UCP2 Overexpression Activates SIRT3 To Regulate Mitochondrial Unfolded Protein Response And Reduce Hypoxia Reoxygenation Mitochondrial Damage In H9C2 Cardiomyocytes

Objective Using H9C2 cardiomyocytes,the hypoxia reoxygenation（HR）injury model was established to determine whether the mitochondrial unfolded protein response(UPR^mt)was activated and the protective effect of UCP2 in the hypoxia reoxygenation injury of cardiomyocytes.Further verify that UCP2 regulates UPR^mtthrough sirtuin-3（SIRT3）to play a protective role in myocardial mitochondria,and open up new ideas for the study of UCP2 playing a protective role in myocardial mitochondria.Methods1 HR injury model exploration H9C2 cardiomyocytes were cultured for 48 hours,and HR injury treatment could be performed when the cell confluency reached about 80%.Detection of UPR^mtmarker proteins in cardiomyocytes:heat shock protein 10（HSP10）,heat shock protein 60（HSP60）,mitochondrial ATP-dependent LON protease 1（LONP1）,mitochondrial casein hydrolyzed protese P（CLPP）protein expression level to explore whether UPR^mtis activated,and to explore the appropriate HR injury model time in this experiment.2 Evaluation of cardiomyocyte injury and mitochondrial damage CCK-8（Cell Counting Kit-8）was used to detect myocardial cell viability;Dihydroethidium（DHE）fluorescent probe was used to detect reactive oxygen species（ROS）;JC Mitochondrial membrane potential（ΔΨm）was detected with-1 probe;ATP content was determined with Adenosine triphosphate（ATP）detection kit,Observation of mitochondrial ultrastructure under electron microscope.3 Detection of mitochondrial unfolded protein response marker proteins Western blot was used to detect the protein expressions of HSP10,HSP60,LONP1 and CLPP.Results1 HR injury induced the occurrence of UPR^mtin H9C2 cardiomyocytes;After HR treatment of cells,it was found that compared with the Normal group,the expression levels of UPR^mtmarker proteins（HSP10,HSP60,LONP1,CLPP）were significantly higher when hypoxia was 3 h and reoxygenated for 0.5 h.At the same time,cell viability was significantly decreased,ROS production was significantly increased,ΔΨm decreased significantly（all p<0.05）.It shows that HR treatment of cardiomyocytes affects UPRmt,that is,HR injury can induces UPR^mt.2 UCP2 overexpression was involved in the regulation of UPR^mtand improved the mitochondrial structure and function of H9C2 cardiomyocytes;HR treatment after UCP2 overexpression,compared with the HR group,ROS production decreased,ΔΨm increased,and cellular ATP production also increased,and the expression levels of HSP10,HSP60,LONP1,and CLPP proteins were significantly increased（both p<0.05）.Compared with the HR group,after UCP2 silencing followed by HR treatment,the production of ROS was significantly increased,the loss ofΔΨm was more obvious,the production of ATP was significantly decreased,and the protein expression levels of HSP10,HSP60,LONP1,and CLPP were significantly decreased（all p<0.05）.3 UCP2 overexpression regulated UPR^mtby activating SIRT3 and played a protective effect on H9C2 cardiomyocytes mitochondria.H9C2 cardiomyocytes were subjected to SIRT3 overexpression and then HR treatment.Compared with the pure HR treatment group,ROS production was significantly reduced,ΔΨm was recovered to a certain extent,and the production of ATP was also increased.The protein expression levels of HSP10,HSP60,LONP1,and CLPP increased significantly（all p<0.05）。The SIRT3 silencing group was treated with HR,compared with the simple HR treatment,ROS increased,ΔΨm was further lost,and the content of ATP also decreased.The expression of HSP10,HSP60,LONP1,and CLPP also decreased significantly（all p<0.05）.When SIRT3 silenced and then overexpressed UCP2 and then performed HR treatment,compared with SIRT3 silencing followed by HR treatment,ROS production,loss ofΔΨm,and ATP production,HSP10,HSP60,LONP1,and CLPP protein expression levels were not significantly reversed（All p>0.05）.Conclusions HR injury induced the occurrence of UPR^mtin H9C2 cardiomyocytes;UCP2 overexpression was involved in the regulation of UPR^mtand improved the mitochondrial structure and function of H9C2 cardiomyocytes;UCP2 overexpression regulated UPR^mtby activating SIRT3 and played a protective effect on H9C2cardiomyocytes mitochondria.