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Research ArticleActivation of ALDH2 with Low Concentration ofEthanol Attenuates Myocardial IschemiaReperfusion Injury inDiabetes Rat Model

Pin-Fang Kang1 Wen-Juan Wu2 Yang Tang1 Ling Xuan1

Su-Dong Guan3 Bi Tang1 Heng Zhang1 Qin Gao3 and Hong-Ju Wang1

1Department of Cardiovascular Disease The First Affiliated Hospital of Bengbu Medical College Bengbu 233004 China2Department of Biochemistry and Molecular Biology Bengbu Medical College Bengbu 233030 China3Department of Physiology Bengbu Medical College Bengbu 233030 China

Correspondence should be addressed to Qin Gao bbmcgq126com and Hong-Ju Wang docwhj1101163com

Received 10 May 2016 Revised 9 August 2016 Accepted 24 August 2016

Academic Editor Ryuichi Morishita

Copyright copy 2016 Pin-Fang Kang et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The aim of this paper is to observe the change of mitochondrial aldehyde dehydrogenase 2 (ALDH2) when diabetes mellitus(DM) rat heart was subjected to ischemiareperfusion (IR) intervention and analyze its underlying mechanisms DM rat heartswere subjected to 30min regional ischemia and 120min reperfusion in vitro and pretreated with ALDH2 activator ethanol(EtOH) cardiomyocyte in high glucose (HG) condition was pretreated with ALDH2 activator Alda-1 In control IR groupmyocardial tissue structure collapse appeared Compared with control IR group left ventricular parameters SOD activity thelevel of Bcl-2Bax mRNA ALDH2 mRNA and protein expressions were decreased and LDH and MDA contents were increasedmeanwhile the aggravation of myocardial structure injury in DM IR group When DM IR rats were pretreated with EtOH leftventricular parameters SOD Bcl-2Bax and ALDH2 expression were increased LDHMDA andmyocardial structure injury wereattenuated Compared with DM+ EtOH IR group cyanamide (ALDH2 nonspecific blocker) atractyloside (mitoPTP opener) andwortmannin (PI3K inhibitor) groups all decreased left ventricular parameters SOD Bcl-2Bax and ALDH2 and increased LDHMDA andmyocardial injury When cardiomyocyte was under HG condition CCK-8 activity and ALDH2 protein expression weredecreased Alda-1 increased CCK-8 and ALDH2 Our findings suggested enhanced ALDH2 expression in diabetic IR rats playedthe cardioprotective role maybe through activating PI3K and inhibiting mitoPTP opening

1 Introduction

Diabetes mellitus (DM) is a common metabolic disorderthat can affect patient life and survival quality with acuteand chronic complications [1ndash3] It is associated with a highcardiovascular mortality and it is also the most commoncause of end-stage heart disease [4] The morbidity andmortality of diabetes show an increasing trend and the inci-dence of heart failure after myocardial infarction in diabeticpatients is more than 2-3 times than nondiabetic Myocardialischemiareperfusion (IR) injury can cause cardiac glucosemetabolism disorders calcium overload and cardiac fibrosisresulting in accumulation of harmful products easily leadingto apoptosis and necrosis [5] Therefore how to protect

ischemic myocardium effectively promote myocardial func-tional recovery and reduce myocardial apoptosis become aresearch hotspot

Mitochondrial aldehyde dehydrogenase 2 (ALDH2) isone of the key enzymes of alcohol metabolism whichcatalyzes the conversion of aldehyde to acetic acid [6]ALDH2 plays a crucial metabolic role in the detoxificationand oxidation of aldehyde such as inhibiting the produc-tion of 4-hydroxynon-2-enal [7ndash9] ALDH2 was found toattenuate ethanol exposure-inducedmyocardial dysfunctionand activation of ALDH2 led to cardiac protection againstischemia and reperfusion injury [10ndash15] Previous studiesalso indicated that with the development of diabetes cardiacALDH2 activity was further decreased and inhibition of

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2016 Article ID 6190504 12 pageshttpdxdoiorg10115520166190504

2 Oxidative Medicine and Cellular Longevity

ALDH2 by oxidative stress leads to cardiac dysfunction indiabetes mellitus [16] What changes of ALDH2 expressionthat appeared and what mechanisms involved in diabetic ratmyocardial IR injury spark our interest

At the beginning of IR the signal transduction pathwayswere activated by various means of myocardial protec-tion proactively especially phosphatidylinositol-3-kinase ser-inethreonine kinase (PI3K-Akt) PI3K-Akt signaling path-way is an important pathway which regulates cell prolifera-tion cell division apoptosis and other activities Heart wasprotected by ischemic preconditioning via activated PI3K-Akt signaling pathway however the effect of myocardialprotection was abolished by PI3K inhibitor wortmannin Itis suggested that PI3K-Akt signal transduction pathway wasinvolved in myocardial protection

Mitochondrial permeability transition pore (mitoPTP)is located in the inner and outer mitochondrial membranemultiprotein complexes which plays an important role inmaintainingmitochondrial membrane potential and protect-ing the structure and function of mitochondrion [17 18]Under physiological condition mitoPTP is closed and it hasalmost no permeability for all mitochondrial metabolites andions Opening of mitoPTP is involved in cell death inducedby a variety of causes for example IR endotoxin andanticancer agents [19] Opening mitoPTP leads to mitochon-drial swelling and outer membrane rupture the proapoptoticfactors and cytochrome C were released into the cytoplasminducing energymetabolismunbalance intracellular calciumoverload and exacerbated ischemic cell damageThe openingof mitoPTP in reperfusion is a sign of myocardial injury fromreversible to irreversible [20 21] mitoPTP is considered tobe the terminal effector of cell death in IR injury whiledelaying or blocking the opening of mitoPTP may reduceIR injury [22 23] A large number of reports suggestedthat ischemic preconditioning ischemic postconditioningand drug pretreatment could protect heart against injury byinhibiting of the opening of mitoPTP [24ndash26]

Based on above background we were prompted to inves-tigate the effects of pretreatment with ethanol (EtOH) as atool to induce ALDH2 activity in myocardial IR injury ofdiabetic rats and give drug intervention including ALDH2inhibitor cyanimide PI3K inhibitor wortmannin and theopener of mitoPTP atractyloside to determine its underlyingmechanisms

2 Materials and Methods

21 Animals Male Sprague-Dawley rats (200sim250 g) werepurchased from the Animal Center of Bengbu MedicalCollege Anhui The rats were fed normal chow and hadfree access to water Housing was at a constant temperatureof (21 plusmn 1)∘C with a fixed 12 h lightdark cycle All animalprocedures were in accordance with United States NationalInstitutes of Health Guide and were approved by the AnimalUse and Care Committee of Bengbu Medical College

22 Chemicals and Reagents Streptozotocin (STZ) cyana-mide (CYA) wortmannin (Wor) atractyloside (Atr) and

Alda-1 were purchased from Sigma (St Louis MO USA)Ethanol (EtOH) was obtained from Bengbu New ChemicalReagent Factory China 10 fetal bovine serumwas obtainedfrom Zhe Jiang Tianhang Biological corporation China Lac-tate dehydrogenase (LDH) Malondialdehyde (MDA) andsuperoxide dismutase (SOD) assay kits were purchased fromNanjing Jiancheng Bioengineering Institute China CCK-8assay kit was from Shanghai Bestbio Life Technology ChinaThe primers used were as follows for ALDH2 forward 51015840-GTG TTC GGA GAC GTC AAA GA-31015840 and reverse 51015840-GCAGAG CTT GGG ACA GGT AA-31015840 the amplified fragmentlengthwas 187 bp for Bcl-2 forward 51015840-CTGGTGGACAACATCGCTCTG-31015840 and reverse 51015840-GGTCTGCTGACCTCACTT GTG-31015840 the amplified fragment length was 227 bp forBax forward 51015840-GGA TCG AGC AGA GAG GAT GG-31015840and reverse 51015840-GCT CAT TGC CGA TAG TGA TGA CT-31015840 the amplified fragment length was 464 bp for 120573-actinforward 51015840-GAT GGT GGG TAT GGG TCA GAA-31015840 andreverse 51015840-GGC CAT CTC TTG CTC GAA GTC-31015840 theamplified fragment length was 630 bp Mouse anti-ALDH2anti-Bcl-2 anti-Bax and anti-120573-actin monoclonal antibodieswere purchased from Santa Cruz Biotechnology (CA) Goatanti-mouse secondary antibodies were fromBostonCo LtdWuhan China

