The Effect And Mechanism Study On Aldehyde Dehydrogenase 2 Protects Against Post-cardiac Arres Myocardial Dysfunction

BackgroundSudden cardiac arrest(CA)is the abrupt loss of heart pumping function and can cause sudden death without immediate treatment.It was estimated that-500,000 people have cardiac arrest every year in China.While the success rate of cardiopulmonary resuscitation(CPR)and the rate of return of spontaneous circulation(ROSC)have increased,the mortality thereafter remains > 50%.Therefore,it is urgent to find out the method to improve the survival rate of patients with cardiac arrest and ROSC.Post-cardiac arrest myocardial dysfunction is an important cause of early mortality after ROSC.The mechanisms for post-cardiac arrest myocardial dysfunction include ischemia/reperfusion injury,asphyxia,drug and mechanical damage during resuscitation.However,there is currently no effective therapeutic approach for post-cardiac arrest myocardial dysfunction as well as ischemia/reperfusion injury.It is important to explore the strategy to improve cardiac function after cardiac arrest.Recently,mitochondrial reactive oxygen species(ROS)have been shown to be an important factor in contributing sudden cardiac death and myocardial dysfunction.Mitochondrial ROS were able to drive both acute emergent events,such as electrical instability responsible for sudden cardiac arrest,and chronic heart failure remodeling.However,the mechanism of mitochondrial ROS production and mitochondria-targeted treatments designed to ameliorate mitochondrial oxidative stress are still under study.Aldehydes are generated through lipid peroxidation on mitochondrial and plasma membranes in response to oxidative stress and have been observed in the heart after cardiac arrest.Among them,4-hydroxy-2-nonenal(4-HNE)is the most abundant and reactive carbonyl species.Aldehydes can impair mitochondria by attacking amino acid residues of mitochondrial proteins,but the underlying mechanism is still unclear(14,15).Therefore,clarifying the role of aldehydes in mitochondrial injury and effectively clearing these highly harmful aldehydes is crucial to protect mitochondria from ischemia/reperfiasion injury and alleviate post-cardiac arrest myocardial dysfunction.Aldehyde dehydrogenase 2(ALDH2)is one of the ALDH2 family and primarily located in mitochondria.Previous studies have suggested that ALDH2 plays a central protective role in several types of cardiac diseases and global cellular oxidative stress mainly through metabolizing various aldehydes,such as 4-HNE.ALDH2 could regulate many pathological progressions such as mitophagy,endoplasmic reticulum stress and apoptosis.However,the influence of ALDH2 on post-cardiac arrest myocardial dysfunction and mitochondrial ROS has not been investigated yet.In this study,we investigated the effect and mechanism of ALDH2 on myocardial dysfimction after cardiac arrest.Objective1.To evaluate the effect of enhanced activity or expression of ALDH2 on myocardial dysfunction and survival after cardiac arrest.2.To examine the importance of ALDH2 in reducing cardiomyocyte death and mitochondrial injury.3.To elucidate the underlying mechanisms by which ALDH2 exerts cardioprotection with a focus on mitochondrial ROS production.MethodsWe used the animal cardiac arrest and CPR model to investigate the effect of enhanced activity or expression of ALDH2 on myocardial dysfunction,survival,cardiomyocyte death and mitochondrial injury after cardiac arrest.We used the rat cardiomyoblasts cell line(H9c2)and primary cardiomyocytes with hypoxia/reoxygenation(H/R)procedure to investigate the mechanisms by which ALDH2 exerted cardioprotection.1,Asphyxia-cardiac Arrest and CPR ProcedureWistar rats(375g-425g)were anesthetized with pentobarbital sodium(45 mg/kg,intraperitoneal injection).The oral trachea intubation,ventilator,femoral artery and veinintubation were administrated.The Millar pressure-volume catheter was inserted into the left ventricle,as appropriate.Blood pressure,left ventricular pressxire,and electrocardiogram data were recorded with the PowerLab acquisition system(ADInstruments).CA was induced by asphyxia via turning off the ventilator and clamping the endotracheal tube.CA was defined as the femoral mean arterial pressure(MAP)<30 mm Hg.After 8 minutes of asphyxia,the mechanical ventilator was reinitiated.The epinephrine(2 (?)g/100 g,once every 3 minutes)was administered,and chest compression(200 beats/minute)was performed,attempting for 10 minutes at most.ROSC was defined as the return of sinus rhj^thm with a MAP (?) 