Study On Activiation Of UPRmt And Mitophagy In Skeletal Muscle During About Of Incremental Exercise

Objective:Mitochondria is a sensitive organelle with complex structure and function.It has a genome independent of the nucleus.The morphology,quantity and quality of mitochondria are highly plasticity under different phases,physiological processes and environmental conditions of the cell.Normally,the number,form and function of mitochondria remain relatively stable,known as mitochondrial homeostasis.Mitochondrial quality control system is an important mechanism for homeostasis maintenance.These include mitochondrial unfolded protein response（UPRmt）and mitophagy.Under low levels of mitochondrial stress,mitochondria activate UPRmt to increase organelle proteases,molecular chaperones,and antioxidants.In severe mitochondrial defects,fusion promotes mt DNA mixing,and fission leads to irreversible mitochondrial isolation,which is eliminated by mitophagy.In this study,we investigated the temporal changes of skeletal muscle UPRmt and mitophagy and their correlations with mitochondrial respiratory efficiency and reactive oxygen species（ROS）production during a bout of incremental exercise pre-and pro-endurance exercise,to explore the potential biological effects of these two mitochondrial quality control mechanisms in exercise response.Method:In this study,a rat model of a bout of incremental exercise pre-and pro-endurance exercise was established to explore the relationship between the two protective mechanisms of mitochondrial quality control.Mitochondrial respiratory control rate（RCR）was measured using the Oroboros Oxygraph-2K respirometer.The level of mitochondrial ROS production was measured by AMR probe method.The transcription levels of UPRmt（c-Jun,CHOP,HSP60,CLPP）and mitophagy（PINK1,Parkin,BNIP3）related genes were determined by Quantitative Real-time PCR.The expression levels of UPRmt（c-Jun,CHOP,HSP60,CLp P）and mitophagy（PINK1,Parkin,BNIP3,LC3II/I）related proteins were determined by western blot and Simple Western^TM.SPSS Statistics 21 was used for data analysis.Results:1 Effects of a bout of incremental exercise pre-and pro-endurance exercise on rat skeletal muscle mitochondrial RCRBefore endurance training,RCR showed a trend of increasing firstly and then decreasing in a bout of incremental exercise,which was significantly increased at90min and reached the peak value（p<0.05）,and then returned to the quiet state at150min.After endurance training,the RCR showed a trend of increasing firstly and then decreasing in a bout of incremental exercise,which was significantly increased at90min（p<0.05）,reached the peak at 150min（p<0.05）,and returned to the quiet state at3h after exercise.Compared with the time points before endurance training,the post-training RCR was significantly increased at 150min and 12h after exercise（p<0.05）.2.Effects of a bout of incremental exercise pre-and pro-endurance exercise on mitochondrial ROS production rate in skeletal muscle of ratsBefore endurance exercise,the ROS production rate was firstly increased and then decreased in a bout of incremental exercise,which was significantly increased at 150min and reached the peak value（p<0.05）,and returned to the quiet state at 3h after exercise.After endurance exercise,the ROS production rate increased firstly and then decreased in a bout of incremental exercise,which significantly increased at 90min and reached the peak value（p<0.01）,and returned to the quiet state at 3h after exercise.Compared with the time points before endurance exercise,the ROS production rate after training was significantly decreased at rest and 150min of exercise（p<0.05）.3.Effects of a bout of incremental exercise pre-and pro-endurance exercise on expression of UPRmt-related proteins in skeletal muscle of ratsBefore endurance exercise,c-Jun increased firstly and then decreased in a bout of incremental exercise,and increased significantly in 45min（p<0.01）,reached the peak value in 90min（p<0.01）,and returned to the quiet state 12h after exercise.CHOP showed a trend of first increase and then decrease,and significantly increased at 150min and reached the peak value（p<0.05）,and returned to the quiet state at 3h after exercise.HSP60 increased at first and then decreased,and was significantly increased at 90min（p<0.01）,and reached the peak value at 12h after exercise（p<0.001）.CLp P increased first and then decreased,and was significantly increased at 150min（p<0.05）,reached the peak at 12h rest（p<0.001）,and returned to the quiet state at 24h after exercise.After endurance exercise,c-Jun increased firstly and then decreased in a bout of incremental exercise,and increased significantly at 45min（p<0.05）,reached the peak value at 150min（p<0.001）,and returned to the quiet state at 3h after exercise.CHOP showed a trend of first increase and then decrease,and significantly increased at 150min and reached the peak value（p<0.05）,and returned to the quiet state at 3h after exercise.HSP60 increased first and then decreased,and was significantly increased at150min（p<0.01）,and reached the peak value at 12h after exercise（p<0.01）.CLp P increased first and then decreased,and increased significantly at 150min and reached the peak value（p<0.001）,and returned to the quiet state 12h after exercise.Compared with the time points before endurance exercise,c-Jun was significantly increased at 150min after training（p<0.05）.CHOP was significantly increased at 45,90 and 150min after exercise（p<0.05～p<0.01）.HSP60 was significantly increased at 3 and 12h after rest（p<0.01～p<0.01）.CLp P was significantly increased at 45,90,150min of exercise and 3h of rest（p<0.05～p<0.01）.4.Effects of a bout of incremental exercise pre-and pro-endurance exercise on the expression of mitophagy-related proteins in skeletal muscle mitochondria of ratsBefore endurance exercise,PINK1 and Parkin had no significant changes in a bout of increamental exercise.LC3II/I decreased first,then increased,and then gradually decreased,and increased significantly at 150min and reached the peak value（p<0.05）,and returned to the quiet state at 3h after exercise.BNIP3 showed a trend of decreasing first and then increasing,and significantly decreased and reached a trough value at150min（p<0.05）.After endurance exercise,PINK1 increased firstly and then decreased in a bout of incremental exercise,and increased significantly at 150min and reached the peak value（p<0.05）,and returned to the quiet state at 3h after exercise.Parkin had no significant change.LC3II/I decreased first,then increased,and then gradually decreased,and increased significantly at 150min and reached the peak value（p<0.01）,and returned to the quiet state at 3h after exercise.BNIP3 showed a trend of decreasing first and then increasing,and significantly increased 24h after exercise compared with 150min after exercise（p<0.05）.Compared with the rats before endurance exercise,there was no significant difference in PINK1 and Parkin after training,LC3II/I was significantly increased at150min after exercise（p<0.05）,and BNIP3 was significantly increased at 3h after exercise（p<0.05）.Conclusions:1.UPRmt activation was earlier than mitophagy in the process of a bout of incremental exercise,and UPRmt was consistent with the upregulation of mitochondrial respiratory efficiency,suggesting that UPRmt was involved in maintaining mitochondrial energy metabolism response to exercise.mitophagy is consistent with ROS upregulation,suggesting that mitophagy is involved in the protective response to oxidative stress during exercise.2.Eight weeks of endurance exercise did not change the levels of UPRmt and mitophagy in the quiet state,but increased the activation degree of UPRmt and mitophagy in the acute exercise,suggesting that the up-regulation of the stress capacity of mitochondrial quality control system is one of the ways to achieve the health effects of endurance exercise.