| BackgroundWith the aging of the population and the change of people's lifestyle,the incidence of ischemic heart disease is increasing year by year.As the most common cardiovascular diseases in clinic,ischemic heart disease has become a serious threat to human life and health.It is the best treatment for acute ischemic heart disease to restore blood flow to ischemic tissue as soon as possible.However,clinical studies and animal experiments show that with the recovery of perfusion,the function and structure of some hearts are aggravated,causing more serious dysfunction and structural damage,known as myocardial ischemia/reperfusion(I/R)injury.How to restore blood flow perfusion as soon as possible while avoiding or even reducing the occurrence of ischemia-reperfusion injury has become the key for the treatment of ischemic heart disease.Therefore,the first prerequisite is to identify the pathogenesis of I/R injury.The damage caused by excessive Reactive Oxygen Species(ROS)produced from mitochondria is considered as the most fundamental mechanism of I/R injury in the light of existing studies.Excessive ROS leads to the damage of mitochondria themselves,and the damaged mitochondria will produce more ROS,eventually leading to a vicious circle between damaged mitochondria and ROS.To maintain mitochondrial steady-state equilibrium,cells selectively remove damaged mitochondria through the mitophagy mechanism,so as to ensure the normal continuity for mitochondrial function.Poly(adp-ribose)polymerase(PARP)is a protease family that performs DNA repair by poly-ADP-ribosylation of damaged DNA.Parp-1 accounts for about 90%of the PARP family.Studies have confirmed that excessive activation of PARP-1 due to extensive DNA strand damage caused by ROS plays an important role in myocardial I/R injury.Overactivation of PARP-1 will lead to depletion of intracellular NAD+and ATP and formation of insufficient cell energy supply,resulting in cardiomyocyte apoptosis or necrosis.Parp-1 is an NAD+dependent ADP-ribozyme polymerase,whose activity depends on the level of the cofactor NAD+.As an important substrate of metabolic process in mitochondrial respiratory chain.excessive consumption of NADH/NAD+will affect oxidative respiration and ATP synthesis of mitochondria,causing and aggravating mitochondrial dysfunction.In order to maintain normal mitochondrial function and its steady state,mitophagy mechanism was further activated to eliminate dysfunctional mitochondria and achieve mitochondrial quality control.Nevertherless,studies have shown that mitophagy has a double-edged sword effect.Moderate mitophagy can promote cell survival,while excessive mitophagy can lead to cell death.Therefore,we speculate that hyperactive activation of PARP-1 can induce excessive mitophagy,thereby exacerbating myocardial I/R injury.This project aims to study the cardioprotective effect of PARP inhibition in myocardial I/R injury from the perspective of regulation of mitophagy and the mechanism of PARP regulating mitophagy.ObjectiveThe objectives of this experiment is to investigate:1.The presence of PARP regulating mitophagy pathway in myocardial ischemia-reperfusion.2.The role of PARP regulating mitophagy pathway in myocardial ischemia-reperfusion.3.The mechanism of PARP regulating mitophagy in myocardial ischemia-reperfusion.Methods1.Establishment and grouping of mouse heart I/R model:The model of mouse heart I/R injury was established by thoracotomy ligation and loosening of the anterior descending branch of the left coronary artery(Half an hour for ischemia time,2 hours for reperfusion time).It was divided into Sham group(Sham),ischemia-reperfusion group(I/R)and ischemia-reperfusion+DPQ group(DPQ+I/R).2.Establishment and grouping in H/R model of H9C2 cells and AC 16 cells:The model of H9C2 cells H/R injury was constructed after 10 hours of hypoxic culture in a hypoxic box(1%O2/5%CO2/94%N2)and reoxygenated for 2 hours in a cell culture box(5%CO2/95%Atmosphere):it was divided into control group,H/R group,DPQ+H/R group.AC 16 cells H/R model was established and grouped in the same way as H9C2 cells.3.The lentiviral plasmid of shRNA with Parkin as a target(referred to as Lv-Parkin-RNAi)was transfected into H9C2 cells to reduce the expression level of protein Parkin and the transfection efficiency was detected by western blot.4.The electron microscope was used to observe the mitophagy level of each group including mouse cardiomyocytes,H9C2 cells and H9C2 cells infected by Lv-Parkin-RNAi.5.The co-location of MitoTracker Deep Red FM(mitochondrial fluorescence probe)and the key protein Parkin on the pivotal mitophagy pathway of H9C2 cells in each group was detected by immunofluorescence assay.6.Western blot was used to detect the Parkin protein