ALDH2 Alleviates Myocardial Ischemia-Reperfusion Injury Via Regulating PARP1-Dependent Cell Death

Background:Acute myocardial infarction is one of the most deadly diseases in the world.Percutaneous coronary intervention can restore coronary blood flow by removing coronary artery stenosis or obstruction.However,the restoration of blood flow results in acute myocardial ischemia-reperfusion(M1/R)injury,causing myocardial cell death and other irreversible injuries.Parthanatos,which is activated by oxidants,alkylating agents,and MI/R,is a type of programmed cell death characterized by the overactivation of PARP1(poly(ADP-ribose)(PAR)polymerase-1),subsequent accumulation of PAR polymers in cytoplasm and apoptosis inducing factor(AIF)dependent DNA fragments,leading to energy collapse.4-Hydroxynonenal(4-HNE)and other toxic active aldehydes are produced during lipid peroxidation in myocardial ischemia-reperfusion,heart failure,and other diseases.Aldehyde dehydrogenase 2(ALDH2)is a mitochondrial protein present in various organs and plays a protective role by removing 4-HNE and alleviating necrotic apoptosis of injured cardiomyocytes.There is no literature to report whether ALDH2 regulates the role and mechanism of Parthanatos in myocardial ischemia-reperfusion injury.Objectives:1.To verify Parthanatos induced by acute myocardial ischemia-reperfusion injury;2.To identify the specific mechanism of Parthanatos in acute myocardial ischemia-reperfusion injury;3.To investigate whether ALDH2 regulates Parthanatos induced by acute myocardial ischemia-reperfusion injury by scavenging 4-HNE.Methods:1.Establishment of acute MI/R model in mice:C57BL/6 adult male mice at the age of 8 weeks were selected.After the left anterior descending branch of the coronary artery was ligated with thorax for 45min,the mouse heart tissues were collected after the slip-knot was opened and reperfused for 2 hours and 7 days,respectively.The sham operation group did not tie the thread after threading,and the other operations were consistent;2.Cardiac function test of mice:The changes in cardiac function of mice in each group were detected by small animal ultrasound;3.Test for myocardial infarction size:After MI/R sampling,Evans blue and 2,3,5triphenyl tetrazole chloride(Evans blue-TTC)double staining was used to observe myocardial infarction size;4.Detection of myocardial injury markers:Lactate dehydrogenase(LDH)and Creatine kinase isoenzyme MB(CKMB)were detected by biochemical analyzer after mouse serum collection,4-HNE and Malondialdehyde(MDA)were evaluated by Elisa kit;5.Cell anoxia and reoxygenation treatment:cardiomyocytes starved for 12h were placed in the anoxic chamber,cells starved for 12h were incubated in the normal incubator for 2h,4h,8h;6.Cell 4-HNE stimulation treatment:After 12h of starvation,cardiomyocytes were given 4-HNE(20μM,40μM,60μM,80μM)to incubate for 0.5h,1h,2h,4h;7.DDX21 expression in cardiomyocytes was reduced by small interference:cardiomyocytes transfected with small interference mRNA of DDX21;8.Histopathology and immunofluorescence:The expression changes of PAR,γ-H2AX,and 4-HNE were detected by immunohistochemistry in paraffin-embedded sections of mouse hearts,or by immunofluorescence in myocardial cells of each group;9.Protein immunoblot:The myocardial tissue or myocardial cell protein samples were analyzed by western blot PAR,PARP1,Cleaved PARP1(C-PARP1),γ-H2AX,Caspase-3,BAX,BCL-2,DDX21,AIF,and MIF;10.Immunoco-precipitation:Detect the interaction between DDX21-PAR.After extracting proteins,incubate primary antibodies and beads,centrifugally discard the supernatant,and add 2x loading buffer,after the metal bath is cooked,the same as the above method of protein immunoblotting detection;11.The level of ATP and NAD+detected by the kit;12.mRNA detection:cardiomyocyte RNA was extracted DDX21 expression for RT-qPCR detection;13.Statistical analysis:The experimental data were represented by Mean±standard error(Mean±SEM)and Graphpad Prism 6 statistical analysis,including t-test between two groups and single-factor analysis of variance between multiple groups.Results:1.Myocardial ischemia-reperfusion injury induces Parthanatos and 4-HNE accumulation;2.Cardiac anoxia and reoxygenation induces Parthanatos and 4-HNE accumulation;3.Parthanatos is induced by 4-HNE stimulation;4.DDX21-PAR binding to mediate Parthanatos caused by hypoxia and reoxygenation of myocardial cells;5.DDX21-PAR binding to mediate Parthanatos of myocardial cells induced by 4-HNE stimulation;6.ALDH2 improves Parthanatos induced by DDX21-PAR binding in acute myocardial ischemia-reperfusion injury by scavenging 4-HNE.Conclusion:ALDH2 improves Parthanatos induced by DDX21-PAR binding in acute myocardial ischemia-reperfusion injury by clearing 4-HNE.