(arrininscular NeSeArYI ELSEVIER Cudiovoculu Rocud 55 (2002)681-689 The relative order of mKT channels,free radicals and p38 MAPK in preconditioning's protective pathway in rat heart Yuankun Yue",Qining Qin".Michael V.Cohen James M.Downey Stuart D.Critz* 'Deporrewr af Cei Molgy aN?Newrosctance.N 3742.Uatrersiry of Sov Altoa.Callege ar Medcive.Mfote.AL 36688.UYA Drpunueer of Plycio.Lnmeray f Souh Mlatu,Collegr of Mnkeme.Mobie,AL3668 LS "prnon oy Mvhete.(intverxiy gf Soutl Aiohow.Calpe of Mivfcire.Mohuv.Al.if 1SA Rertivu 18 Dmler 2001,uxxpkal I Mury 2002 Ahstract Ohjectives:Ischcmic proconditioning (PC)reduces myucandial infarclion hy a mechanism tha involes opening of mitochundrial AIP-dependent potassiam charnels (K.),reactiwe oxygen species (ROS)and posibly activation of pi8 mitogen-activated protein kinase (p38 MAPK).The actual order o'these steps.however,is a mamer od current debate.This study ecamined whether protection arded by merdicne,which proteets byg mitochondrroue RS.reres mKopening In addition,we ested whether protection from anisomycin,a p38 MAPK activator,is dependent on ROS production.Methods and Results:Isolated,buffer-perfued ra. hcarts were peetreaed with memadione.and infarction wasd afr 30min of regional ischemia ad 120 min of repcrfusion. Menadione reduced inthrction in a dose-dependest manner with E'o of'270 nM.Menadioee's infarct-limmting ettect was isensitnve to 200 HM 5-hdroxydecanomte (5HD),an mK chareel blocker,wherens peotection by diazoocide and PC were blocked by SHD. Anisumyein esal hearls to resi infrcliun and this prolecliv cleel wars ahmgaled hy SB203580.p38 MAPK inhihike,and 2.merapopropiamlghcine (MPG)a fe radical vr.Concusos:These results indcate that mKoperng occurs uptream of mitochondrial ROS generation in the protective pathway.Furthermore,procection afforded by anisomycin was p38 MAPK.and RCS-dependent. 2002 Elsever Science BV.All nghts reserved 1.Introduction kinase was specifically activated by PC.and SB203580,a p38 MAPK inhibitur,abeogated prutection 19-13].Fur Ischemic preconditioning (PC)is a cellular process thermore.anisomycin.a p38 MAPK activator 1141. tngeered by brief ischemia which enables hearts to resist protected in situ mbbet hearts apninst infaretion It has infaretion during a subsequent prolonged period of is- been known for some time that resctive oxygen species chemia [12].The opening of mitochondrial ATP-depen- (ROS)nre involved in PC's protective effeet [13.16]The dent pocissium channels (mK)contributes to PC's release of ROS from mitochondria reduced infarction in protection becautse channel openers proteet ngainst infhre- isolated,huffer-perfused rat hearts [17].while free radical tion whereas channel blockers inhibit provection [3-7]. scavengers blocked protection from PC 15].This studhy There is considerable debste,however.whether mKarr examined whether there is a protective effect of low levels channels act as signal transduction elements or as end- of ROS that is dependent upon opening of mK chan- eflectors of cardioprotection [8].Activation of p38 MAPK nels.That would only be sen if mK channels reside may also be requrred for PC's protection because this upstream from the ROS step in PC's sigral transduction pathway.We also examined whether proxection from Comesponing asthor.Tel:+1-34-460-74:frs:+-4 unisumycin involved ROS. 677刀. E-mai akino xctilauuthal au (S.D.Crila. Time fur primary revirw 27 days 0008-6363/017/$-ere front mrter 20812 lilowvir Science I All nghts reserved P11:s0008-636302)00452-2
Cardiovascular Research 55 (2002) 681–689 www.elsevier.com/locate/cardiores T he relative order of mK channels, free radicals and p38 MAPK in ATP preconditioning’s protective pathway in rat heart a b b,c b a, Yuankun Yue , Qining Qin , Michael V. Cohen , James M. Downey , Stuart D. Critz * a Department of Cell Biology and Neuroscience, MSB 2342, University of South Alabama, College of Medicine, Mobile, AL 36688, USA b Department of Physiology, University of South Alabama, College of Medicine, Mobile, AL 36688, USA c Department of Medicine, University of South Alabama, College of Medicine, Mobile, AL 36688, USA Received 18 December 2001; accepted 1 May 2002 Abstract Objectives: Ischemic preconditioning (PC) reduces myocardial infarction by a mechanism that involves opening of mitochondrial ATP-dependent potassium channels (mK ), reactive oxygen species (ROS), and possibly activation of p38 mitogen-activated protein ATP kinase (p38 MAPK). The actual order of these steps, however, is a matter of current debate. This study examined whether protection afforded by menadione, which protects by causing mitochondria to produce ROS, requires mK opening. In addition, we tested whether ATP protection from anisomycin, a p38 MAPK activator, is dependent on ROS production. Methods and Results: Isolated, buffer-perfused rat hearts were pretreated with menadione, and infarction was assessed after 30 min of regional ischemia and 120 min of reperfusion. Menadione reduced infarction in a dose-dependent manner with an EC of 270 nM. Menadione’s infarct-limiting effect was insensitive 50 to 200 mM 5-hydroxydecanoate (5HD), an mK channel blocker, whereas protection by diazoxide and PC were blocked by 5HD. ATP Anisomycin caused hearts to resist infarction and this protective effect was abrogated by SB203580, a p38 MAPK inhibitor, and 2-mercaptopropionylglycine (MPG), a free radical scavenger. Conclusions: These results indicate that mK opening occurs upstream ATP of mitochondrial ROS generation in the protective pathway. Furthermore, protection afforded by anisomycin was p38 MAPK- and ROS-dependent. 2002 Elsevier Science B.V. All rights reserved. Keywords: Free radicals; Infarction; Ischemia; Mitochondria; Preconditioning; Signal transduction 1. Introduction kinase was specifically activated by PC, and SB203580, a p38 MAPK inhibitor, abrogated protection [9–13]. FurIschemic preconditioning (PC) is a cellular process thermore, anisomycin, a p38 MAPK activator [14], triggered by brief ischemia which enables hearts to resist protected in situ rabbit hearts against infarction [5]. It has infarction during a subsequent prolonged period of is- been known for some time that reactive oxygen species chemia [1,2]. The opening of mitochondrial ATP-depen- (ROS) are involved in PC’s protective effect [15,16]. The dent potassium channels (mK ) contributes to PC’s release of ROS from mitochondria reduced infarction in ATP protection because channel openers protect against infarc- isolated, buffer-perfused rat hearts [17], while free radical tion whereas channel blockers inhibit protection [3–7]. scavengers blocked protection from PC [15]. This study There is considerable debate, however, whether mK examined whether there is a protective effect of low levels ATP channels act as signal transduction elements or as end- of ROS that is dependent upon opening of mK chan- ATP effectors of cardioprotection [8]. Activation of p38 MAPK nels. That would only be seen if mK channels reside ATP may also be required for PC’s protection because this upstream from the ROS step in PC’s signal transduction pathway. We also examined whether protection from anisomycin involved ROS. *Corresponding author. Tel.: 11-334-460-7324; fax: 11-334-460- 6771. E-mail address: scritz@usouthal.edu (S.D. Critz). Time for primary review 27 days. 0008-6363/02/$ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S0008-6363(02)00452-2