23 Induction of Diabetes and Experimental Protocol Dia-betes was induced in overnight fasted rats by administeringa single intraperitoneal (ip) injection of 55mgkg strepto-zotocin (STZ) freshly dissolved in 01molL sodium citratebuffer (pH 45)The rats in control group were injected with asimilar volume of sodium citrate buffer aloneThe rats whosefasting blood glucose level was more than 167mmolL after72 h of injection were as diabetic [27] All rats were fed foreight weeks Animals were randomly divided into controldiabetes (DM) and DM + EtOH groups respectively In DM+ EtOH group DM rats were fed with 25 EtOH in theirdrinking water for one week to initiate drinking and thenchanged to 5EtOH continuous access through sevenweeks

24 Ischemia and Reperfusion and Drugs Intervention inIsolated Perfused Heart All rat hearts were subjected toregional ischemia and reperfusion intervention (IR) in vitroAfter the rats were anesthetized (chloral hydrate 4 gkgip) hearts were excised rapidly placed in ice-cold Krebs-Henseleit (K-H) buffer mounted on a Langendorff apparatusand perfused at 37∘C with K-H buffer The buffer wasequilibrated with 95 O25 CO2 (pH 74) and had thefollowing composition (mmolL) NaCl 1180 KCl 47 CaCl2125 KH2PO4 12 NaHCO3 250 and glucose 110 A latexfluid-filled balloon was introduced into the left ventriclethrough the left atrial appendage and the balloon catheterwas linked to a pressure transducer connected to a dataacquisition system (Medlab Nanjing China) to assess con-tractile function The left ventricular end diastolic pressure(LVEDP) was adjusted to 4sim8mmHg for 30min Myocardialinfarction (MI) was created by ligation of the left anteriordescending artery (LAD) MI was induced by LAD ligation2sim3mm from the origin with 5-0 silk suture Then 30min of

Oxidative Medicine and Cellular Longevity 3

C IR

DM IR

DM + EtOH + Atr IR

DM + EtOH + CYA IR

DM + EtOH + Wor IR

DM + EtOH IR

CYA

Atr

Wor

Ischemia Reperfusion

minus20 minus10 0 30 40 15025(min)

Figure 1 Protocol of various drugs intervention on rat myocardialischemiareperfusion model in vitro C IR control ischemia andreperfusion DM IR diabetes rats subjected tomyocardial ischemiaand reperfusion DM + EtOH IR diabetes + ethanol subjectedto myocardial ischemia and reperfusion DM + EtOH + CYA IRdiabetes + ethanol + cyanamide subjected to myocardial ischemiaand reperfusion DM + EtOH + Atr IR diabetes + ethanol +atractyloside subjected tomyocardial ischemia and reperfusionDM+ EtOH + Wor IR diabetes + ethanol + wortmannin subjected tomyocardial ischemia and reperfusion

regional myocardial ischemia (ie MI) followed by 120minof reperfusion was done in all rats The cardiac parametersincluding HR left ventricular developed pressure (LVDPdifference between left ventricular end systolic pressure andend diastolic pressure) maximal risefall rate of left ven-tricular pressure (plusmn119889119901119889119905max) left ventricular end diastolicpressure (LVEDP) and rate-pressure product (RPP LVDPtimes HR) were monitored continuously The rats were dividedinto control IR (119899 = 6) DM IR (119899 = 6) and DM + EtOHIR (119899 = 6) Other EtOH treated rats were subjected to threedifferent drugs intervention respectively (119899 = 6group) TheALDH2 inhibitor cyanamide (CYA) at 1mmolL was givenfrom 10min before ischemia to the first 10min of reperfusionwith total perfusing for 50min (DM + EtOH + CYA IR)the mitoPTP opener atractyloside (Atr) at 20mmolL wasgiven from the last 5min of ischemia to the early 10min ofreperfusion with total perfusing for 15min (DM + EtOH+ Atr IR) The inhibitor of PI3K 100 nmolL wortmannin(Wor) was given at the end ischemia for 5min and earlyreperfusion for 10min (DM + EtOH +Wor IR) (Figure 1)

25 Detection of LDH Release in Coronary Flow Coronaryflow was collected at 5min and 10min of reperfusion andLDH release was measured by commercially available kitsaccording to the manufacturerrsquos instructions

26 Detection ofMDAContent and SODActivity in Heart Tis-sue At the end of the experimental period 01 g heart tissuewas homogenized in ice-cold PBS buffer Malondialdehyde(MDA) content and superoxide dismutase (SOD) activitywere measured by commercially available kits according tothe manufacturerrsquos instructions

27 Detection of Cardiac ALDH2 Bax and Bcl-2 at mRNALevel by RT-PCR RT-PCR was used to detect the levelsof ALDH2 Bax and Bcl-2 mRNA expression in heartBriefly total RNAwas extracted with TRIzol according to themanufacturerrsquos instructions Two micrograms of total RNAwas reverse-transcribed to cDNA andPCRwas performedbya routinemethod PCRproducts were analyzed on 1 agarosegel Densitometry results for ALDH2 Bax and Bcl-2 genewere compared with corresponding 120573-actin levels to accountfor loading differences

28 Ultrastructure Observation of Myocardial Cell by Trans-mission Electron Microscope Cardiac tissue was dissectedand small pieces were fixed with 25 glutaraldehyde in 01molL cacodylate buffer for 1 h Ultrathin sections were cutand contrasted with uranylacetate buffer by lead citrate andobserved with JEM-1230 transmission electron microscope(JEOL Japan)

29 Cell Culture and Drug Treatment Rat neonatal car-diomyocytes were isolated from 1sim2 d old Sprague-Dawleyrats by digestion with trypsin Briefly ventricular tissue wasaseptically removed from neonatal rats minced and thendigested with mixed protease (025 trypsin 02 collage-nase II 100120583gmL DNA enzyme I and D-Hanks solution)(Solarbio Beijing China) at 37∘C in a shaking water bathThe cells were released after the first digestion was discardedwhereas the cells from subsequent digestion were added toan equal volume of Dulbeccorsquos modified Eaglersquos medium(DMEM) supplemented with 10 fetal bovine serum untilall cells were collected Cells were pelleted by centrifugationat 1000 rpm for 6min and resuspended in DMEM supple-mented 10 fetal bovine serum The isolated cells were firstplated in culture disks of 95 air and 5 CO2 at 37

∘C for 2 hto exclude nonmuscle cells The suspended cells were thencollected and plated at a density of 5 times 105 cellscm2 andmaintained under the same conditions as above The cellswere used for outlined experiments after 4sim5 days later

210 Immunofluorescence Analysis Cardiomyocytes culturedon coverslips were seeded in 6-well plates and fixed in 4paraformaldehyde in PBS for 15min at room temperaturefollowed by washing with PBS three times per 5min Thencardiomyocyteswere incubatedwith 01TritonX-100 in PBSfor 30min followed by washing with PBS three times per5min again After blocking with 1 bovine serum albuminin PBS for 1 h the cells were incubated in BSA blockingbuffer containing primary antibody-120572-SMA (1 200 BosterChina) and the cells were washed and then incubated insecondary anti-mouse antibody (1 200 Boster China) for1 h Furthermore cardiomyocytes were incubated for 15minwith 46-diamidino-2-phenylindole (DAPI ZSGB BeijingChina) for nuclear staining and fluorescence micrographswere obtained using Olympus FSX100 microscope

211 Grouping The cardiomyocytes were divided into 3groups as follows control (C) group HG group and Alda-1 + HG group In C group cardiomyocytes were incubated


in DMEMmedium including 55mmolL glucose for 48 h InHG group cardiomyocytes were incubated in high glucosemedium (25mmolL glucose) for 48 h and in Alda-1 + HGgroup cardiomyocytes were pretreated with HG and ALDH2activator Alda-1 (20 120583molL) for 48 h

212 Cell Counting Kit- (CCK-) 8 Assay Cardiomyocytes(100 120583Lwell) were seeded into 96-well plate at a densityof (05sim10) times 104 cellsmL and incubated at 37∘C 5 CO2overnight A CCK-8 assay kit was used to detect cell viabil-ities according to the manufacturerrsquos instructions Followingtreatment test samples and CCK-8 were added to eachwell 10 120583L respectively After 1sim4 h incubation cells viabilitywas determined by measuring the absorbance at 450 nmusing microplate reader The activity levels were determinedaccording to the manufacturerrsquos instructions

213 Detection of Cardiac and Cardiomyocyte ALDH2 ProteinExpression by Western Blot The ALDH2 protein in cardiactissue and cardiomyocyte was detected by western blot [1]Anti-ALDH2 (1 500) antibody was used Mouse anti-120573-actin antibody (1 500) was used as an internal control Theimmunoblots were exposed to X-ray film and analyzed witha digital image system

