of LC3-?/LC3-? in E.cansus was extremely significantly higher than that of SD rats at three oxygen concentrations.3?Expression of HIF-1?.BNIP3?BNIP3L?BECLIN1 mRNA and protein in skeletal muscle of E.cansus and SD ratsHif-1? mRNA expression:Compared with 21%oxygen concentration,the expression level of Hif 1? mRNA in E.cansus and SD rats did not show a significant difference under 10.5%oxygen concentration,and the expression level increased significantly under 6.5%oxygen concentration.Bnip3?Bnip3l?Beclin1 mRNA expression:There were no significant differences in the expression levels of Bnip3?Bnip3l?Beclinl mRNA under three oxygen concentrations in E.cansus;The expression of Bnip3?Bnip3l?Beclin1 mRNA in SD rats increased gradually with the decrease of oxygen concentration.HIF-1a protein expression:Compared with 21%oxygen concentration,the expression of HIF-1? protein in E.cansus and SD rats increased significantly at both 10.5%and 6.5%oxygen concentrations.The expression of HIF-1? in E.cansus was higher than that of SD rats at 21%and 6.5%oxygen concentration,but lower than that of SD rats at 10.5%oxygen concentration.BNIP3?BNIP3L?BECLIN1 protein expression:Compared with the expression of 21%oxygen concentration in E.cansus,the expression levels of BNIP3?BNIP3L and BECLIN1 decreased at 10.5%oxygen concentration,and increased significantly at 6.5%oxygen concentration;BNIP3 in SD rats gradually increased with decreasing oxygen concentration.The expression of BNIP3L was significantly higher than 21%oxygen concentration at 10.5%oxygen concentration,but 6.5%oxygen concentration was significantly lower than 21%oxygen concentration.BECLIN1 expression increased under hypoxia(10.5%and 6.5%oxygen concentration).It shows that:(1)The skeletal muscle fibers of E.cansus were not damaged as the oxygen concentration decreased,and the mitochondrial structure was intact without large-scale damage.Autophagy occurred under all three oxygen concentrations,and the number of mitochondria didn't change significantly.(2)E.cansus had no significant change in autophagy under chronic hypoxia(10.5%oxygen concentration 44h),and acute hypoxia(6.5%oxygen concentration 6h)up-regulated autophagy.The autophagy level of E.cansus was higher than that of SD rats under three oxygen concentrations.(3)E.cansus and SD rats showed significant differences in mitochondrial autophagy regulation.At the transcription level,hypoxia did not affect the transcription of Bnip3?Bnip3l and Beclinl in E.cansus,but up-regulated Bnip3?Bnip3l?Beclinl transcription;At the protein level,BNIP3?BNIP3L and BECLIN1 proteins of E.cansus were down-regulated under chronic hypoxia(10.5%oxygen concentration 44h),and acute hypoxia(6.5%oxygen concentration 6h)were all up-regulated.It indicated that chronic hypoxia(10.5%oxygen concentration 44h)mitochondrial autophagy was down-regulated in E.cansus,while acute hypoxia(6.5%oxygen concentration 6h)up-regulated mitochondrial autophagy.The expression levels of BNIP3 and BECLIN1 in SD rats increased under hypoxia,but BNIP3L decreased significantly under acute hypoxia(6.5%oxygen concentration 6h),indicating that SD rats can induce enhanced mitochondrial autophagy under hypoxia,mainly by BNIP3 is mediated.It is shown that the hypoxia-tolerant animal E.cansus mitochondrial autophagy and its regulation are significantly different from the ground oxygen-sensitive animals,which reflects the unique energy metabolism mechanism of E.cansus.