682 了了rgk./Cardlmutuar Rexcarch5226s!-5s9 2.Methods o index ischemia and reperfusion.Drug-treated hearts were perfused for 20 min prior to index ischemia and All experiments were conducled in accordance with the reperfusion with either mensdione (0.1-10.0 uM).diazox- Guide for the Care and Use of Laboratory Animafs ide (50 uM)or anisomycin (100 ng/ml).The perfusion published by the Naticeal Institutes of Health (NIH apparatus was shielded from room light with aluminum Puhlicntinn No 85-23,revised 1996) foil for the experiments with menadione.Drgs were solubilized in dimethylsulfoxide (DMSO)or saline slu- 2 1 Infaret are sdies tion as 1000-fold stock solutions and diluted as indicaced prior to experiments DMSO alune (0.2%)did not affect Male Wistar rats (250-350 g)were anesthetized with hemodyramics ar infarct size in control studies (data not sodium pentobarbital (60 mg/kg 1.p.).Hearts were sur- shown).Some hearts were pretreated with 5-hydroy- pically removed and moumod on a LangendorlT apparatus decanoate (SHD](200 M).N-(2-mcrcaptop- ns previously described 118]Isolmed hearts were perfused ropionylglyeine (MPG)(1 mM).or SB203580(10 uM) with waned (37-C)Kreb-Henscleit buffer (118.5 mM for 5 min before the above indicated drups were perflsed NoCl,4.7 mM KCI.1.2 mM MgSO..1.25 mM CnCl2. in combinntion for the next 20 min.Risk zones were 248 mM NaHCO..1.2 mM KH,PO,.10 mM glucose) marked by retrogrnde infusion of 1-10 pm fluorescent which was bubbled with 95%O,-5%CO,to maintain pll micruspberes (Duke Scientific.Palo Alto.CA)into the 7.4.A latex balloon placed in the left ventricle measured acrtic root after religatiun of the coronary tery at the end develeped ventricular pressure and a snare placed around of esch experiment..目earts were frozen cut in】mm he lell coronary artery was used to produce ischems Fig. slices.and incubsted with 1%triphemyltetrazolium chlo- I summarizes the protocol that were used for each nide in phosphate buller foc 15 min to vsualize the experimental group.All hearts were stabilired for 30 min unstained zone of infirction as previoushy repored [17] treated as indicated,and then subjected to 30 min of Infirction wus measured by planimetry and expressed as a regional (index)ischemia followed by 120 min of reperfu percentage of the risk zone.Anisomycin,diazoxide, sion.PC-treated hearts received three cycles precondition. SB203580,5-hydroxydecanoule,MPG,menadione,and ing ischemia,each consisting of 5 min of globul ischemia laboratary reagents were ubeained from Sigma Chemical and 5 min of reperfusion after which they were subected Co ischomia 22.p38 MAPK ocliration in isoluled hearts Control Isolated rat hearts.nffixed to n Langendorrf'apparatus as descrbed above.were treated with 3.0 uM mendione and left ventricular biopsy samples were removed at indicsted 3xPC times (basal,10 min,20 min.and 30 mink.Buopsies were frozen in liquid nitrogen and homogenized following methods reported previcusly 117].After homogenization in 2'0J9F 300 Hl of cold homogeniztion buffer (75 mM B-glycerol Protocol 1 phosphate,20 mM HEPES,2 mM EGTA,2 mM EDTA,I mM Na VO.0.05%Triton X-100,4 Hg/ml leupepein,I mM PMSF,pH 7.2)lysates were centrifuged at 13,000xg 2'u里R: for 15 min at 4'C and protein was determined in super. Protocol 2 pee-Rx natarts usng a modified Lowry method (BioRad.500- 0116).Cytosolie samples (25 g)were processed for SDS-PAGE/Westem blotting and phosphorylated p38 Time MAPK wns detected with a phospho-spocifie antibody 0 (9211.Cell Signaling Technology)using HRP-linked chemiluminescence (Phototope 771:Cell Signaling Fi.1.Experimentil proleenis.All hearts were wthilinod Sor 30 rin Technology)following the marufacturer's specibications. pnor to exxpenntems.Comtrol heants reoened 30 min of regionol ischemia fulluol by 2 k uf iupefinion.PC he b roocivel tuee tycko uf S min p38 MAPK phosphorylation was normalized to the basal iohal ischeni and min of mperfesinn prier to the indes inchema and a止Sorbance observed in un山eedc0n山ol samples. reperfusion Protoool I,heunts were perduasod wih vest Egenrs Irenodione. 100M-l00μ4 direonitle 50dw可in100r用/fa20 23.H9c2 carioc celf citre and drug frealment min pnoe to chema and reperfision Proocel 2.heans were pretreated fw5 is wih eiher20间MS-rdbuxydcraak (SHD人1.2fuM N-2-meremopmpinmd chrine (MI1.or 10 uM SH201581 (SB)ior H9c2 myoblast cells (ATCC,#CRL-1446)were used to o treatmen:with the dnugs in protcol I and then drugs usod in pictocol evaluste the effects of meradione on p38 MAPK.Cells 】rd2ees-nirirod for Be ae过20ein were plated at 5X 10'cells/35-mm culture dish in DMEM
682 Y. Yue et al. / Cardiovascular Research 55 (2002) 681 –689 2. Methods to index ischemia and reperfusion. Drug-treated hearts were perfused for 20 min prior to index ischemia and All experiments were conducted in accordance with the reperfusion with either menadione (0.1–10.0 mM), diazoxGuide for the Care and Use of Laboratory Animals ide (50 mM), or anisomycin (100 ng/ml). The perfusion published by the National Institutes of Health (NIH apparatus was shielded from room light with aluminum Publication No. 85-23, revised 1996). foil for the experiments with menadione. Drugs were solubilized in dimethylsulfoxide (DMSO) or saline solu- 2 .1. Infarct size studies tion as 1000-fold stock solutions and diluted as indicated prior to experiments. DMSO alone (0.2%) did not affect Male Wistar rats (250–350 g) were anesthetized with hemodynamics or infarct size in control studies (data not sodium pentobarbital (60 mg/kg i.p.). Hearts were sur- shown). Some hearts were pretreated with 5-hydroxygically removed and mounted on a Langendorff apparatus decanoate (5HD) (200 mM), N-(2-mercaptopas previously described [18]. Isolated hearts were perfused ropionyl)glycine (MPG) (1 mM), or SB203580 (10 mM) with warmed (37 8C) Krebs-Henseleit buffer (118.5 mM for 5 min before the above indicated drugs were perfused NaCl, 4.7 mM KCl, 1.2 mM MgSO , 1.25 mM CaCl , in combination for the next 20 min. Risk zones were 4 2 24.8 mM NaHCO , 1.2 mM KH PO , 10 mM glucose) marked by retrograde infusion of 1–10 mm fluorescent 3 24 which was bubbled with 95% O –5% CO to maintain pH microspheres (Duke Scientific, Palo Alto, CA) into the 2 2 7.4. A latex balloon placed in the left ventricle measured aortic root after religation of the coronary artery at the end developed ventricular pressure and a snare placed around of each experiment. Hearts were frozen, cut in 1 mm the left coronary artery was used to produce ischemia. Fig. slices, and incubated with 1% triphenyltetrazolium chlo- 1 summarizes the protocols that were used for each ride in phosphate buffer for 15 min to visualize the experimental group. All hearts were stabilized for 30 min, unstained zone of infarction as previously reported [17]. treated as indicated, and then subjected to 30 min of Infarction was measured by planimetry and expressed as a regional (index) ischemia followed by 120 min of reperfu- percentage of the risk zone. Anisomycin, diazoxide, sion. PC-treated hearts received three cycles precondition- SB203580, 5-hydroxydecanoate, MPG, menadione, and ing ischemia, each consisting of 5 min of global ischemia laboratory reagents were obtained from Sigma Chemical and 5 min of reperfusion after which they were subjected Co. 2 .2. p38 MAPK activation in isolated hearts Isolated rat hearts, affixed to a Langendorrf apparatus as described above, were treated with 3.0 mM menadione and left ventricular biopsy samples were removed at indicated times (basal, 10 min, 20 min, and 30 min). Biopsies were frozen in liquid nitrogen and homogenized following methods reported previously [17]. After homogenization in 300 ml of cold homogenization buffer (75 mM b-glycerol phosphate, 20 mM HEPES, 2 mM EGTA, 2 mM EDTA, 1 mM Na VO , 0.05% Triton X-100, 4 3 4 mg/ml leupeptin, 