214 Statistical Analysis All values are expressed as meanplusmn SD Statistical comparisons were performed by one-wayanalysis of variance and the Newman-Keuls test Differencesof 119901 lt 005 were regarded as significant

3 Results

31 Changes of Ventricular Hemodynamic Parameters In C IR group comparedwith baseline LVDPplusmn119889119901119889119905max and RPPwere decreased and LVEDP was increased during ischemiaperiod during reperfusion period LVDP plusmn119889119901119889119905max andRPPwere lower and LVEDPwas higher During ischemia andreperfusion period in contrast to C IR rat in DM IR ratLVDP plusmn119889119901119889119905max and RPP were decreased and LVEDP wasincreased compared with DM IR group LVDP plusmn119889119901119889119905maxand RPP were increased and LVEDP was decreased in DM+ EtOH IR group compared with DM + EtOH IR groupin DM + EtOH + CYA IR group LVDP plusmn119889119901119889119905max andRPPwere furtherly decreased and LVEDPwas increased thechanges in DM + EtOH + Atr IR and DM + EtOH + WorIR groups were similar to DM + EtOH + CYA IR group(Table 1) There were no differences among the three groupsin DM + EtOH + cyanamide + IR versus DM + EtOH +wortmannin + IR versus DM + EtOH + atractyloside + IR

32 Changes of LDH Content The changes of LDH contentin different groups were shown in Table 2 In contrast to CIR group in DM IR group rats LDH release was increasedCompared with DM IR group LDH release was decreasedin DM + EtOH IR group Compared with DM + EtOH IRgroup LDH release was increased in DM+ EtOH + CYA IRDM + EtOH + Atr IR and DM + EtOH + Wor IR groups(Table 2)

33 Changes of MDA Content and SOD Activity in HeartTissue Compared with C IR group in DM IR group MDAcontent was increased and SOD activity was decreasedCompared with DM IR group in DM + EtOH IR groupMDA content was decreased accompanied with the increaseof SODactivity ComparedwithDM+EtOHIR groupMDAcontentwas increased and SODactivitywas decreased inDM+ EtOH + CYA IR DM + EtOH + Atr IR and DM + EtOH+Wor IR groups (Figure 2)

34 Change of Bax and Bcl-2 at mRNA Level in Heart Com-pared with C IR group Bax mRNA was increased Bcl-2 mRNA and the ratio of Bcl-2Bax were significantlydecreased in DM IR group Compared with DM IR groupin DM + EtOH IR group Bax mRNA was decreased Bcl-2mRNA and the ratio of Bcl-2Bax were increased Comparedwith DM + EtOH IR group Bax mRNA was decreased Bcl-2 mRNA and the ratio of Bcl-2Bax were decreased in DM +EtOH + CYA IR DM + EtOH + Atr IR and DM + EtOH +Wor IR groups (Figure 3)

35 Change of ALDH2 mRNA and Protein Level in HeartCompared with C IR group ALDH2 mRNA and proteinexpressions were significantly decreased in DM IR groupCompared with DM IR group ALDH2 mRNA and proteinexpressions were increased significantly in DM + EtOH IRgroup compared with DM + EtOH IR group ALDH2mRNA and protein expressions were decreased in DM +EtOH +CYA IR and DM+ EtOH +Atr IR and DM+ EtOH+Wor IR groups (Figures 4(a) 4(b) 4(c) and 4(d))

36 CCK-8 Assay and ALDH2 Protein Level in Cardiomy-ocyte Compared with C group cardiomyocyte activity andALDH2 protein expressions were significantly decreased inHGgroup ComparedwithHGgroup cardiomyocyte activityand ALDH2 protein expression were increased significantlyin Alda-1 + HG group (Figures 5(a) 5(b) and 5(c))

37 Ultrastructural Changes of Myocardial Cell In C IRgroup mitochondrial dense membrane was uncompletedmild swelling andmyocardial myofilament was fractured nolarge tracts of dissolution in DM IR group mitochondrialswellingwas aggravatedmyocardial cytoplasmicmuscle fiberwas ruptured and large areas of dissolution and vacuolationoccurred Compared with DM IR group in DM + EtOH IRgroup the degree of derangement of the cardiac myofilamentandmitochondrial swellingwas lessened comparedwithDM+ EtOH IR group in DM + EtOH + CYA IR DM + EtOH+ Atr IR and DM + EtOH +Wor IR groups the injury wasincreased (Figure 6)

4 Discussion

In the current study we mimicked myocardial ischemiareperfusion (IR) injury in diabetic rats Compared withC IR group LDH levels were increased cardiac systolicand diastolic dysfunction were aggravated as indicated bythe decreases of LVDP plusmn119889119901119889119905max and RPP and increases


Table 1 Hemodynamic parameters in the isolated perfused rat hearts subjected to ischemia and reperfusion (IR) (mean plusmn SD 119899 = 5)

Variable Baseline Ischemia Reperfusion30min 5min 10min 120min

LVDP ( of baseline )C IR 100 plusmn 0 59 plusmn 13 63 plusmn 13 65 plusmn 14 58 plusmn 4DM IR 100 plusmn 0 33 plusmn 7lowastlowast 42 plusmn 12lowast 48 plusmn 5lowastlowast 32 plusmn 5lowastlowast

DM + EtOH IR 100 plusmn 0 59 plusmn 15 93 plusmn 12lowastlowast 90 plusmn 7lowastlowast 58 plusmn 13

DM + EtOH + CYA IR 100 plusmn 0 36 plusmn 7lowastlowastandand 44 plusmn 8lowastlowastandand 44 plusmn 8lowastlowastandand 35 plusmn 9lowastlowastandand

DM + EtOH + Atr IR 100 plusmn 0 38 plusmn 8lowastlowastand 48 plusmn 8lowastandand 47 plusmn 8lowastlowastandand 30 plusmn 4lowastlowastandand

DM + EtOH +Wor IR 100 plusmn 0 39 plusmn 6lowastlowastandand 47 plusmn 8lowastandand 44 plusmn 7lowastlowastandand 32 plusmn 3lowastlowastandand

LVEDP ( of baseline)C IR 100 plusmn 0 101 plusmn 1 116 plusmn 2 120 plusmn 10 177 plusmn 11DM IR 100 plusmn 0 151 plusmn 15lowastlowast 177 plusmn 14lowastlowast 159 plusmn 9lowastlowast 262 plusmn 27lowastlowast



DM + EtOH + Atr IR 100 plusmn 0 147 plusmn 20lowastlowastandand 166 plusmn 3lowastlowastandand 166 plusmn 5lowastlowastandand 251 plusmn 16lowastlowastandand

DM + EtOH +Wor IR 100 plusmn 0 168 plusmn 19lowastlowastandand 176 plusmn 7lowastlowastandand 170 plusmn 11lowastlowastandand 281 plusmn 45lowastlowastandand

+119889119901119889119905max ( of baseline)C IR 100 plusmn 0 64 plusmn 10 80 plusmn 15 78 plusmn 12 55 plusmn 8DM IR 100 plusmn 0 48 plusmn 8 43 plusmn 9lowastlowast 46 plusmn 5lowastlowast 34 plusmn 6lowastlowast

DM + EtOH IR 100 plusmn 0 60 plusmn 14 91 plusmn 17 91 plusmn 19 62 plusmn 11

DM + EtOH + CYA IR 100 plusmn 0 47 plusmn 7 46 plusmn 6lowastlowastandand 47 plusmn 6lowastlowastandand 32 plusmn 7lowastlowastandand

DM + EtOH + Atr IR 100 plusmn 0 48 plusmn 6 45 plusmn 2lowastlowastandand 47 plusmn 4lowastlowastandand 34 plusmn 7lowastlowastandand

DM + EtOH +Wor IR 100 plusmn 0 49 plusmn 9 43 plusmn 6lowastlowastandand 44 plusmn 4lowastlowastandand 33 plusmn 5lowastlowastandand

minus119889119901119889119905max ( of baseline)C IR 100 plusmn 0 59 plusmn 13 59 plusmn 10 59 plusmn 1 41 plusmn 5DM IR 100 plusmn 0 42 plusmn 9lowastlowast 42 plusmn 6lowastlowast 46 plusmn 9lowastlowast 31 plusmn 4lowastlowast

DM + EtOH IR 100 plusmn 0 62 plusmn 5 85 plusmn 10lowastlowast 84 plusmn 12lowastlowast 48 plusmn 6lowast

DM + EtOH + CYA IR 100 plusmn 0 45 plusmn 4lowastandand 46 plusmn 7lowastandand 44 plusmn 4lowastlowastandand 35 plusmn 4lowastandand