expression of mitochondrial protein in each group including H9C2 cells,AC 16 cells,H9C2 cells infected by Lv-Parkin-RNAi and H9C2 cells incubated with CsA and FCCP respectively;meanwhile Western blot was also used to detect the expressions of COX ? protein,TOM 20 protein and VDAC protein of total proteins of H9C2 cells in each group.7.DCFH-DA method was used to determine the ROS levels in each group including H9C2 cells and H9C2 cells incubated with NAC.8.JC-1 staining tested the ??m levels of each group including H9C2 cells and H9C2 cells incubated with CsA,NAC,NAD+,FCCP respectively.9.NAD+and ATP kits were used to detect the levels of NAD+and ATP of H9C2 cells before and after transfection with Lv-Parkin-RNAi in H9C2 cells in each group respectively.10.Western blot was used to detect the PAR protein expression in each group including mouse cardiomyocytes,H9C2 cells,AC 16 cells and the PAR protein expression of mitochondria in each group of H9C2 cells.11.TTC staining was used to determine the myocardial infarction area of mice in each group.12.Echocardiographic measurements of cardiac function in mice of each group.13.TUNEL assay was used to detect the apoptosis rates in each group including mouse cardiomyocytes,H9C2 cells,AC 16 cells,H9C2 cells infected by Lv-Parkin-RNAi and flow cytometry was also used to detect the apoptosis rate of H9C2 cells in each group.14.The immunoprecipitation method was used to detect separately the interaction between protein CypD,TSPO and protein PAR or PARP of H9C2 cells in each group.Results1.Inhibition of PARP activity reduces the occurrence of mitophagy during myocardial ischemia-reperfusion.(1)Inhibition of PARP activity reduced the mitophagy level in I/R-treated mouse cardiomyocytes.?PARP was significantly activated in I/R-treated mouse cardio-myocytes,and its activity was decreased after DPQ was applied.There was only weak protein PAR expression in the control group.The expression of PAR in H/R group was significantly increased.After applying DPQ,PAR expression is reduced.?Inhibition of PARP activity reduced the mitophagy level in I/R-induced mouse cardiomyocytes.Inhibition of PARP activation prevented the reduction of the number of mitochondria and the formation of autophagic vacuoles containing mitochondria induced by I/R.(2)Inhibition of PARP activity reduced mitophagy in H/R-treatd H9C2 Cells and Inhibition of Parkin protein expression also decreased mitophagy in H/R-treatd H9C2 Cells.?There was only weak protein PAR expression in the control group.The expression of PAR in H/R group was significantly increased.After applying DPQ,PAR expression is reduced.?Under electron microscopy,autophagy vacuoles containing mitochondria in the H/R group increased significantly compared with the control group,while autophagy vacuoles containing mitochondria in the DPQ+H/R group decreased obviously compared with the H/R group.?Under laser confocal microscope,the co-localization of protein Parkin and MitoTracker Deep Red FM increased after H/R.while the co-localization of Parkin protein and MitoTracker Deep Red FM decreased after DPQ was applied.?The expressions of COX ? protein,TOM 20 protein and VDAC protein of the total protein in the H/R group were decreased compared with the control group,while the expressions of DPQ+H/R group were increased compared with the H/R group.The expression of protein Parkin of mitochondrial protein in the H/R group increased significantly compared with the control group,while the expression of DPQ+H/R group decreased obviously compared with the H/R group.?The protein Parkin expression of 1v-Parkin-RNAi+H/R group was significantly decreased compared with that in 1v-NC+H/R group.?The number of autophagy vacuoles containing mitochondria in the H/R group and 1v-NC+H/R group increased significantly compared with the control group,and the change between the H/R group and the 1v-NC+H/R group was not significant.However,the number of autophagy vacuoles containing mitochondria in the 1v-Parkin-RNAi+H/R group decreased obviously compared with the H/R group and 1v-NC+H/R group.?The protein Parkin expression in the H/R group and 1v-NC+H/R group increased significantly compared with that in the control group.There was no significant difference in the protein Parkin expression between the H/R group and the 1v-NC+H/R group.However,the expression of protein Parkin in 1v-Parkin-RNAi+H/R group decreased obviously compared with that in H/R group and 1v-NC+H/R group.