1 mM PMSF, pH 7.2), lysates were centrifuged at 13,0003g for 15 min at 4 8C and protein was determined in supernatants using a modified Lowry method (BioRad, [500- 0116). Cytosolic samples (25 mg) were processed for SDS–PAGE/Western blotting and phosphorylated p38 MAPK was detected with a phospho-specific antibody ([9211, Cell Signaling Technology) using HRP-linked chemiluminescence (Phototope [7071; Cell Signaling Fig. 1. Experimental protocols. All hearts were stabilized for 30 min Technology) following the manufacturer’s specifications. prior to experiments. Control hearts received 30 min of regional ischemia p38 MAPK phosphorylation was normalized to the basal followed by 2 h of reperfusion. PC hearts received three cycles of 5 min global ischemia and 5 min of reperfusion prior to the index ischemia and absorbance observed in untreated control samples. reperfusion. Protocol 1, hearts were perfused with test agents (menadione, 100 nM–10.0 mM; diazoxide, 50 mM; anisomycin, 100 ng/ml) for 20 2 .3. H9c2 cardiac cell culture and drug treatment min prior to ischemia and reperfusion. Protocol 2, hearts were pretreated for 5 min with either 200 mM 5-hydroxydecanoate (5HD), 1.0 mM H9c2 myoblast cells (ATCC, [CRL-1446) were used to N-(2-mercaptopropionyl)glycine (MPG), or 10 mM SB203580 (SB) prior evaluate the effects of menadione on p38 MAPK. Cells to treatment with the drugs in protocol 1 and then drugs used in protocols 5 1 and 2 were co-administered for the next 20 min. were plated at 5310 cells/35-mm culture dish in DMEM
Y.Yur el al.Cardhumuuar Rracarch 55 (20021 681-689 63 supplemented with 10%fetal calf serum,5%horse serum. control smples Tiss culture supplies were abtained 0.5 unit penicillin,and 1.0 uit streptomycin and grown in from GIBCO unless otherwise indicated. a 5%CO:incubalur at 37C.At 75-80%cunfluence,cells were washed in serum-free DMEM and incubated in 1.0 2..Statistics ml serum-free DMEM fer 30 min at 37'C with or without meradione at concentrations mnging from 1 0 to 10,000 All data are presented ns means+S EM A one-wav nM.In some cells expased to 10,000 nM menadione,I ANOVA with Tukey's post hoc test was performed on mM MPG was added 5 min prior to drug treatment.After infaret size and haseline hemodynamics,an ANOVA with menadione treatment,p38 MAPK activity was determined repeated measures was performed on the p38 MAPK by conventional Western blatting hy modification of phosphorylation data,and Student's t-test was perfommed methods deseribed above.H9e2 cell were lysed in 300 ul to compare group treatment effects.Data were corsidered of cold homogenzation bunler (75 mM B-glycerol phos- signiticant at the P0.05 level. phate.20 mM HEPES.2 mM ETA.2 mM EDTA.1 mM NaVO.D.05%Triton X-10风，4/ml leupeptin..1mM PMSF.pH 7.2)Cell lysates were centrifuged at 13.000xg 3.Results for 15 min in the cold (4 and protein was desemmined in the superratants by a modified Lowry assany (BioRad. 王.OIIs *500-0116).Samples (25 Hg)were processed for SDS- PAGE/Westem blotting and pbosphorylsted p38 MAPK There were no significant differences in the bassl heart was detected as described above with phospho-specific p38 rate.developed pressure,o coronary flow among any of MAPK antibody (9211,Cell Signaling Technology)and the experimental groups (Table 1).Averaged group data HRP-linked chemiluminescence (Phototope 071:Cell show that menadione treatment did not decrense developed Signaling Technology)p38 MAPK phosphorylation wns pressure more than 20%,which is comparable to effects normalized to the hasal absorhance ohserved in untreated ohserved during preconditioning Tahie 1 Henodytunie du上fu iolall,peifioed net bea山 G联界 Bncie Pynnockoin Codoi Pepofmn ® 同 C'mntrol 71 6241 7=1a M2±7 7社13 项052 1161350 3709 38上0g 17.4t12 172±05 260115 经611 发4=01 3106107 品86行 91108 0.1pM 1开土49 29 94t司 3μ VIN 200上1 11132.1 14714 217±0 8363 157=t5 311±115 611生4J 5‘504 1光1±102 6M624E K士A 10M 上 10生4 4293 6地2线1 0 415 42.139 33496 151411 344兰73 %2±I33 m4】3 3424k5 稀8上96 R5421 30:24 2:5% 113417 40上：121 025, 151=3 只4±0 6s0+9 12法18 6士t2 “看4兰51 dl2 1划重±9 410=6J 0±3 VEX+ 3450上56 1"84 113 对山法s 18女05 1球士4标 4141 2光±日2 618243 00壮3 知 ：25 97356 174 1311 36T±T6 6595 x0上5 1015 4士法 为24 1线4杜1山 145 114杜12 重 厦+ 34机0上1 7±1 2地3 1±5s 55 315±1b5 4s±l21 9±10 VG T行 00:93 1770 150 2951 w41247 213t71 线714E 天天10想 A+ 2上出5 加1生9 15= 出 2出1 150始13 士 60=6 中 2±5 61051 家) ANTS+ 3刘3：5 150=2 749 22 6 2城7 6251 0 H 3150-95 天2 角17甲 来0t11 0上16 15A .8=4.7 161±245 4±4 社昌 量a4yf王里量8r血d道应4Er0里星3国f3道Hp里证土5M
Y. Yue et al. / Cardiovascular Research 55 (2002) 681 –689 683 supplemented with 10% fetal calf serum, 5% horse serum, control samples. Tissue culture supplies were obtained 0.5 unit penicillin, and 1.0 unit streptomycin and grown in from GIBCO unless otherwise indicated. a 5% CO incubator at 37 8C. At 75–80% confluence, cells 2 were washed in serum-free DMEM and incubated in 1.0 2 .4. Statistics ml serum-free DMEM for 30 min at 37 8C with or without menadione at concentrations ranging from 1.0 to 10,000 All data are presented as means6S.E.M. A one-way nM. In some cells exposed to 10,000 nM menadione, 1 ANOVA with Tukey’s post hoc test was performed on mM MPG was added 5 min prior to drug treatment. After infarct size and baseline hemodynamics, an ANOVA with menadione treatment, p38 MAPK activity was determined repeated measures was performed on the p38 MAPK by conventional Western blotting by modification of phosphorylation data, and Student’s t-test was performed methods described above. H9c2 cells were lysed in 300 ml to compare group treatment effects. Data were considered of cold homogenization buffer (75 mM b-glycerol phos- significant at the P,0.05 level. phate, 20 mM HEPES, 2 mM EGTA, 2 mM EDTA, 1 mM Na VO , 0.05% Triton X-100, 4 3 4 mg/ml leupeptin, 1 mM PMSF, pH 7.2). Cell lysates were centrifuged at 13,0003g 3. Results for 15 min in the cold (4 8C) and protein was determined in the supernatants by a modified Lowry assay (BioRad, 3 .1. Hemodynamics [500-0116). Samples (25 mg) were processed for SDS– PAGE/Western blotting and phosphorylated p38 MAPK There were no significant differences in the basal heart was detected as described above with phospho-specific p38 rate, developed pressure, or coronary flow among any of MAPK antibody ([9211, Cell Signaling Technology) and the experimental groups (Table 1). Averaged group data HRP-linked chemiluminescence (Phototope [7071; Cell show that menadione treatment did not decrease developed Signaling Technology). p38 MAPK phosphorylation was pressure more than 20%, which is comparable to effects normalized to the basal absorbance observed in untreated observed during preconditioning. Table 1 Hemodynamic data for isolated, perfused rat hearts Group Baseline Preocclusion Occlusion Reperfusion HR DP CF HR DP CF HR DP CF HR DP CF (beats/min) (mmHg) (ml/min) (beats/min) (mmHg) (ml/min) (beats/min) (mmHg) (ml/min) (beats/min) (mmHg) (ml/min) Control 329.3620.0 113.863.2 13.760.7 – – – 313.3610.0 65.264.1 8.761.0 299.2617.8 66.267.3 9.761.2 MEN 328.0616.2 116.465.0 17.260.5 318.0620.8 107.468.2 17.260.5 296.067.5 55.663.2 8.460.7 310.0620.7 63.866.7 9.260.8 (0.1 mM) MEN 324.068.1 111.866.1 16.260.7 309.0611.2 101.065.6 14.860.5 292.065.8 64.666.1 7.460.7 297.064.9 66.265.5 9.460.8 (0.3 mM) MEN 300.062.6 113.365.1 14.760.4 291.763.0 98.366.5 13.760.5 261.7611.9 61.064.3 6.560.4 276.7610.2 68.664.6 8.060.4 (1.0 mM) MEN 326.0617.7 118.466.0 16.060.4 310.0618.4 94.269.5 14.060.3 278.0616.6 60.269.1 7.060.4 294.0622.4 59.065.2 9.460.5 (3.0 mM) 3PC 359.2623.9 123.369.6 16.362.0 344.0617.8 96.2613.3 20.362.3 334.2616.9 69.869.6 8.562.0 340.0612.4 81.265.8 