DM + EtOH + Atr IR 100 plusmn 0 43 plusmn 5lowastlowastandand 49 plusmn 5lowastandand 45 plusmn 4lowastlowastand 33 plusmn 4lowastlowastandand

DM + EtOH +Wor IR 100 plusmn 0 46 plusmn 4lowastand 44 plusmn 5lowastlowastandand 43 plusmn 4lowastlowastandand 30 plusmn 5lowastlowastandand

RPP ( of baseline)C IR 100 plusmn 0 59 plusmn 6 62 plusmn 17 62 plusmn 8 33 plusmn 3DM IR 100 plusmn 0 26 plusmn 7lowastlowast 32 plusmn 8lowastlowast 28 plusmn 7lowastlowast 18 plusmn 4lowastlowast

DM + EtOH IR 100 plusmn 0 55 plusmn 13 76 plusmn 7lowast 79 plusmn 19lowast 37 plusmn 6



DM + EtOH +Wor IR 100 plusmn 0 32 plusmn 8lowastlowastandand 30 plusmn 3lowastlowastandand 25 plusmn 4lowastlowastandand 18 plusmn 2lowastlowastandandlowast119901 lt 005 lowastlowast119901 lt 001 versus C IR 119901 lt 001 versus DM IR and119901 lt 005 andand119901 lt 001 versus DM + EtOH IR

Table 2 Effect of different drugs on LDH release in the coronary effluent in the isolated rat hearts subjected to ischemia and reperfusion(IR) (mean plusmn SD 119899 = 5)

Group 5min (UL) 10min (UL)C IR 6608 plusmn 760 6050 plusmn 693DM IR 15458 plusmn 1650lowastlowast 12958 plusmn 2223lowastlowast

DM + EtOH IR 7792 plusmn 1162 6158 plusmn 563

DM + EtOH + CYA IR 13983 plusmn 2041lowastlowastandand 10792 plusmn 2742lowastlowastandand

DM + EtOH + Atr IR 13717 plusmn 2214lowastlowastandand 11267 plusmn 2402lowastlowastandand

DM + EtOH +Wor IR 13625 plusmn 2942lowastlowastandand 11375 plusmn 1840lowastlowastandandlowastlowast119901 lt 001 versus C IR 119901 lt 001 versus DM IR andand119901 lt 001 versus DM + EtOH IR


lowastlowast

lowastlowast

lowast

lowastlowast

lowastlowastandand

andandandand

C IRDM IRDM + EtOH IR

DM + EtOH + CYA IRDM + EtOH + Atr IRDM + EtOH + Wor IR

0

1

2

3

4

5M

DA

(nm

olm

g-pr

ot)

(a)

SOD

(Um

g-pr

ot) lowastlowast

lowastlowastlowastlowast lowastlowast

lowastlowastandandandand

andand



0

5

10

15

(b)

Figure 2 The levels of MDA (a) and SOD (b) of rats myocardium in each group (mean plusmn SD 119899 = 5) lowast119901 lt 005 lowastlowast119901 lt 001 versus C IR119901 lt 001 versus DM IR andand119901 lt 001 versus DM + EtOH IR

of LVEDP cardiac oxidative stress injury was aggravatedas indicated by the decrease of SOD activity and increaseof MDA content and the expression of Bcl-2 mRNA wasdecreased while Bax mRNA was increased also indicatingthat myocyte apoptosis was aggravated Meanwhile we alsofound that myocardial ultrastructure was damaged in DMIR group Myocardial ALDH2 at mRNA and protein levelswere decreased in DM IR group When diabetes rats werepretreated with low concentration of EtOH which was usedas a tool to induce ALDH2 activity LDH level was decreasedwhereas LVDPplusmn119889119901119889119905max RP SODactivity andBcl-2mRNAlevel were increasedMDA content and BaxmRNA level weredecreased Also the injury to myocardial ultrastructure wasattenuated The effect of EtOH was associated with increasedmyocardial ALDH2 mRNA and protein levels When thediabetic rats were treated with CYA Wor or Atr afterEtOH intervention the injury was increased and ALDH2activity was reduced and LDH level was decreased whereasLVDP plusmn119889119901119889119905max RPP SOD activity and Bcl-2 mRNAlevel were increased MDA content and Bax mRNA levelswere decreased At the same time the injury to myocardialultrastructure was aggravated in DM + EtOH + CYA IRgroup and the changes in DM + EtOH + Atr IR group andDM + EtOH + Wor IR group were similar to DM + EtOH+ CYA IR group These results suggested that decreases inALDH2 expression may play a key role in myocardial IRinjury in diabetic rats Upregulation of ALDH2 can protectthe heart against myocardial IR injury and the mechanismmaybe through activation of PI3K-Akt signaling pathway andinhibiting mitoPTP opening

Cardiovascular complications remain the leading cause ofdiabetes related mortality and morbidityThe oxidative stressof diabetes can increase myocardial IR injury myocardialIR injury produced excessive amounts of reactive oxygenspecies and increasingly aggravated the injury of myocardialreperfusion Several reports in recent years had discussed theassociation between ALDH2 and myocardial injury Over-expression of ALDH2 offers myocardial protection maybeagainst alcohol-induced cardiac tissue and cellular injuryEnhanced ALDH2 activity by the direct effect of ALDH2activator-1 or ethanol preconditioning led to cardiac pro-tection against IR injury on the other hand myocardialIR injury may be exacerbated after ALDH2 knockout inmice [28 29] Earlier findings from our group also indicatedthat activation of ALDH2 with ethanol attenuated diabetes-induced myocardial injury in rats [27] However there arefew researches on the relationship of myocardial ALDH2 anddiabetes myocardial ischemiareperfusion injury Recent evi-dence revealed that ALDH2preventedROS-induced vascularcontraction in angiotensin-II induced hypertensive mice[30] ALDH2 overexpression attenuated hyperoxia-inducedcell death in lung epithelial cells through reduction of ROSactivation of ERKMAPK and PI3K-Akt signaling pathways[31]Thus far there have been no reports about the changes ofALDH2 in diabetesmyocardial IR injury and the underlyingcellular mechanisms are not clear

Therefore in the present study we firstly sought to deter-mine whether ALDH2 expression also changed in diabetesmyocardial IR injury model In previous study we observedthat with the progression of diabetes myocardial ALDH2


M 1 65432

Bcl-2

M 1 65432

Bax

lowastlowastlowastlowastandand

lowastlowastandand

lowastlowastandand

0

1

2

Bax

Bcl-2

mRN

A



120573-Actin

120573-Actin

Figure 3 The expressions of Bcl-2 and Bax mRNA of heart tissuein rats (mean plusmn SD of 5 separate experiments) lowastlowast119901 lt 001 versus CIR 119901 lt 001 versus DM IR andand119901 lt 001 versus DM + EtOH IR(1) C IR (2) DM IR (3) DM + EtOH IR (4) DM + EtOH + CYAIR (5) DM + EtOH + Atr IR (6) DM + EtOH +Wor IR

expression was further decreased accompanying decreasedventricular function and activation of ALDH2 can decreasediabetes- induced myocardial injury In the present studywe used diabetic IR model and observed cardiac systolicand diastolic function was destroyed and myocardial ultra-structure was damaged accompanied with the aggravationof oxidative stress At the same time myocardial ALDH2 atmRNA and protein levels were decreased and LDH releasein the coronary effluent was increased It is suggested that theexpression of ALDH2 was related to the cardiac IR injuryinduced by diabetes