60 mm Hg lasting for at least 5 minutes.2.Animal ProtocolRats were assigned to 1 of 3 animal study frameworks:(1)protocol 1 of the ALDH2 activation study(n=53)— Alda-1 was administered via intraperitoneal injection 30 minutes before cardiac arrest;(2)protocol 2 of the ALDH2 activation study(n=28)— Alda-1(10 mg/kg)was administered via intraperitoneal injection at the start of resuscitation;and(3)cardiac overexpression of ALDH2 study(n=45)— adeno-associated virus(serotype 9)(AAV9)-ALDH2 or AAV9-Veh was delivered via tail vein injection at 2.5 x 10 n vector genomes/rat 4weeks before cardiac arrest.Furthermore,rats with ROSC were assigned to 1 of 3 tissue collection time points in each protocol.In protocol 1,rats were euthanized for assessing mitochondrial morphology of heart at 1 hour post-ROSC,At 4 hours post-ROSC,rats were euthanized for myocardial functional and histological assessment.At 72 hours post-ROSC,rats were euthanized for assessing survival rate,myocardial function and histology.In protocol 2,rats with ROSC were followed up for 72 hours for survival rate analysis.The myocardial function was detected within 4 hours and at 72 hours post-ROSC.In protocol 3? at 1 hour post-ROSC,rats were euthanized for assessing mitochondrial morphology of heart.At 4 hours post-ROSC,rats were euthanized for myocardial functional and histological studies.3.Cell ProtocolTo study the effect of ALDH2 on mitochondria,we detected the level of mitochondrial ROS,mitochondrial respiratory function,ATP and 4-HNE using the H9c2 cell under H/R.To further investigate the mechanism of ALDH2 on mitochondrial ROS? 4-HNE was added to cell media and the mitochondrial ROS,succinate were detected.Then,we assessed the specificity of Alda-1 on ALDH2,using primary cardiomyocj^es obtained from ALDH2 KO mice and its background WT mice.4.Measurement of Myocardial FunctionIn protocol 1, Millar pressure-volume was used to monitor myocardial function 4 hours post-ROSC.In protocol 2 and 3,echocardiography was used to detect the myocardial function at 1 hour,2 hour,3 hour,4 hour or 72 hour post-ROSC.5.Measurement of Myocardial ApoptosisMyocardial tissues were fixed in 4% paraformaldehyde,embedded in paraffin and cut into4 ixm sections.Apoptosis was assessed by transferase mediated dUTP nick-end labeling(TUNEL)staining using ApopTag? In Situ Apoptosis Detection Kits.6.Examination of Mitochondrial MorphologyLeft ventricular tissue were fixed with glutaraldehyde overnight for the transmission electron microscope(TEM)exanimation.The severity of mitochondrial structural damage was semi-quantified using Flameng grading of 1 through 5 as described previously.7.Measurement of Cardiac ROSFrozen sections(6 |xm)of myocardium were incubated with DHE and incubated with DAPI to label nuclei.Tissue sections were observed under a fluorescence microscope and the intensity of fluorescence was quantified by ImageJ software.8.Determination of Plasma Creatine Kinase-MB(CK-MB)Blood samples were centrifuged at 1000 x g for 20 minutes to collect plasma.The levels of CK-MB were measured using enzyme-linked immunosorbent assay(ELISA).9.Determination of Blood GasArterial blood was obtained at 15 minutes,1 hour and 4 hours post-ROSC and blood gas profiles(pH? PaO2,PaCO2,glucose and lactate)were measured immediately using automated blood gas analyzer.10.Measurement of ALDH2 ActivityThe mitochondria were isolated from myocardial tissue.Acetaldehyde as the substrate of ALDH2 was oxidized to acetic acid,whereas NAD+ was reduced to NADH which was used to determine ALDH2 activity.11.Protein Expression DetectionWestern blot analysis was used to detect the expression level of ALDH2 and 4-HNE.12.Hypoxia/Reoxygenation(H/R)ProcedureCells were cultured normally in 95% air/5% CO2 at 37 °C.H/R procedure was induced by exposing cells in a hypoxic workstation containing 94% N2? 5% CO2 and 1% O2 at 37 ? with serum-deprived media for 4 hours and then culturing cells under normal conditions with complete media for another 2 hours.13.Measurement of Cellular ROS and Mitochondrial ROSThe dihydroethidium(DHE)and MitoSOX Red reagent were respectively used to detect the level of cellular ROS and mitochondrial ROS.After incubation,cells were observed under a fluorescence microscope and the intensity of fluorescence was quantified by ImageJ software.14.Measurement of Mitochondrial Respiratory FunctionMitochondrial respiratory function evaluated by oxygen consumption rate(OCR)was assessed using a Seahorse XFe24 analyzer with the Seahorse XF Cell Mito Stress Test Kit.15.Measurement of ATP and SuccinateCells were schizolysised and analyzed by a firefly luciferase-based ATP assay kit and the established