11.261.7 3PC1 334.0612.1 102.065.1 16.860.9 314.0616.9 68.064.9 21.261.6 246.0617.2 50.466.2 6.660.2 295.068.9 51.066.8 10.860.7 5HD MEN1 346.0616.6 110.862.8 17.261.3 332.0613.6 79.064.8 13.860.9 268.0614.6 54.068.1 6.860.9 296.0613.2 60.864.3 10.060.3 5HD DZ 333.3612.5 97.369.6 16.061.0 336.6617.4 90.068.7 18.561.6 298.369.1 60.068.5 9.260.7 316.7617.6 68.569.9 11.360.7 DZ1 368.0621.3 102.066.04 18.061.5 384.0615.0 96.264.8 18.461.3 320.0615.2 53.463.5 8.261.3 378.0618.3 51.663.2 11.461.2 5HD DZ1 340.0620.4 130.768.95 16.761.0 337.5619.3 117.067.0 20.061.3 290.065.8 76.067.1 8.260.8 332.5616.5 64.8612.1 9.361.0 MPG ANIS 320.069.3 111.767.0 15.061.1 295.067.2 94.764.9 14.360.8 283.4624.7 57.563.4 7.560.9 303.367.1 60.764.6 8.860.8 ANIS1 352.0621.5 103.069.9 15.660.7 318.0618.8 95.0610.7 16.061.5 278.0620.0 61.066.6 7.860.7 298.0626.9 63.065.8 8.860.7 MPG ANIS1 328.3614.5 93.266.7 15.061.2 303.3613.3 71.764.9 12.361.2 294.364.8 67.766.6 7.261.5 299.066.7 60.265.1 8.060.6 SB 5HD 315.0619.5 100.066.1 15.261.1 318.0622.4 96.467.9 15.261.0 267.0623.4 56.465.1 6.660.5 304.0612.5 56.065.9 8.061.4 MPG 370.0614.6 88.064.4 15.660.4 360.8620.4 79.862.7 15.360.9 316.7624.6 46.761.9 6.860.6 340.0615.7 44.863.4 8.860.8 HR, heart rate; DP, developed pressure; CF, coronary flow; 3PC, three cycles of preconditioning; MEN, menadione; 5HD, 5-hydroxydecanoate (200 mM); DZ, diazoxide (50 mM); ANIS, anisomycin (100 ng/ml); MPG, N-(2-mercaptopropionyl)glycine (1.0 mM). Values for baseline were measured after 30 min stabilization. Values for preocclusion, occlusion, and reperfusion were measured before the 30-min ischemia, at the end of 30 min of ischemia, and at the end of 60 min of reperfusion. Values represent mean6S.E.M
684 Y.Yvr el al.Cardunutwlar fescarch 55 (2002)68!-689 3.2 Infaret sice 40 EC 270 nH There were po significant differences in body weight. 0 heart weight,or size of nsk zooes belween any of the 0 experimental groups (Table 2).As illustrated in Fg.2. ·P0.05 treatment with menadione caused a dose-dependent reduc- 0 tion of infarction Infarction was 32.6+3.7%in control 0 1010001000 hearts (u=8)Treatment with 100 nM menadione slighcly 40 0 reduced infarction to 286+4.5%(n=5).Infarction was 0 MENADIONC [nNI significantly reduced to15.5±4.2%，8.7±l.6%,and 8 10.92.7%(P005)in hearts treated with 300 nM (n- 8 0 8 8 5).10100 (m-6).and 3000 nM mensdione (n-6).respee- o 0 4 trvely.This protective cffoct was attenated at 10 uM B+ menadione (infaretion=223+45%w=3).possibly a re- 0 台 sult of toxiciry at higher doses (dnta not shown).These 190 00 1030 3000 datn were fit by a dose-response nlgorithm (Origin 6.1. Rockware,Giolden.CO)which yielded an ECo of 270 nM MFNADIONE [nM] for the prodective effect of menadione (inset,Fig 2).The infaret-limiting ellect of menadione was observed at much Meradione caned a doe-dependert prtection agairat irfaresns with an lower doses than previously reported for prolection in beart EC of 270 rM and soouration between 1000 and 3020 nM.Open circles tssues 19.201. e inivitlal eprinerlal olocivutiums.fillod citlo w:te guu In a previous report we showed that protection induced meons+S IM..and asteridos indicare signticance vs comrol heans a by 3.0 HM menadione was hlocked by myxothiazl,a mitochondrial electron transport inhibitor,indicating that [21,22],hut reproducible signals could not be detected mitochondria were the sourte of protective free radicals below 30 HM meradione using DCF (data nut shown). [17].We attempted to use 2',7'-dichluruftuaresein (DCF) Menadione can activate p38 MAPK in various cardiac Huorescence to measure ROS in solated cnrdiomyocytes cells [17.23241.Therefore,in this study we evaluated the TaNe 2 Infnet xi图山由rode且r6 ed rt heartx Group Bedy weigll Heart weighn 民kG lafirer Infuctisn (gl (g) (cmn] (an of risk xure) Coztrol 308.8±2.1 1.37±010 0250占00 000=001 326±37 MEN 060±1.9 135±00@ 0309±02 0059=002 186±45 0.1MD MEN 305.0±32 1.35±0.03 0307±0.0l 0.047=001 155±42 (03M) MEN 295.8±4.4 129±004 024±0.g 0025=001 87±16° 4l0=M0 MIN 313.0±128 1.10±0W 02%±001 0029=01 109±27” 30=M0 C 2859±R4 1.10±023 02i0±0g 0085±0001 26±08 3PC+ 3刘收0±66 1.34±0.01 0345±0G 0057=0见 248±26 SHD MIN 050+7.7 1.30+00的 025+0e 0015-09 52+12 SHD z 29k3±107 1.36±005 022±003 0029±0006 1m1±16 DZ+ 257.0士k2 126±0.05 02s5±00川 00%1=009 234±38 SHD h/+ 2k名+197 1AR+01 025+0倍 016欲-005 09+57 MIG ANIS 2T2.5±R5 1.1R±004 026±02 0.029=0006 10.1±469 ANIS 200±1.2 1260g 02T4±0tC 0064二u010 232±25 MPG ANIS 35±142 1.3±00 02%士0码 0124=020 将2+45 SB SHD 2900±&1 128±006 0317±006 0079±0009 249±19 MIG 2w.2+20 131±g 0H5±01 019=020 287±56 Fur usbuvir.ura se:Tilsle I Values tepreeal tan 5.E.M.P5005 va contrel infaction
684 Y. Yue et al. / Cardiovascular Research 55 (2002) 681 –689 3 .2. Infarct size There were no significant differences in body weight, heart weight, or size of risk zones between any of the experimental groups (Table 2). As illustrated in Fig. 2, treatment with menadione caused a dose-dependent reduction of infarction. Infarction was 32.663.7% in control hearts (n58). Treatment with 100 nM menadione slightly reduced infarction to 28.664.5% (n55). Infarction was significantly reduced to 15.564.2%, 8.761.6%, and 10.962.7% (P,0.05) in hearts treated with 300 nM (n5 5), 1000 (n56), and 3000 nM menadione (n56), respectively. This protective effect was attenuated at 10 mM menadione (infarction522.364.5%; n53), possibly a result of toxicity at higher doses (data not shown). These data were fit by a dose–response algorithm (Origin 6.1, Rockware, Golden, CO) which yielded an EC of 270 nM 50 for the protective effect of menadione (inset, Fig. 2). The Fig. 2. The effect of menadione on infarct size in isolated rat hearts. infarct-limiting effect of menadione was observed at much Menadione caused a dose-dependent protection against infarction with an EC of 270 nM and saturation between 1000 and 3000 nM. Open circles lower doses than previously reported for protection in heart 50 are individual experimental observations, filled circles are the group tissues [19,20]. means6S.E.M., and asterisks indicate significance vs. control hearts (0 In a previous report we showed that protection induced nM). by 3.0 mM menadione was blocked by myxothiazol, a mitochondrial electron transport inhibitor, indicating that [21,22], but reproducible signals could not be detected mitochondria were the source of protective free radicals below 30 mM menadione using DCF (data not shown). [17]. We attempted to use 29,79-dichlorofluorescein (DCF) Menadione can activate p38 MAPK in various cardiac fluorescence to measure ROS in isolated cardiomyocytes cells [17,23,24]. Therefore, in this study we evaluated the Table 2 Infarct size data for isolated, perfused rat hearts Group Body weight Heart weight Risk zone Infarct Infarction 3 3 (g) (g) (cm ) (cm ) (% of risk zone) Control 308.862.1 1.3760.10 0.28060.02 0.09060.01 32.663.7 MEN 306.061.9 1.3560.02 0.30960.02 0.08960.02 28.664.5 (0.1 mM) MEN 305.063.2 1.3560.03 0.30760.01 0.04760.01 15.564.2* (0.3 mM) MEN 295.864.4 1.2960.04 0.28460.02 0.02560.01 8.761.6* (1.0 mM) MEN 313.0612.8 1.4060.09 0.29560.01 0.02960.01 10.962.7* (3.0 mM) 3PC 285.968.4 1.1060.23 0.21060.08 0.00560.001 2.660.8* 3PC1 308.066.6 1.3460.01 0.34360.03 0.08760.02 24.862.6 5HD MEN1 305.067.7 1.3060.06 0.28560.02 0.01560.009 5.261.2* 5HD DZ 298.3610.7 1.3660.05 0.27260.03 0.02960.006 10.161.6* DZ1 287.068.2 1.2660.05 0.28560.01 0.06160.009 23.463.8 5HD DZ1 328.8619.7 1.4860.1 0.32560.03 0.10260.025 30.965.7 MPG ANIS 272.568.5 1.1860.04 0.27660.02 0.02960.008 10.164.6* ANIS1 290.064.2 1.2660.02 0.27460.02 0.06460.010 23.262.5 MPG ANIS1 335.3614.2 1.1360.07 0.32860.04 0.12460.020 38.264.5 SB 5HD 290.066.1 1.2860.06 0.31760.03 0.07960.009 24.961.9 MPG 299.262.0 1.3160.02 0.34360.01 0.09960.020 28.765.6 For abbreviations see Table 1. Values represent mean6S.E.M. *P,0.05 vs. control infarction