ALDH2 as an antioxidant enzyme may be easily inac-tivated by free radicals Recently ALDH2 was identified asa target for oxidative modification during glyceryl trini-trate tolerance [32 33] and ALDH2 activity was correlatedinversely with cardiac infarct size in rat hearts subjectedto ischemia and reperfusion ex vivo [34] We used low

concentration of EtOH as a tool to induce ALDH2 activity toinvestigate whether upregulation of ALDH2 expression couldbenefit recovery from myocardial function it is likely thatincreasing ALDH2 expression can attenuate the happeningof oxidative stress and the destroying of myocardial functionThese findings suggest that compared with diabetic IRgroup ALDH2 overexpression can exert the protective effectagainst cardiac IR injury of diabetes However the ALDH2inhibitor CYA which was involved in ALDH2 conferredprotection against cardiac IR injury and LDH release wasincreased and the expressions of ALDH2 at mRNA andprotein level were decreased The result showed that ALDH2overexpression reconciled diabetes-induced contractile dys-function whereas cardioprotection elicited by ALDH2 wasblocked by the inhibitor CYA Further study is required tobetter elucidate the mechanism of ALDH2 Mitochondrialpermeability transition pore (mitoPTP) is a nonselectivehighly conductive channel which exists between mitochon-drial inner and outer membranes playing an important rolein apoptosis The opening of mitoPTP is an ultimate target ofall kinds of damage ischemiareperfusion induced mitoPTPto open and also induced the overload of intracellularcalcium accumulation of ROS and increase in PH and otherfactors [35] Argaud et al reported in the rabbit myocardialischemia model that when the rabbits were given precon-ditioning postconditioning and mitoPTP inhibitor respec-tively during reperfusion the effects were similar in reducingmyocardial infarct size and the tolerance of myocardialmitochondria was decreased for calcium overload in IRinjury However the myocardial mitochondria resistancewas increased after postconditioning or mitoPTP inhibitortreatment [36]These studies indicated that postconditioningmay be via inhibiting the opening of mitoPTP resulting inreducing the myocardial damage by overload of calcium It issuggested that the myocardial protection of preconditioningand postconditioning was involved in suppression of openmitoPTP In the present paper we further investigate therole of ALDH2 in the diabetic rat hearts In our study weused the specific openers of mitoPTP atractyloside (Atr)Theresult suggested that Atr canceled the protection of EtOHindeed we found the ALDH2 mRNA and protein levelswere reduced meanwhile cardiac LV contractile functionwas declining and myocardial ultrastructure was damagedaccompanied with the aggravation of oxidative stress Zhouet al reported on hydrogen peroxide-induced myocardialinjury model Low concentration of ethanol can inhibit theopening of mitoPTP induced by oxidative stress via GSK-3120573 pathway and PI3KAkt however high concentration ofethanol-fed rats can induce the cell apoptosis and openingof mitoPTP [37 38] These results displayed that promotingthe opening of mitoPTP might be via inhibiting ALDH2generation against its protection

It is well known that PI3KAkt signaling is a keymediatorof cell survival Akt is also implicated in the cardioprotectionfor cardiac myocyte survival [39] However the role of Aktsignaling in regulation of diabetic rat myocardial IR injuryis not clear Activation of Akt was found to rescue thecardiacmechanical function defect induced by ER stress [40]In the study we used the PI3KAkt inhibitor wortmannin


M 1 2 3 4 5 6

ALDH2

120573-Actin

(a)

lowastlowast

lowastlowast

andand lowastlowastandand

lowastlowastandand

00

01

02

03

04

05

06

07

08

09

10

11

ALD

H2

120573-a

ctin

at m

RNA

leve

l



(b)

1 2 3 4 5 6

ALDH2

120573-Actin

(c)

lowastlowastlowastlowast andand lowastlowast

andandlowastlowastandand

ALD

H2

120573-a

ctin

at p

rote

in le

vel

00

05

10

15


DM + EtOH + Atr IRDM + EtOH + CYA IR

DM + EtOH + Wor IR(d)

Figure 4 Expressions of ALDH2 at mRNA (a b) and protein (c d) level of heart tissue in rats (mean plusmn SD of 5 separate experiments)lowastlowast119901 lt 001 versus C IR 119901 lt 001 versus DM IR andand119901 lt 001 versus DM + EtOH IR (1) C IR (2) DM IR (3) DM + EtOH IR (4) DM +EtOH + CYA IR (5) DM + EtOH + Atr IR (6) DM + EtOH +Wor IR

(Wor) compared with DM + EtOH IR group ALDH2mRNAand protein levels were decreasedmeanwhile cardiacLV contractile function and myocardial ultrastructure weredamaged accompanied with the aggravation of oxidativestress Data depicted that ALDH2 induced cell survival wasblocked by inhibition of PI3K These results suggested thatthe protection of ALDH2 was at least partially mediated by

PI3KAkt pathway However further study is required tobetter elucidate themechanismofALDH2 cardiac protection

IR injury is a complex process and results in oxidativestress in myocardium such as O2

minus and H2O2 accumulatingand resulting in cellular toxicity finally due to imbalancebetween production and removal of ROS The survivalmyocardial cells after myocardial infarction were more prone


120572-SMA staining DAPI staining Merged (times200)

(a)

lowastlowast

lowastlowast

00

05

10

15

Card

iom

yocy

te ac

tivity

C Alda-1 + HGHG(b)

C HG Alda-1

A

LDH2

120573-a

ctin

pro

tein

leve

l

lowast

lowast

ALDH2

120573-Actin

C HG Alda-1 + HG0

500

1000

1500

2000

2500

(c)

Figure 5 Identify cardiomyocytes by immunofluorescence (a) CCK-8 activity (b) and expressions of ALDH2 protein level of cardiomyocytein rats (c) (mean plusmn SD of five separate experiments) lowast119901 lt 005 lowastlowast119901 lt 001 versus C 119901 lt 005 and 119901 lt 001 versus HG

to calcium overload and therefore more susceptible to IRinjury accumulating evidence proved that overproduction ofROS triggered myocyte apoptosis by upregulating proapop-totic members of Bcl-2 family In addition investigators haveshown that Bcl-2 gene family is known to modulate thepermeability of the mitochondrial membrane and the releaseof cytochrome C [41] Therefore we investigated whether theexpressions of Bax and Bcl-2 genes are related to ALDH2induced protection in diabetic myocardial IR injury In ourstudy we detected Bax and Bcl-2 at mRNA levels The resultshowed that the expression of cardiac Bcl-2 at mRNA levelwas decreased the expression of Bax at mRNA level wasincreased Bcl-2Bax mRNAwas decreased in DM IR groupand LDH release in the coronary effluent of diabetic ratswas increased Meanwhile ALDH2 mRNA and protein weredecreased However compared with DM IR group the ratioof Bax to Bcl-2 mRNA and LDH levels were decreased inDM + EtOH IR group and the expressions of ALDH2mRNA and protein were increased which suggested that the

increase of myocardial cells apoptosis may be associated withthe decrease of ALDH2 To further prove our hypothesisvia given drug intervention ALDH2 inhibitor CYA PI3Kinhibitor Wor and the opener of mitoPTP Atr the resultsuggests the ratio of Bax to Bcl-2 mRNA was decreasedmeanwhile ALDH2 levels were decreased accompanied withthe changes of cardiac LV contractile function oxidativestress and myocardial ultrastructure

To verify whether cardiac ALDH2 directly participates indiabetes-induced cardiomyocyte injury in this study we alsoapplied high glucose induced cardiomyocyte injury to mimicdiabetesWe found that in cardiomyocytes model comparedwith C group CCK-8 activity and theALDH2 protein expres-sionwere decreased inHG group when cardiomyocytes werepretreatedwithAlda-1 the specific activator ofALDH2 inHGcondition CCK-8 activity was increased accompanied withthe increase of ALDH2 protein expression It is suggested thatdirect activation of cardiomyocyte ALDH2 attenuated highglucose induced myocardial injury It is worthwhile to note


(a) C IR (b) DM IR

(c) DM + EtOH IR (d) DM + EtOH + CYA IR

(e) DM + EtOH + Atr IR (f) DM + EtOH +Wor IR

Figure 6 Changes of rat myocardium ultrastructural organization in each group (magnification 10K)

that there were some limitations in our study In cardiomy-ocyte model we only observed the protection of enhancedALDH2 expression in HG condition To better understandthe mechanism involved we will investigate the effect ofcardiomyocytes ALDH2 in hypoxia and reoxygenation statusand further validate our hypothesis

In conclusion activation of ALDH2 participates in theregulation of diabetic IR injury induced cardiac myocyteapoptosis via mitochondrial pathway ALDH2 may play anantiapoptotic effect through decreasing the ratio of BaxBcl-2 activation of PI3KAkt signaling pathway and inhibit-ing mitoPTP opening Promoting ALDH2 expression may


become a new pathway for the clinical treatment DM withCHD patients and further studies are necessary to confirmthis hypothesis

Competing Interests

The authors declare that they have no competing interests

Authorsrsquo Contributions

Pin-FangKang andWen-JuanWu contributed equally to thiswork

Acknowledgments

This work was supported by research grants from NationalChina Natural Science Foundation (nos 81000074 and81550036) and from Anhui Province Natural Science Foun-dation (no 1508085MH169) and Anhui Province EducationKey Projects (no KJ2016A484) China and Key Project ofTop-Notch Talent of Discipline (specialty) of the higher Edu-cation Institute of Anhui Province 2016 (no gxbjZD2016072)China

References

[1] E Adeghate P Schattner and E Dunn ldquoAn update on theetiology and epidemiology of diabetes mellitusrdquo Annals of theNew York Academy of Sciences vol 1084 pp 1ndash29 2006