(3)Inhibition of PARP activity also reduced mitophagy in H/R-treated AC16 cells.?The protein PAR expression of AC 16 cells in H/R group was significantly increased compared with that in control group.After applying DPQ,PAR expression was significantly reduced compared to the H/R group.?The expression of protein Parkin of mitochondrial protein in AC 16 cells increased in H/R group compared with that in control group and decreased in DPQ+H/R group compared to the H/R group.2.PARP activation promoted the production of ROS in H/R-treated H9C2 cells,ROS reduced the level of ATm in H/R-treated H9C2 cells.The ROS level of the H/R group was significantly higher than that of the control group,and the ROS level of NAC+H/R group and DPQ+H/R group were significantly lower than that of the H/R group.The ??m level of the H/R group was reduced obviously compared with that in control group,the ??m level of the NAC+H/R group increased significantly compared to the H/R group.3.Inhibition of PARP activity increased the levels of NAD+and ATP in H/R-treated H9C2 cells;inhibition of mitophagy activation did not change the NAD+and ATP levels in H/R-treated H9C2 cells;the reduction of ??Pm in H/R-induced H9C2 cells could be reversed by exogenous NAD+.(1)The levels of NAD+and ATP in H/R group?1v-NC+H/R group,and 1v-Parkin-RNAi+H/R group were significantly lower than those in control group,and the NAD+and ATP levels in the DPQ+H/R group were significantly higher than those in the H/R group.However,there was no difference in the levels of NAD+and ATP of the H/R group,1v-NC+H/R group,and 1v-Parkin-RNAi+H/R group.(2)H/R significantly induced a reduction of ??m,while exogenous NAD+prevented the reduction.4.Inhibition of PARP activity has a protective effect for heart and cardiomyocyte.(1)Inhibition of PARP activity can reduce the size of myocardial Infarc-tion induced by I/R,the apoptosis rate of myocardial cells and improve the cardiac function of mice.Compared with the I/R group,the area of myocardial infarction in DPQ+I/R group was significantly reduced.I/R significantly reduced the cardiac ejection fraction of the mice,and the cardiac ejection fraction of the mice was significantly improved after DPQ was applied.Compared with sham group,the apoptosis rate of myocardial cells in I/R group increased significantly.The apoptosis rate of cardiomyocytes in DPQ+I/R group was significantly lower than that of I/R group.(2)Inhibition of PARP activity alleviates H/R-induced apoptosis of H9C2 cells.The apoptosis rate of H9C2 cells in each group detected by TUNEL assay and flow cytometry:H/R significantly induced apoptosis,while DPQ reduced apoptosis,and the difference was statistically significant.(3)Inhibition of PARP activity alleviates H/R-induced apoptosis of AC16 cells.TUNEL assay was used to determine the apoptosis rate of AC 16 cells in each group:H/R significantly induced apoptosis,while DPQ reduced apoptosis,and the difference was statistically significant.S.Overactivation of mitophagy promotes H/R-induced H9C2 cells apoptosis.The apoptosis rate of H9C2 cells in each group was determined by TUNEL assay.The apoptosis rate of H/R group and 1v-NC+H/R group was significantly higher than that of the control group,and there was no significant difference between H/R group and 1v-NC+H/R group.However,the apoptosis rate of Iv-Parkin-RNAi+H/R group was significantly lower than that of H/R group and lv-NC+H/R group,and the difference was statistically significant.6.Inhibition of PARP activity regulates H/R-induced mitophagy by adjusting ??m.(1)For ??m,the H/R group was significantly lower than the control group,yet CsA+H/R group and the DPQ+H/R group was significantly higher than the H/R group,while the FCCP+DPQ+H/R group was obviously lower than the DPQ+H/R group.(2)The protein Parkin expression significantly increased in the H/R group compared with the control group,and obviously decreased in the DPQ+H/R group or CsA+H/R group compared with the H/R group,while markedly increased in the FCCP+DPQ+H/R group compared with the DPQ+H/R group.7.PARP may regulate the ??m by poly-ADP-ribosylation of CypD and TSPO proteins.(1)The protein PAR expression in mitochondria significantly increased in the H/R group compared with the control group,while obviously decreased in the DPQ+H/R group compared with the H/R group.(2)Protein CypD and TSPO can be modified by poly-ADP-ribosylation.The application of DPQ significantly prevents this modification.(3)Protein CypD and TSPO have no direct interaction with protein PARP.Conlusion1.The overactivation of PARP promoted the overoccurrence of mitophagy in myocardial ischemia-reperfusion.2.Inhibition of PARP activity has a protective effect on heart and cardiomyocytes.3.The overactivation of mitophagy is involved in H/R-induced apoptosis of H9C2 cells.4.PARP participates in H/R-induced apoptosis of H9C2 cells by regulating mitophagy.5.PARP regulates mitophagy by adjusting ??m via poly-ADP-ribosylation of protein CypD and TSPO of mPTP. |
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