Y.Tur e al.Cardhunurular fescarch 55 (2002)681-689 665 A ph-p38 basal 10 min 20 min 30 min B 2.5 *px0.05 2 1.5 1 ￥ 0.5 0 basal 10 min 20 min 30 min Fig.3.Aclivutin u pl&MAPK by mesadine in iaulad tal huts.(A)Reptserlalive Wodem bkt showing tul perfipcmn cf 3.0 pM mrralion mupc a time-dependet increse in pl MAIK phogheryltion (ph-p()Gmup dr o41 show that 1.0 aM renddione teatmert increased ps MAPK hoophrylalio0%fich reached或out twofdld over bes1 ctivity an20 n Astensks indis.取starilieance3.t色1eo组 effects of 3.0 uM meradione on p38 MAPK phosphoryls anisumycin.As shown in the representative Western hloc in tionn isolaed rat hearts.The representotive Western blot 像.6A.pers0箱with anis0 nycin caus0d8tume-dcpe- in Fip 3A shows that 3.0 uM menadione increased p38 den increa5emp3移MAPK phosphorylati心n whach peaked MAPK phosphorylation.an indicator of kinase activation. after 20 min Furthermore.as shown in Fie.6B 100 ng/ml in a time-dependent manner.The group data show that ansomcin was sipnificantly proccctive in isolated rat perfusion with 30 M menadione for 20 min increased hearts (intaretion=10.14.6%=6)compared to control p38 MAPK activity significantly to a level that was hearts.A role for p38 MAPK activation was further 20740%over basal activity in isolated rat hearts (Fig. confirmed in heerts pretrealed for 5 min with 10 uM 3B).The dose-response relationship of mensdione's acti- SB203580,a widely used p38 MAPK inhibitor vation of p38 MAPK was further investigated in H9c2 112,13.17.26].Pretreatment with SB203580 prior to cardise cells.Fig.4A is a representalive Western blot anisomycin treatment blocked protection,irxcrexsing in- showing a dose-dependent activation of p38 MAPK hy farction significantly to 38 245%w=6)when compared meradione in these cells.The average of five experiments to anisomycin-treated bearts We next examined whether for each group of H9e2 cells is shown in Fig.4B.Note anisomycin's effect might involve generation of ROS. also that p38 MAPK activation by 10 HM menadione in Anisomycin's protection wus significantly attenuated in [19c2 cells could he blocked hy 10 mM MPG. hearts pretreated with 1.0 mM MPG for 5 min Experiments were performed in iolated rat bearts to (infaretion-2322.5%.m-6)as shown in Fig.6B.These evaluate whether mK channel opening contribuned to resuhs compare with MPG's attenuation of the protection menadsone's protective effoct.Fig shows that 5HD (200 aftorded by dia0xdc(intarcti0m-28.少±5.7%，t-51 M).n mK channel blocker了，25.did no bloek indicating that anisomyein's proccetion wns due.at least in protection by 30 HM menndione Infarction wns part,to relense of ROS 2+12%(=3)and not significantly different from that in hearts treated with menadione alone (10.92.7%)PC and treatment with 50 uM dizoxide reduced infarction 4.Discussion ni5cly,o2.6±08%u-7)nd10.1±1.6%m-6. respectively.Protection was sersitive to 200 uM 5HD.as This study demonstrates that the protective effect of infarction increased to 24.842.6%(n-5)in PC and menadicne does not require mK channel opening which 2343.8%(=5)in diazoxide-treated hearts. is an indication that the channels are upstream of The mechanism of p38 MAPK activation hy anisomycin menadione's site of action That contrasts with the effects is not fully understood.To examine this issue further,we of PC and diazoxide which were blocked by 5HD. treated isolated hearts with 100 ng/ml (-300 nM) Menadione disrupts electron transport at sive III in the
Y. Yue et al. / Cardiovascular Research 55 (2002) 681 –689 685 Fig. 3. Activation of p38 MAPK by menadione in isolated rat hearts. (A) Representative Western blot showing that perfusion of 3.0 mM menadione caused a time-dependent increase in p38 MAPK phosphorylation (ph-p38). (B) Group data (n54) show that 3.0 mM menadione treatment increased p38 MAPK phosphorylation which reached a peak about twofold over basal activity after 20 min. Asterisks indicate significance vs. basal time point. effects of 3.0 mM menadione on p38 MAPK phosphoryla- anisomycin. As shown in the representative Western blot in tion in isolated rat hearts. The representative Western blot Fig. 6A, perfusion with anisomycin caused a time-depenin Fig. 3A shows that 3.0 mM menadione increased p38 dent increase in p38 MAPK phosphorylation which peaked MAPK phosphorylation, an indicator of kinase activation, after 20 min. Furthermore, as shown in Fig. 6B 100 ng/ml in a time-dependent manner. The group data show that anisomycin was significantly protective in isolated rat perfusion with 3.0 mM menadione for 20 min increased hearts (infarction510.164.6%; n56) compared to control p38 MAPK activity significantly to a level that was hearts. A role for p38 MAPK activation was further 207640% over basal activity in isolated rat hearts (Fig. confirmed in hearts pretreated for 5 min with 10 mM 3B). The dose–response relationship of menadione’s acti- SB203580, a widely used p38 MAPK inhibitor vation of p38 MAPK was further investigated in H9c2 [12,13,17,26]. Pretreatment with SB203580 prior to cardiac cells. Fig. 4A is a representative Western blot anisomycin treatment blocked protection, increasing inshowing a dose-dependent activation of p38 MAPK by farction significantly to 38.264.5% (n56) when compared menadione in these cells. The average of five experiments to anisomycin-treated hearts. We next examined whether for each group of H9c2 cells is shown in Fig. 4B. Note anisomycin’s effect might involve generation of ROS. also that p38 MAPK activation by 10 mM menadione in Anisomycin’s protection was significantly attenuated in H9c2 cells could be blocked by 1.0 mM MPG. hearts pretreated with 1.0 mM MPG for 5 min Experiments were performed in isolated rat hearts to (infarction523.262.5%; n56) as shown in Fig. 6B. These evaluate whether mK channel opening contributed to results compare with MPG’s attenuation of the protection ATP menadione’s protective effect. Fig. 5 shows that 5HD (200 afforded by diazoxide (infarction528.965.7%; n55), mM), an mK channel blocker [7,25], did not block indicating that anisomycin’s protection was due, at least in ATP protection by 3.0 mM menadione. Infarction was part, to release of ROS. 5.261.2% (n55) and not significantly different from that in hearts treated with menadione alone (10.962.7%). PC and treatment with 50 mM diazoxide reduced infarction 4. Discussion significantly, to 2.660.8% (n57) and 10.161.6% (n56), respectively. Protection was sensitive to 200 mM 5HD, as This study demonstrates that the protective effect of infarction increased to 24.862.6% (n55) in PC and menadione does not require mK channel opening which ATP 23.463.8% (n55) in diazoxide-treated hearts. is an indication that the channels are upstream of The mechanism of p38 MAPK activation by anisomycin menadione’s site of action. That contrasts with the effects is not fully understood. To examine this issue further, we of PC and diazoxide which were blocked by 5HD. treated isolated hearts with 100 ng/ml (|300 nM) Menadione disrupts electron transport at site III in the