[2] C Gremizzi A Vergani V Paloschi and A Secchi ldquoImpact ofpancreas transplantation on type 1 diabetes-related complica-tionsrdquo Current Opinion in Organ Transplantation vol 15 no 1pp 119ndash123 2010

[3] S Boudina and E D Abel ldquoDiabetic cardiomyopathy revisitedrdquoCirculation vol 115 no 25 pp 3213ndash3223 2007

[4] M Kooistra M I Geerlings W P T M Mali K L Vincken YVan Der Graaf and G J Biessels ldquoDiabetes mellitus and pro-gression of vascular brain lesions and brain atrophy in patientswith symptomatic atherosclerotic disease The SMART-MRstudyrdquo Journal of the Neurological Sciences vol 332 no 1-2 pp69ndash74 2013

[5] Y Zhang and J Ren ldquoALDH2 in alcoholic heart diseasesmolecular mechanism and clinical implicationsrdquo PharmacologyandTherapeutics vol 132 no 1 pp 86ndash95 2011

[6] J Ren ldquoAcetaldehyde and alcoholic cardiomyopathy lessonsfrom the ADH and ALDH2 transgenic modelsrdquo NovartisFoundation Symposium vol 285 pp 69ndash76 2007

[7] G R Budas M-H Disatnik and D Mochly-Rosen ldquoAldehydedehydrogenase 2 in cardiac protection a new therapeutictargetrdquo Trends in Cardiovascular Medicine vol 19 no 5 pp158ndash164 2009

[8] H J Forman J M Fukuto T Miller H Zhang A Rinnaand S Levy ldquoThe chemistry of cell signaling by reactiveoxygen and nitrogen species and 4-hydroxynonenalrdquo Archivesof Biochemistry and Biophysics vol 477 no 2 pp 183ndash195 2008

[9] D R Petersen and J A Doorn ldquoReactions of 4-hydroxynonenalwith proteins and cellular targetsrdquo Free Radical Biology andMedicine vol 37 no 7 pp 937ndash945 2004

[10] H Ma J Li F Gao and J Ren ldquoAldehyde dehydrogenase 2ameliorates acute cardiac toxicity of ethanol role of protein

phosphatase and forkhead transcription factorrdquo Journal of theAmerican College of Cardiology vol 54 no 23 pp 2187ndash21962009

[11] T A Doser S Turdi D P Thomas P N Epstein S-Y Li andJ Ren ldquoTransgenic overexpression of aldehyde dehydrogenase-2 rescues chronic alcohol intake-induced myocardial hypertro-phy and contractile dysfunctionrdquoCirculation vol 119 no 14 pp1941ndash1949 2009

[12] W Ge R Guo and J Ren ldquoAMP-dependent kinase andautophagic flux are involved in aldehyde dehydrogenase-2-induced protection against cardiac toxicity of ethanolrdquo FreeRadical Biology and Medicine vol 51 no 9 pp 1736ndash1748 2011

[13] E N Churchill M H Disatnik and D Mochly-RosenldquoTime-dependent and ethanol-induced cardiac protection fromischemia mediated by mitochondrial translocation of varep-silonPKC and activation of aldehyde dehydrogenase 2rdquo JournalofMolecular and Cellular Cardiology vol 46 no 2 pp 278ndash2842009

[14] C-H Chen G R Budas E N Churchill M-H DisatnikT D Hurley and D Mochly-Rosen ldquoActivation of aldehydedehydrogenase-2 reduces ischemic damage to the heartrdquo Sci-ence vol 321 no 5895 pp 1493ndash1495 2008

[15] G R Budas M-H Disatnik C-H Chen and D Mochly-Rosen ldquoActivation of aldehyde dehydrogenase 2 (ALDH2)confers cardioprotection in protein kinase C epsilon (PKC120576)knockout micerdquo Journal of Molecular and Cellular Cardiologyvol 48 no 4 pp 757ndash764 2010

[16] J Wang H Wang P Hao et al ldquoInhibition of aldehydedehydrogenase 2 by oxidative stress is associated with cardiacdysfunction in diabetic ratsrdquoMolecular Medicine vol 17 no 3-4 pp 172ndash179 2011

[17] H-J Wang P-F Kang W-J Wu et al ldquoChanges in cardiacmitochondrial aldehyde dehydrogenase 2 activity in relationto oxidative stress and inflammatory injury in diabetic ratsrdquoMolecular Medicine Reports vol 8 no 2 pp 686ndash690 2013

[18] S-B Ong S Subrayan S Y Lim D M Yellon S M Davidsonand D J Hausenloy ldquoInhibiting mitochondrial fission protectsthe heart against ischemiareperfusion injuryrdquo Circulation vol121 no 18 pp 2012ndash2022 2010

[19] A Rasola and P Bernardi ldquoThe mitochondrial permeabilitytransition pore and its involvement in cell death and in diseasepathogenesisrdquo Apoptosis vol 12 no 5 pp 815ndash833 2007

[20] D B Zorov M Juhaszova Y Yaniv H B Nuss S Wang and SJ Sollott ldquoRegulation and pharmacology of the mitochondrialpermeability transition porerdquo Cardiovascular Research vol 83no 2 pp 213ndash225 2009

[21] C Wang and R J Youle ldquoThe role of mitochondria in apopto-sisrdquo Annual Review of Genetics vol 43 pp 95ndash118 2009

[22] S M Davidson D Hausenloy M R Duchen and D MYellon ldquoSignalling via the reperfusion injury signalling kinase(RISK) pathway links closure of themitochondrial permeabilitytransition pore to cardioprotectionrdquo International Journal ofBiochemistry and Cell Biology vol 38 no 3 pp 414ndash419 2006

[23] S Y Lim S M Davidson D J Hausenloy and D M YellonldquoPreconditioning and postconditioning the essential role ofthemitochondrial permeability transition porerdquoCardiovascularResearch vol 75 no 3 pp 530ndash535 2007

[24] ZH Li C R JiangM L Xia et al ldquoActivation ofmitochondrialaldehyde dehydrogenase 2 and inhibition of mitochondrialpermeability transition pore involved in cardioprotection ofethanol postconditioningrdquo Zhe Jiang Da Xue Xue Bao Yi XueBan vol 39 no 6 pp 566ndash571 2010


[25] J Liao A Sun Y Xie et al ldquoAldehyde dehydrogenase-2 defi-ciency aggravates cardiac dysfunction elicited by endoplasmicreticulum stress inductionrdquo Molecular Medicine vol 18 no 5pp 785ndash793 2012

[26] E K Iliodromitis C Gaitanaki A Lazou et al ldquoDifferentialactivation of mitogen-activated protein kinases in ischemicand nitroglycerin-induced preconditioningrdquo Basic Research inCardiology vol 101 no 4 pp 327ndash335 2006

[27] Q Gao H-J Wang X-M Wang et al ldquoActivation of ALDH2with ethanol attenuates diabetes induced myocardial injury inratsrdquo Food and Chemical Toxicology vol 56 pp 419ndash424 2013

[28] N S Chandel D S McClintock C E Feliciano et al ldquoReactiveoxygen species generated at mitochondrial complex III stabilizehypoxia-inducible factor-1120572during hypoxia amechanismofO2sensingrdquoThe Journal of Biological Chemistry vol 275 no 33 pp25130ndash25138 2000

[29] R Aikawa M Nawano Y Gu et al ldquoInsulin prevents car-diomyocytes from oxidative stress-induced apoptosis throughactivation of PI3 kinaseAktrdquo Circulation vol 102 no 23 pp2873ndash2879 2000

[30] H Choi R C Lee H Y Wong et al ldquoLipid peroxidationdysregulation in ischemiac strokeplasma 4-HNE as a potentialbio-markerrdquo Biochemical and Biophysical Research Communi-cations vol 425 no 4 pp 842ndash847 2012

[31] D Xu J R Guthrie S Mabry T M Sack and W E TruogldquoMitochondrial aldehyde dehydrogenase attenuates hyperoxia-induced cell death through activation of ERKMAPK andPI3K-Akt pathways in lung epithelial cellsrdquo American Journal ofPhysiologymdashLung Cellular and Molecular Physiology vol 291no 5 pp L966ndashL975 2006

[32] K Sydow A Daiber M Oelze et al ldquoCentral role of mito-chondrial aldehyde dehydrogenase and reactive oxygen speciesin nitroglycerin tolerance and cross-tolerancerdquo The Journal ofClinical Investigation vol 113 no 3 pp 482ndash489 2004