Succinate Assay Kit.Results1.Baseline and Procedural Characteristics of the Animal StudyIn 3 protocols,compared with Con group,there were no signijBcant differences in baseline and procedural characteristics after enhancing ALDH2 activity or expression,including body weight,heart rate,MAP,left ventricular CO,EF or FS,cardiac arrest duration,CPR duration or ROSC rate.2.Activation of ALDH2 Effectively Improves Post-cardiac Arrest Myocardial Dysfunction and Survival RateWe found that compared with Con group,enhancing ALDH2 activity significantly improved myocardial function within 4 hour post-ROSC,72 hour post-ROSC and 72-hour survival rate.We also determined the activity and expression levels of ALDH2 in myocardium in rats subjected to CA-CPR procedure.The results showed that the activity of ALDH2 was reduced by 58.8% at 4 hours and 64.8% at 72 hours post-ROSC,respectively,which was significantly elevated by Alda-1;however,the expression levels of ALDH2 did not alter during the observation periods.These findings can explain why upregulation of ALDH2 activity has a protective effect on post-cardiac arrest myocardial dysfunction.3.Activation of ALDH2 Attenuates Cardiac Arrest-Induced Cardiomyocyte Death and Mitochondrial InjuryThe rats in protocol 1 of the ALDH2 activation study were used for the following mitochondrial morphology and biomedical investigations.Cardiac myocyte apoptosis was increased at both 4 hours and 72 hours post-ROSC in the CA-CPR group,which was significantly attenuated by Alda-1.Obvious mitochondrial structural damage was observed at1 hour post-ROSC in the CA-CPR group,which was partially reversed by Alda-1.The elevated cellular ROS levels and 4-HNE-protein adducts were also inhibited by Alda-1.4.Enhanced Expression of ALDH2 Improves Post-cardiac Arrest Myocardial DysfunctionTo further validate the cardioprotective effect of ALDH2 activation,we examined whether enhancing the expression of ALDH2 had protective effects on post-cardiac arrest myocardial dysfunction.Cardiac specific overexpression of ALDH2 significantly improved left ventricular EF5 cellular ROS levels,4-HNE-protein adducts and mitochondrial structure.These findings indicate that enhancement of ALDH2 through both enzymatic activation and protein overexpression attenuates post-cardiac arrest myocardial dysfunction.5.Alda-1 Specifically Activates ALDH2 and Suppresses Hypoxia/ReoxygenationInduced Mitochondrial ROSThe levels of mitochondrial ROS were increased 5.7-fold during H/R,the extent of which was significantly reduced by Alda-1.Alda-1 also improved the cellular ATP levels and mitochondrial respiratory dysfunction.Furthermore,we assessed the specificity of Alda-1 on ALDH2 by primary cardiomyocytes obtained from ALDH2 KO mice and its background WT mice.ALDH2 KO cardiomyocytes generated more mitochondrial ROS under H/R compared with the cardiomyocytes from WT mice;however,Alda-1 had no effect on the levels of mitochondrial ROS in ALDH2 KO cardiomyocytes.Similar findings were observed for H/Rinduced mitochondrial respiratory dysfunction.These results indicate that Alda-1 has no other targets in mitochondrial protection except interacting with ALDH2.6.4-HNE is Increased Under Hypoxia/Reoxygenation and Promotes Mitochondrial ROS ProductionWe further examined whether the effect of ALDH2 on inhibiting mitochondria ROS production during H/R was attributed to its enzymatic function against toxic aldehyde overload.The 4_HNE-protein adducts were increased after H/R,and 4-HNE increased the levels of mitochondrial ROS.Since previous evidence has shown that the accumulated succinate is the main driver of mitochondrial ROS production,we hypothesized that 4-HNE may interfere with this process.We observed that 4-HNE stimulation resulted in the elevated succinate accumulation in a concentration-dependent manner and increased mitochondrial membrane potential.Additionally,augmented SDH carbonylation was observed with 4-HNE treatment.Treatment with DMM inhibited 4-HNE-induced increase of mitochondrial ROS levels.Taken together,these data suggest that aldehydes play an essential role in mediating succinate accumulation and mitochondrial ROS production,which may explain why enhancement of ALDH2 inhibits mitochondrial ROS production during H/R.Conclusion1.Enhancing activity or expression of ALDH2 improved myocardial dysfunction and survival after cardiac arrest,reduced cardiomyocyte death and mitochondrial injury.2.4-HNE increased after ischemia/reperflision injury which promotes succinate accumulation and mitochondrial ROS production.By clearing 4-HNE,ALDH2 suppressed mitochondrial ROS.