683 了了ar./Cardhumuwar Rracarch 5s226S!-S89 A ph-p38 100 3001000 30001000010000 B +MPG 10 D40.05v80nM 9 7 5 0 100 300100030001000010000 MENADIONE [nM] +MPG og-Tekt3 waton of pig MAPK1 he actnratian o时8 MAPK y10 M meredigre uas hlo女对yI0n1.I)agd内a-5 indcue hal msalione's te hold foe p38 MAPK aetialion ws-300 aM ud uctivalin ous sxtive lo fice alcdl scuveairg by MPG. inner membrane of'mnochondria leading to the production by myxothiazol.a site llI mitociondral inhsbitor.and by of mitochondrinl ROS 117.19.20].Menadione may cause MPG.a free rodical scaerger.indicating the inolvement other effects.however.Bellomo ct al.127]reported that of mitochondrial ROS 117]MeCormick et al.9]re- menadione inhibitod plutathione redoctase thus causing porod that menadione was photochemically activated and increased levels of GSII.Since that effect required a therefure our experiments were performed with light- concentration of menadione greaer than 25 uM,while shielding.Furthermore.400 uM menadione had to be prutection was seen below 3.0 uM in the present study,it exposed to 340 nM (UV)light for 6i0 min to observe seems unlikely that altered GSH levels contrbuted to cur photoactivstion,while our studies ued low concentration findings.Menadione also causes sulthydryl arylation in of menadione and low levels of visible light.We,therefore, platelets [28],but that required 250 HM.which wns ngnin conclude that menadione most likely protects the hearts by much higher than corcentrations that we used.Moreover inducing ROS genertion from mitochondria meradione's proection against infarction could be blocked The possihility that ROS act is sigraling molecules in myocardial tissues has recently gained considerable atien- tion [21,24,30-35].Yao et al.[21]showed that ROS were 5 gencrated by acetylholme which then protected chick 4s销9 Fus wth的lH0 eardiomyocytes against simulited ischema in an mK dependent manner.Cohen et al.132]found that mamy G-coupled receptor systems mimic preconditioning 6 ,* throuph a KOS-dependent mochanism in the rabbit hesrt 8 8 8 While the mechanism of this protection is unkrown.we speculae that mK channel opening releases mitocbon- 8 drial ROS which are then capable of activating p38 MAPK o mediste protection.Interestingly.Carroll et al.[33] ohserved that 30 uM diapoxide evoked ROS which 3PC 3PC MEH NON 02 G 45H0 +340 +5H0 protected cultured human atrial myocytes against simulsed Fi.5.The effect uf ak cluneel wtiviy u ptuautiut.TIw polee- ischemia-reperfursion injury.Furthermore,scavenging free mdicals with W-acetylcysteine blocked diazooride's abdlity to improve post-ischemic left ventricular function in rats Hocked by 200 pM 5HD 34]Menadione treatment can be detrimental in cardiac
686 Y. Yue et al. / Cardiovascular Research 55 (2002) 681 –689 Fig. 4. Activation of p38 MAPK by menadione in H9c2 cells. (A) Representative Western blot showing that 30 min of treatment with menadione caused a dose-dependent activation of p38 MAPK. The activation of p38 MAPK by 10 mM menadione was blocked by 1.0 mM MPG. (B) Group data (n55) indicate that menadione’s threshold for p38 MAPK activation was |300 nM and activation was sensitive to free radical scavenging by MPG. inner membrane of mitochondria leading to the production by myxothiazol, a site III mitochondrial inhibitor, and by of mitochondrial ROS [17,19,20]. Menadione may cause MPG, a free radical scavenger, indicating the involvement other effects, however, Bellomo et al. [27] reported that of mitochondrial ROS [17]. McCormick et al. [29] remenadione inhibited glutathione reductase thus causing ported that menadione was photochemically activated and increased levels of GSH. Since that effect required a therefore our experiments were performed with lightconcentration of menadione greater than 25 mM, while shielding. Furthermore, 400 mM menadione had to be protection was seen below 3.0 mM in the present study, it exposed to 340 nM (UV) light for 60 min to observe seems unlikely that altered GSH levels contributed to our photoactivation, while our studies used low concentration findings. Menadione also causes sulfhydryl arylation in of menadione and low levels of visible light. We, therefore, platelets [28], but that required 250 mM, which was again conclude that menadione most likely protects the hearts by much higher than concentrations that we used. Moreover, inducing ROS generation from mitochondria. menadione’s protection against infarction could be blocked The possibility that ROS act as signaling molecules in myocardial tissues has recently gained considerable attention [21,24,30–35]. Yao et al. [21] showed that ROS were generated by acetylcholine which then protected chick cardiomyocytes against simulated ischemia in an mK - ATP dependent manner. Cohen et al. [32] found that many G -coupled receptor systems mimic preconditioning i through a ROS-dependent mechanism in the rabbit heart. While the mechanism of this protection is unknown, we speculate that mK channel opening releases mitochon- ATP drial ROS which are then capable of activating p38 MAPK to mediate protection. Interestingly, Carroll et al. [33] observed that 30 mM diazoxide evoked ROS which protected cultured human atrial myocytes against simulated ischemia–reperfusion injury. Furthermore, scavenging free Fig. 5. The effect of mK channel activity on protection. The protec- ATP radicals with N-acetylcysteine blocked diazoxide’s ability tive effect of menadione (3.0 mM) was not sensitive to 200 mM 5HD. The protection afforded by 50 mM diazoxide and PC were significantly to improve post-ischemic left ventricular function in rats blocked by 200 mM 5HD. [34]. Menadione treatment can be detrimental in cardiac
了.了wE cla./Caraism山ww frscarch Fs2265!-6$g 687 A ph-p38 basal 10 min 20 min 30 min 100 ng/ml ANISOMYCIN B 60 0 50 pc0.05 vs ANIS 正 40 8 30 9 % 20 0 0 n 10 8 0 ANIS ANIS ANIS DZ MPG +SB +MPG +MPG Fi2.6.The effect of anisomycin in isdlued rat heants.(A)Repoesemauve Wesiem blt showing thet perfusion or 100 ng/ril ensomyen ceused a time-depercknt activalion of p38 MAPK which peiked afr 20 min.(B)[tat heebs trenadl with 100 ag/nl (ANIS)for 20 rin wre pednPreteatment with 1M S3 (S)IM MIi toemn scatth hlocked the proectie eftect of ans MPG al sigiiculy boueled the pruxeaou uffudedl by 50 pM disoooide cells [19.201.but these ellects were seen al higher doses Furthermore.low doses of meradione (1-3 uM)caused a than we used in this study.Since ROS production in rapid protective ellect medated by p38 MAPK m isolsed isolated rat cardiomyocytes wus not detectahle below 30 nt hearts.The mechanism of p3&MAPK's provection is uM menadione with DCF,we suspect that the level of not completely understood.Studies in CCL39 lung epi. mitochondrial ROS required for procection is quite low c thelial cells indicate that p38 MAPK ativation causes confined to a small microcompurtment.The protective phosphorylation of HISP27,a dowrstream target of p38 effect of menadione diminished at 10 uM.as might be MAPK associated with stress fiber stabiliration.which expected if higher levels of ROS were toxic in cardine modiates resistance ngainst oxidative stress 38391 As cells This bephnsie etteet has been documented before. shown in Fip 7.mitochondrial ROS could netivate p38 Tumer e al.[23]reported that menodione levels up to 20 MAPK and stnbilize stress fibers in a similnr mnnner. M cnused resistance to apoptosis while this protective thereby conferring resistance to osmotic swelling and effect disnppenred ahove 40 uM in H9c2 cnrdine cells attendant damnpe nssocaated with ischemia in the heart Hy Interestingly,low doses of the ROS generaor bilinhin contrast,some studies have shown that activntion of p38 protected neurons while high doses were toxic 361.and MAPK may be detrimental in the heart [40-431 Schneider low lvels of ROS enhanced survival of human aorbe et al.[43]observed that SB202190,a p38 MAPK inhibitor. smooth muscle oells while higher levels were taxie [371. reduced infaret ste and enhanced ventricular function in Menadione can activate p38 MAPK in many cell types rat hearts subjected to ischemia.Saurn et al 44]also ncluding H9c2 cardsac oclls 123.241.Treatment with reported that inhibition of p38 MAPK reduced cell death m meradione (5-40 uM)for 4 h caused the activation of p38 rat ventricular myocytes subjected to simulated ischemia. MAPK and c-jun NH,-terminal kinase (JNK)in H9c2 cells While the reason for differences between the latter findings 23].Ou studies indicate that p38 MAPK is activated hy and the present results and others previously documenting brief expasure to lower doses of menadione in 119c2 cells. a prutective action of p38 MAPK [9-13]remains unclear