[33] P Wenzel U Hink M Oelze et al ldquoRole of reduced lipoicacid in the redox regulation of mitochondrial aldehyde dehy-drogenase (ALDH-2) activity implications for mitochondrialoxidative stress and nitrate tolerancerdquo The Journal of BiologicalChemistry vol 282 no 1 pp 792ndash799 2007

[34] W Ge and J Ren ldquoMTOR-STAT3-notch signalling contributesto ALDH2-induced protection against cardiac contractile dys-function and autophagy under alcoholismrdquo Journal of Cellularand Molecular Medicine vol 16 no 3 pp 616ndash626 2012

[35] F Di Lisa and P Bernardi ldquoMitochondria and ischemia-reperfusion injury of the heart fixing a holerdquo CardiovascularResearch vol 70 no 2 pp 191ndash199 2006

[36] L Argaud O Gateau-Roesch O Raisky J Loufouat D Robertand M Ovize ldquoPostconditioning inhibits mitochondrial per-meability transitionrdquo Circulation vol 111 no 2 pp 194ndash1972005

[37] K Zhou L Zhang J Xi W Tian and Z Xu ldquoEthanol preventsoxidant-induced mitochondrial permeability transition poreopening in cardiac cellsrdquo Alcohol and Alcoholism vol 44 no1 pp 20ndash24 2009

[38] A L King T M Swain D A Dickinson M J Lesort and SM Bailey ldquoChronic ethanol consumption enhances sensitivityto Ca2+-mediated opening of the mitochondrial permeabilitytransition pore and increases cyclophilin D in liverrdquo AmericanJournal of PhysiologymdashGastrointestinal and Liver Physiologyvol 299 no 4 pp G954ndashG966 2010

[39] T L Yuan G Wulf L Burga and L C Cantley ldquoCell-to-cellvariability in PI3K protein level regulates PI3K-AKT pathway

activity in cell populationsrdquo Current Biology vol 21 no 3 pp173ndash183 2011

[40] Y Zhang and J Ren ldquoThapsigargin triggers cardiac contractiledysfunction via NADPH oxidase-mediated mitochondrial dys-function role of Akt dephosphorylationrdquo Free Radical Biologyand Medicine vol 51 no 12 pp 2172ndash2184 2011

[41] RMKluck E Bossy-Wetzel D R Green andDDNewmeyerldquoThe release of cytochrome c frommitochondria a primary sitefor Bcl-2 regulation of apoptosisrdquo Science vol 275 no 5303 pp1132ndash1136 1997


ALDH2 by oxidative stress leads to cardiac dysfunction indiabetes mellitus [16] What changes of ALDH2 expressionthat appeared and what mechanisms involved in diabetic ratmyocardial IR injury spark our interest

At the beginning of IR the signal transduction pathwayswere activated by various means of myocardial protec-tion proactively especially phosphatidylinositol-3-kinase ser-inethreonine kinase (PI3K-Akt) PI3K-Akt signaling path-way is an important pathway which regulates cell prolifera-tion cell division apoptosis and other activities Heart wasprotected by ischemic preconditioning via activated PI3K-Akt signaling pathway however the effect of myocardialprotection was abolished by PI3K inhibitor wortmannin Itis suggested that PI3K-Akt signal transduction pathway wasinvolved in myocardial protection

Mitochondrial permeability transition pore (mitoPTP)is located in the inner and outer mitochondrial membranemultiprotein complexes which plays an important role inmaintainingmitochondrial membrane potential and protect-ing the structure and function of mitochondrion [17 18]Under physiological condition mitoPTP is closed and it hasalmost no permeability for all mitochondrial metabolites andions Opening of mitoPTP is involved in cell death inducedby a variety of causes for example IR endotoxin andanticancer agents [19] Opening mitoPTP leads to mitochon-drial swelling and outer membrane rupture the proapoptoticfactors and cytochrome C were released into the cytoplasminducing energymetabolismunbalance intracellular calciumoverload and exacerbated ischemic cell damageThe openingof mitoPTP in reperfusion is a sign of myocardial injury fromreversible to irreversible [20 21] mitoPTP is considered tobe the terminal effector of cell death in IR injury whiledelaying or blocking the opening of mitoPTP may reduceIR injury [22 23] A large number of reports suggestedthat ischemic preconditioning ischemic postconditioningand drug pretreatment could protect heart against injury byinhibiting of the opening of mitoPTP [24ndash26]

Based on above background we were prompted to inves-tigate the effects of pretreatment with ethanol (EtOH) as atool to induce ALDH2 activity in myocardial IR injury ofdiabetic rats and give drug intervention including ALDH2inhibitor cyanimide PI3K inhibitor wortmannin and theopener of mitoPTP atractyloside to determine its underlyingmechanisms

2 Materials and Methods

21 Animals Male Sprague-Dawley rats (200sim250 g) werepurchased from the Animal Center of Bengbu MedicalCollege Anhui The rats were fed normal chow and hadfree access to water Housing was at a constant temperatureof (21 plusmn 1)∘C with a fixed 12 h lightdark cycle All animalprocedures were in accordance with United States NationalInstitutes of Health Guide and were approved by the AnimalUse and Care Committee of Bengbu Medical College

22 Chemicals and Reagents Streptozotocin (STZ) cyana-mide (CYA) wortmannin (Wor) atractyloside (Atr) and

Alda-1 were purchased from Sigma (St Louis MO USA)Ethanol (EtOH) was obtained from Bengbu New ChemicalReagent Factory China 10 fetal bovine serumwas obtainedfrom Zhe Jiang Tianhang Biological corporation China Lac-tate dehydrogenase (LDH) Malondialdehyde (MDA) andsuperoxide dismutase (SOD) assay kits were purchased fromNanjing Jiancheng Bioengineering Institute China CCK-8assay kit was from Shanghai Bestbio Life Technology ChinaThe primers used were as follows for ALDH2 forward 51015840-GTG TTC GGA GAC GTC AAA GA-31015840 and reverse 51015840-GCAGAG CTT GGG ACA GGT AA-31015840 the amplified fragmentlengthwas 187 bp for Bcl-2 forward 51015840-CTGGTGGACAACATCGCTCTG-31015840 and reverse 51015840-GGTCTGCTGACCTCACTT GTG-31015840 the amplified fragment length was 227 bp forBax forward 51015840-GGA TCG AGC AGA GAG GAT GG-31015840and reverse 51015840-GCT CAT TGC CGA TAG TGA TGA CT-31015840 the amplified fragment length was 464 bp for 120573-actinforward 51015840-GAT GGT GGG TAT GGG TCA GAA-31015840 andreverse 51015840-GGC CAT CTC TTG CTC GAA GTC-31015840 theamplified fragment length was 630 bp Mouse anti-ALDH2anti-Bcl-2 anti-Bax and anti-120573-actin monoclonal antibodieswere purchased from Santa Cruz Biotechnology (CA) Goatanti-mouse secondary antibodies were fromBostonCo LtdWuhan China

23 Induction of Diabetes and Experimental Protocol Dia-betes was induced in overnight fasted rats by administeringa single intraperitoneal (ip) injection of 55mgkg strepto-zotocin (STZ) freshly dissolved in 01molL sodium citratebuffer (pH 45)The rats in control group were injected with asimilar volume of sodium citrate buffer aloneThe rats whosefasting blood glucose level was more than 167mmolL after72 h of injection were as diabetic [27] All rats were fed foreight weeks Animals were randomly divided into controldiabetes (DM) and DM + EtOH groups respectively In DM+ EtOH group DM rats were fed with 25 EtOH in theirdrinking water for one week to initiate drinking and thenchanged to 5EtOH continuous access through sevenweeks

24 Ischemia and Reperfusion and Drugs Intervention inIsolated Perfused Heart All rat hearts were subjected toregional ischemia and reperfusion intervention (IR) in vitroAfter the rats were anesthetized (chloral hydrate 4 gkgip) hearts were excised rapidly placed in ice-cold Krebs-Henseleit (K-H) buffer mounted on a Langendorff apparatusand perfused at 37∘C with K-H buffer The buffer wasequilibrated with 95 O25 CO2 (pH 74) and had thefollowing composition (mmolL) NaCl 1180 KCl 47 CaCl2125 KH2PO4 12 NaHCO3 250 and glucose 110 A latexfluid-filled balloon was introduced into the left ventriclethrough the left atrial appendage and the balloon catheterwas linked to a pressure transducer connected to a dataacquisition system (Medlab Nanjing China) to assess con-tractile function The left ventricular end diastolic pressure(LVEDP) was adjusted to 4sim8mmHg for 30min Myocardialinfarction (MI) was created by ligation of the left anteriordescending artery (LAD) MI was induced by LAD ligation2sim3mm from the origin with 5-0 silk suture Then 30min of