Y. Yue et al. / Cardiovascular Research 55 (2002) 681 –689 687 Fig. 6. The effect of anisomycin in isolated rat hearts. (A) Representative Western blot showing that perfusion of 100 ng/ml anisomycin caused a time-dependent activation of p38 MAPK which peaked after 20 min. (B) Isolated rat hearts treated with 100 ng/ml anisomycin (ANIS) for 20 min were protected against infarction. Pretreatment with 10 mM SB203580 (SB) or 1 mM MPG for 5 min significantly blocked the protective effect of anisomycin. MPG also significantly blocked the protection afforded by 50 mM diazoxide. cells [19,20], but these effects were seen at higher doses Furthermore, low doses of menadione (1–3 mM) caused a than we used in this study. Since ROS production in rapid protective effect mediated by p38 MAPK in isolated isolated rat cardiomyocytes was not detectable below 30 rat hearts. The mechanism of p38 MAPK’s protection is mM menadione with DCF, we suspect that the level of not completely understood. Studies in CCL39 lung epimitochondrial ROS required for protection is quite low or thelial cells indicate that p38 MAPK activation causes confined to a small microcompartment. The protective phosphorylation of HSP27, a downstream target of p38 effect of menadione diminished at 10 mM, as might be MAPK associated with stress fiber stabilization, which expected if higher levels of ROS were toxic in cardiac mediates resistance against oxidative stress [38,39]. As cells. This biphasic effect has been documented before. shown in Fig. 7, mitochondrial ROS could activate p38 Turner et al. [23] reported that menadione levels up to 20 MAPK and stabilize stress fibers in a similar manner, mM caused resistance to apoptosis while this protective thereby conferring resistance to osmotic swelling and effect disappeared above 40 mM in H9c2 cardiac cells. attendant damage associated with ischemia in the heart. By Interestingly, low doses of the ROS generator bilirubin contrast, some studies have shown that activation of p38 protected neurons while high doses were toxic [36], and MAPK may be detrimental in the heart [40–43]. Schneider low levels of ROS enhanced survival of human aortic et al. [43] observed that SB202190, a p38 MAPK inhibitor, smooth muscle cells while higher levels were toxic [37]. reduced infarct size and enhanced ventricular function in Menadione can activate p38 MAPK in many cell types rat hearts subjected to ischemia. Saurin et al. [44] also including H9c2 cardiac cells [23,24]. Treatment with reported that inhibition of p38 MAPK reduced cell death in menadione (5–40 mM) for 4 h caused the activation of p38 rat ventricular myocytes subjected to simulated ischemia. MAPK and c-jun NH -terminal kinase (JNK) in H9c2 cells While the reason for differences between the latter findings 2 [23]. Our studies indicate that p38 MAPK is activated by and the present results and others previously documenting brief exposure to lower doses of menadione in H9c2 cells. a protective action of p38 MAPK [9–13] remains unclear
688 了了rgk./Cardlmutuar Rexcarch5226s!-5s9 G PROTEN blockade and that it is the ROS rather than the p38 MAPK RECEPTOR activation that protected the bearts.That.however.would not explain why the p38 MAPK inhibitor SB203580 blocks anisomycin's or menadione's protection.The most shraopt-H9P27 likely explaration then would be that anisomycin actrvates nos PnoDucnoN 38 MAPK by causing ROS production in the cell Clearly this is an isse for further study. Aeknowledgrment国 +cz·n0 This study was supparted by grant awards to SDC (AHA MTCCHONCRION 9802371 JMD (NIH/HL 206481 and MVC (NIH/ Fig.7.Pmpoued miochondrid ROS sigroling pehrucry We aegeet thot HL50688) G中olein coupled n6co厘另nalatio0asmk4 charnd cpenng and sultayactt rdean:of nilochrdrial 505 Michdi Ro0s release.which cas he mmicked ty dazocide (11/)and irhibned hy S:止o-aUue5HD,acthul小e3 S MAPK《p38cu.T卫e Referenees actiation nf p MAPK fen activates MAPKAPK2 (MKPK2)mhich then phosphoryfotes HSP27 to cause stablizznon of stress hibers during 】M可CR,rnngx Ri,Reirer KA,rconditian尾w的io +1i山 chemea a deln of lethal cell inury in ischemie myocardun. Ciru山m198%,74ll24-1136 recent evidence suggests that p3&MAPKe activation may Cohen MV.llaires C Downey It Isctemc pecendrtionirg:fmm he coupled to apopeasis whereas p38 MAPKB activatiun adennin:ceepuo K c Ama Rer Plyaiol 2000,62.79- 18. may be coupled to pnxective cellular responses [44-48]. ferlid K.Paucrk P.Yev-Yarmey V ct al.Canlopruocsive Isolated rat hearis were procected by 100 ng/ml rtechenism of diaaoode and its merctin wih mtnochondnal ansomycin (-300 nM)and SB203580 abolished this effiect in ths stody.While SB203580 is n widely used p38 tin Cirrilation 1997.81-1072-1088) MAPK inhibitor.other ertects have been reported includ- H1 Liu Y.Soto T.O'Rourke B.Marten E.Mtochondnal ATP.desen de puoaian chuarneb:novel effertoex of prerurdllimning,Citu ing INK inhibition and phospholipnse A (PLA-)activa- n19V72463-2469 tion (49.50].There is no evidence that P1A2 is imolved in |5月w%2ImG浅.rincioglu M et al Ischemic preoandmoting PC's mechanism,but JNK has been implicsted.Thus we Cepends on interction between mixchonerid K. 。chonnels a cannut eliminate the possibility that 5B203580 may have actin cyloskeleien Am J Prysial 1999276 H1351-H1368. acted on JNK rather than p38 MAPK.The mechanism of Pan T.Yarg X.Cntz SD et al.Upenng of mnochondral K anisomycin's activation of p38 MAPK is not known It is chanch uitec3c山e by pcrali%垂c位1d ak1 T Kes 2国，s74-66 beleved to cause nbotoxic stress by disrupting ribosomal I7]Sono T.Sascki N,Scharaseyon J.ORourke B,Marton E.Selocaive translation and inhibiting protein synthesis. Riboeoxic phermacrlogical arts implicale milechonkrial b not suerlem- stress,however,is seen following prolonged expooure ( rtal K. chonels in ischertc cardopronecnon.Crredlanon 20081012418-2423. h)with micramolar concentrations of anisomycin [51 By contrast,we see p38 MAPK activation and cardioprotee. comes frst.K tian after brief treatment with 500-fold lwer dases of the Re2301,516636 drug Furthermore.ansomyemn's protection was sensitive 9]Wenhretrer C.Lin (Cohen MV.DXuner IM.Phosphorylanon to MFG.suggesting a role for ROS.Recently.10 ng/ml o tyvooise 182 of p38 mloeen-octivuxd grotein kies:ouirdues anisomyein (-30 nM)produced ROS which actrvated p38 uih the protectian of precardtirning in the taHhit heart.J Mn Cell Cd81w7:292383-231. MAPK in PC12 oells [52].Our results are the first to [o]Maulik N,Yiohila T,Zu YL.dt al.lahe ni:ptotundilioning higeen suggest that anisomycin's protection,like that of tymoane kmase upnln a porentinl role fnr MAPKAI knase 2. menadione.is mediared hy the production of ROS in mJ,a159g,275.H1457-H1864 cardise cells.This observation raises several ineresting (11]Nikar A.Baires CP.Kim $o ct al.Iszhemic procandismting actrvnes MAPKAPKZ in the isolned ratbt heart evidence for possibilities.Firstly,perhaps it is activation of p38 MAPK invulveone of p38 MAPK.Ci:Re 200086.144-151. that causes ROS production ratber than the other way [17]Nikarn A.Cnhen MV.Crtx S1),Duzey IM S120180 an around.But that would not be compatible with cur ishbor of g38 MAPB,abolishes infarco-lmitin effect of tschemie observation that generalig ROS directly with menadione caused p38 MAPK activation in the H9c2 cells and that 2Kc95:466-471. MPG could block that activation.The second explaration [13]Mocuu M Bev GF,Yue Y.CiL SD.Ydlon DM.The p38 MAPK ithihtnt.SB5s0 abmgntes ischemie pracenditiaring in would be that anisomycin miry produce ROS as a non nat hean bu:timing i crical.Besiz Res Cardol 200095:472-478. specifie side effect which is unrelated to p38 MAPK [14]Cun E.Dua YN,Ben-Levy K,Cuien P.Mihadevan LC.Idetii-