C IR

DM IR

DM + EtOH + Atr IR

DM + EtOH + CYA IR

DM + EtOH + Wor IR

DM + EtOH IR

CYA

Atr

Wor


minus20 minus10 0 30 40 15025(min)
















3 Results








4 Discussion





































lowastlowast

lowastlowast

lowast

lowastlowast

lowastlowastandand

andandandand



0

1

2

3

4

5M

DA

(nm

olm

g-pr

ot)

(a)

SOD

(Um

g-pr

ot) lowastlowast



andand



0

5

10

15

(b)






M 1 65432

Bcl-2

M 1 65432

Bax


lowastlowastandand

lowastlowastandand

0

1

2

Bax

Bcl-2

mRN

A



120573-Actin

120573-Actin







M 1 2 3 4 5 6

ALDH2

120573-Actin

(a)

lowastlowast

lowastlowast


lowastlowastandand

00

01

02

03

04

05

06

07

08

09

10

11

ALD

H2

120573-a

ctin

at m

RNA

leve

l



(b)

1 2 3 4 5 6

ALDH2

120573-Actin

(c)



ALD

H2

120573-a

ctin

at p

rote

in le

vel

00

05

10

15











(a)

lowastlowast

lowastlowast

00

05

10

15

Card

iom

yocy

te ac

tivity

C Alda-1 + HGHG(b)

C HG Alda-1

A

LDH2

120573-a

ctin

pro

tein

leve

l

lowast

lowast

ALDH2

120573-Actin

C HG Alda-1 + HG0

500

1000

1500

2000

2500

(c)






(a) C IR (b) DM IR








Competing Interests




Acknowledgments


References














































C IR

DM IR

DM + EtOH + Atr IR

DM + EtOH + CYA IR

DM + EtOH + Wor IR

DM + EtOH IR

CYA

Atr

Wor


minus20 minus10 0 30 40 15025(min)
















3 Results








4 Discussion





































lowastlowast

lowastlowast

lowast

lowastlowast

lowastlowastandand

andandandand



0

1

2

3

4

5M

DA

(nm

olm

g-pr

ot)

(a)

SOD

(Um

g-pr

ot) lowastlowast



andand



0

5

10

15

(b)






M 1 65432

Bcl-2

M 1 65432

Bax


lowastlowastandand

lowastlowastandand

0

1

2

Bax

Bcl-2

mRN

A



120573-Actin

120573-Actin







M 1 2 3 4 5 6

ALDH2

120573-Actin

(a)

lowastlowast

lowastlowast


lowastlowastandand

00

01

02

03

04

05

06

07

08

09

10

11

ALD

H2

120573-a

ctin

at m

RNA

leve

l



(b)

1 2 3 4 5 6

ALDH2

120573-Actin

(c)



ALD

H2

120573-a

ctin

at p

rote

in le

vel

00

05

10

15











(a)

lowastlowast

lowastlowast

00

05

10

15

Card

iom

yocy

te ac

tivity

C Alda-1 + HGHG(b)

C HG Alda-1

A

LDH2

120573-a

ctin

pro

tein

leve

l

lowast

lowast

ALDH2

120573-Actin

C HG Alda-1 + HG0

500

1000

1500

2000

2500

(c)






(a) C IR (b) DM IR








Competing Interests




Acknowledgments


References


















































3 Results








4 Discussion





































lowastlowast

lowastlowast

lowast

lowastlowast

lowastlowastandand

andandandand



0

1

2

3

4

5M

DA

(nm

olm

g-pr

ot)

(a)

SOD

(Um

g-pr

ot) lowastlowast



andand



0

5

10

15

(b)






M 1 65432

Bcl-2

M 1 65432

Bax


lowastlowastandand

lowastlowastandand

0

1

2

Bax

Bcl-2

mRN

A



120573-Actin

120573-Actin







M 1 2 3 4 5 6

ALDH2

120573-Actin

(a)

lowastlowast

lowastlowast


lowastlowastandand

00

01

02

03

04

05

06

07

08

09

10

11

ALD

H2

120573-a

ctin

at m

RNA

leve

l



(b)

1 2 3 4 5 6

ALDH2

120573-Actin

(c)



ALD

H2

120573-a

ctin

at p

rote

in le

vel

00

05

10

15











(a)

lowastlowast

lowastlowast

00

05

10

15

Card

iom

yocy

te ac

tivity

C Alda-1 + HGHG(b)

C HG Alda-1

A

LDH2

120573-a

ctin

pro

tein

leve

l

lowast

lowast

ALDH2

120573-Actin

C HG Alda-1 + HG0

500

1000

1500

2000

2500

(c)






(a) C IR (b) DM IR








Competing Interests




Acknowledgments


References
















































































lowastlowast

lowastlowast

lowast

lowastlowast

lowastlowastandand

andandandand



0

1

2

3

4

5M

DA

(nm

olm

g-pr

ot)

(a)

SOD

(Um

g-pr

ot) lowastlowast



andand



0

5

10

15

(b)






M 1 65432

Bcl-2

M 1 65432

Bax


lowastlowastandand

lowastlowastandand

0

1

2

Bax

Bcl-2

mRN

A



120573-Actin

120573-Actin







M 1 2 3 4 5 6

ALDH2

120573-Actin

(a)

lowastlowast

lowastlowast


lowastlowastandand

00

01

02

03

04

05

06

07

08

09

10

11

ALD

H2

120573-a

ctin

at m

RNA

leve

l



(b)

1 2 3 4 5 6

ALDH2

120573-Actin

(c)



ALD

H2

120573-a

ctin

at p

rote

in le

vel

00

05

10

15











(a)

lowastlowast

lowastlowast

00

05

10

15

Card

iom

yocy

te ac

tivity

C Alda-1 + HGHG(b)

C HG Alda-1

A

LDH2

120573-a

ctin

pro

tein

leve

l

lowast

lowast

ALDH2

120573-Actin

C HG Alda-1 + HG0

500

1000

1500

2000

2500

(c)






(a) C IR (b) DM IR








Competing Interests




Acknowledgments


References














































M 1 65432

Bcl-2

M 1 65432

Bax


lowastlowastandand

lowastlowastandand

0

1

2

Bax

Bcl-2

mRN

A



120573-Actin

120573-Actin







M 1 2 3 4 5 6

ALDH2

120573-Actin

(a)

lowastlowast

lowastlowast


lowastlowastandand

00

01

02

03

04

05

06

07

08

09

10

11

ALD

H2

120573-a

ctin

at m

RNA

leve

l



(b)

1 2 3 4 5 6

ALDH2

120573-Actin

(c)



ALD

H2

120573-a

ctin

at p

rote

in le

vel

00

05

10

15











(a)

lowastlowast

lowastlowast

00

05

10

15

Card

iom

yocy

te ac

tivity

C Alda-1 + HGHG(b)

C HG Alda-1

A

LDH2

120573-a

ctin

pro

tein

leve

l

lowast

lowast

ALDH2

120573-Actin

C HG Alda-1 + HG0

500

1000

1500

2000

2500

(c)






(a) C IR (b) DM IR








Competing Interests




Acknowledgments


References














































M 1 2 3 4 5 6

ALDH2

120573-Actin

(a)

lowastlowast

lowastlowast


lowastlowastandand

00

01

02

03

04

05

06

07

08

09

10

11

ALD

H2

120573-a

ctin

at m

RNA

leve

l



(b)

1 2 3 4 5 6

ALDH2

120573-Actin

(c)



ALD

H2

120573-a

ctin

at p

rote

in le

vel

00

05

10

15











(a)

lowastlowast

lowastlowast

00

05

10

15

Card

iom

yocy

te ac

tivity

C Alda-1 + HGHG(b)

C HG Alda-1

A

LDH2

120573-a

ctin

pro

tein

leve

l

lowast

lowast

ALDH2

120573-Actin

C HG Alda-1 + HG0

500

1000

1500

2000

2500

(c)






(a) C IR (b) DM IR








Competing Interests




Acknowledgments


References















































(a)

lowastlowast

lowastlowast

00

05

10

15

Card

iom

yocy

te ac

tivity

C Alda-1 + HGHG(b)

C HG Alda-1

A

LDH2

120573-a

ctin

pro

tein

leve

l

lowast

lowast

ALDH2

120573-Actin

C HG Alda-1 + HG0

500

1000

1500

2000

2500

(c)






(a) C IR (b) DM IR








Competing Interests




Acknowledgments


References














































(a) C IR (b) DM IR








Competing Interests




Acknowledgments


References















































Competing Interests




Acknowledgments


References













































activation of aldh2 with low concentration of ethanol ... · researcharticle activation of aldh2...

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