688 Y. Yue et al. / Cardiovascular Research 55 (2002) 681 –689 blockade and that it is the ROS rather than the p38 MAPK activation that protected the hearts. That, however, would not explain why the p38 MAPK inhibitor SB203580 blocks anisomycin’s or menadione’s protection. The most likely explanation then would be that anisomycin activates p38 MAPK by causing ROS production in the cell. Clearly this is an issue for further study. Acknowledgements This study was supported by grant awards to SDC (AHA 980237), JMD (NIH/HL 20648) and MVC (NIH/ Fig. 7. Proposed mitochondrial ROS signaling pathway. We suggest that HL50688). G-protein coupled receptor stimulation causes mK channel opening ATP and subsequent release of mitochondrial ROS. Mitochondrial ROS release, which can be mimicked by diazoxide (DZ) and inhibited by 5-hydroxydecanoate (5HD), activates the p38 MAPK (p38) cascade. The References activation of p38 MAPK then activates MAPKAPK2 (MKPK2), which then phosphorylates HSP27 to cause stabilization of stress fibers during [1] Murry CE, Jennings RB, Reimer KA. Preconditioning with is- ischemia. chemia: a delay of lethal cell injury in ischemic myocardium. Circulation 1986;74:1124–1136. [2] Cohen MV, Baines CP, Downey JM. Ischemic preconditioning: from recent evidence suggests that p38 MAPKa activation may adenosine receptor to K channel. Annu Rev Physiol 2000;62:79– ATP be coupled to apoptosis whereas p38 MAPKb activation 109. may be coupled to protective cellular responses [44–48]. [3] Garlid K, Paucek P, Yarov-Yarovoy V et al. Cardioprotective Isolated rat hearts were protected by 100 ng/ml mechanism of diazoxide and its interaction with mitochondrial 1 ATP-sensitive K channels: possible mechanism of cardioprotec- anisomycin (|300 nM) and SB203580 abolished this effect tion. Circulation 1997;81:1072–1082. in this study. While SB203580 is a widely used p38 [4] Liu Y, Sato T, O’Rourke B, Marban E. Mitochondrial ATP-depen- MAPK inhibitor, other effects have been reported includ- dent potassium channels: novel effectors of preconditioning. Circuing JNK inhibition and phospholipase A (PLA ) activa- 2 2 lation 1998;97:2463–2469. tion [49,50]. There is no evidence that PLA2 is involved in [5] Baines CP, Liu GS, Birincioglu M et al. Ischemic preconditioning PC’s mechanism, but JNK has been implicated. Thus we depends on interaction between mitochondrial K channels and ATP actin cytoskeleton. Am J Physiol 1999;276:H1361–H1368. cannot eliminate the possibility that SB203580 may have [6] Pain T, Yang X, Critz SD et al. Opening of mitochondrial KATP acted on JNK rather than p38 MAPK. The mechanism of channels triggers the preconditioned state by generating free radianisomycin’s activation of p38 MAPK is not known. It is cals. Circ Res 2000;87:460–466. believed to cause ribotoxic stress by disrupting ribosomal [7] Sato T, Sasaki N, Scharaseyon J, O’Rourke B, Marban E. Selective pharmacological agents implicate mitochondrial but not sarcolem- translation and inhibiting protein synthesis. Ribotoxic mal K channels in ischemic cardioprotection. Circulation ATP stress, however, is seen following prolonged exposure (.6 2000;101:2418–2423. h) with micromolar concentrations of anisomycin [51]. By [8] Patel HH, Gross GJ. Diazoxide induced cardioprotection: what contrast, we see p38 MAPK activation and cardioprotec- comes first, K channels or reactive oxygen species. Cardiovasc ATP tion after brief treatment with 500-fold lower doses of the Res 2001;51:633–636. drug. Furthermore, anisomycin’s protection was sensitive [9] Weinbrenner C, Liu GS, Cohen MV, Downey JM. Phosphorylation of tyrosine 182 of p38 mitogen-activated protein kinase correlates to MPG, suggesting a role for ROS. Recently, 10 ng/ml with the protection of preconditioning in the rabbit heart. J Mol Cell anisomycin (|30 nM) produced ROS which activated p38 Cardiol 1997;29:2383–2391. MAPK in PC12 cells [52]. Our results are the first to [10] Maulik N, Yoshida T, Zu YL et al. Ischemic preconditioning triggers suggest that anisomycin’s protection, like that of tyrosine kinase signaling: a potential role for MAPKAP kinase 2. menadione, is mediated by the production of ROS in Am J Physiol 1998;275:H1857–H1864. [11] Nakano A, Baines CP, Kim SO et al. Ischemic preconditioning cardiac cells. This observation raises several interesting activates MAPKAPK2 in the isolated rabbit heart: evidence for possibilities. Firstly, perhaps it is activation of p38 MAPK involvement of p38 MAPK. Circ Res 2000;86:144–151. that causes ROS production rather than the other way [12] Nakano A, Cohen MV, Critz SD, Downey JM. SB203580, an around. But that would not be compatible with our inhibitor of p38 MAPK, abolishes infarct-limiting effect of ischemic observation that generating ROS directly with menadione preconditioning in isolated rabbit hearts. Basic Res Cardiol 2000;95:466–471. caused p38 MAPK activation in the H9c2 cells and that [13] Mocanu M, Baxter GF, Yue Y, Critz SD, Yellon DM. The p38 MPG could block that activation. The second explanation MAPK inhibitor, SB203580, abrogates ischemic preconditioning in would be that anisomycin may produce ROS as a non- rat heart but timing is critical. Basic Res Cardiol 2000;95:472–478. specific side effect which is unrelated to p38 MAPK [14] Cano E, Doza YN, Ben-Levy R, Cohen P, Mahadevan LC. Identifi-
了了rgsl./Cardymuuar Rrxcarch22S-5s9 669 io uf EnDotryc地u山vuled ltpe34SueS4 in aratink celb [34]Fuib>RA,Sie:sberzen C,Muplny E Diouide-iluced cudiop- m MAPKAP-2.nm19912：5-kI2 meection reqires sgaaling hmugh a rdex-onutive mecharisn 115]Baines CP.Goto M.Downey JM.Oxypen mdicals releesed durng Cire Res2m.8-9明 ichenic pecondiliotins cumrkibue tu cadiuetuection in the rHet (5]Olderbang 0,Qin Q Sleama AR cl u.Acrtylchuline leaib tu fioe heart.I Mol Cell Candiel 197:29207-216 radial produchon dependert on K.charnelr,Git proins.phos. l6TEu1,DAMn山G￥DEeN生山.Oxygon Ealicab cen induce phuidhlisooikl 3.and tyrine kirase.Curdiovse Res 2002.in inchemic preemnditioning in rabhit hearts Cine Res 199):80 743 74报 BS]Dor JS.Takahashi M,Femis CD et al Bilinbn.fomed by TI刀YErY,Kemu.M,Culun MV D时MClile SD.Meitalire Ei4iIkw-2,国T山5 t aIDl c山6e mimies the isfarct-imitine eflect of greconditionng in rat sTess injiry Proc Nat Acad Sri USA 1994.962445-2450 h24飞nJsw20128IH590-H595 [37]Bhana AK,Hen H,Sowden A Chaneroe S.Redox-ezulored liuY.阿M.ahenic pex8 nditirin吧td symaling by lactomyeeramidke in the proliferation of human stic infarctkon in nat heart.Am J Pbyuol 1992:26:111107-1112 m3hmse8ekJ4《hm19w72721562-15649 [19]Tong WF.Lee JL,Chiuu TI.The iude of lipid peiusilulion in (35]Hau J.Houle F,SpiLz DR.Laky 1 HSP27 pla:larylaliut meadree-medored teoccty in cardinrtyneytes I Mol Crll Caedinl red ated rsidance agnd acrin fagmeron and cell doth 1595,27.19明-2008 du8oNy61山trs强Cneg民es1966273-279. 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