Cnrdimnsrulnr FeserTh ELSEVIER Curdioveseular Research 57(20031 426-433 wwwebevar mm/l在izs Matrix metalloproteinase-2 mediates cytokine-induced myocardial contractile dysfunction Cindy Qun Gao",Grzegorz Sawicki",Wilma L.Suarez-Pinzon',Tamas Csont", Micczyslaw Wozniak,Peter Ferdinandy°，Richard Schulz,◆ "Deparmew o Pemtrles.Carafowarcnlar Nesearch Croap,4-62 Mfertoge Mecaf Merearch Censer.Uamersity of AMomm Eishwoo Aiberro. Cuenla T6G 252 "Dpursoe Parcolngy.Cardomrcakrerc Grap lierge Mehcaf Berearch Cvrer.Uanarn Arm.Gdeosron.r Cunonla T6G 252 "Dpormar nf Medeiv.Cardouaruar Brarch Grpp.4-62 Hortage Medeal Bere Cmvr.eray Aibvri:.Faewwne.Aibers. C6G252 “t of CAnvcal Chrutry,Mnchcal Lnreraaty用nehe,,Paland Dxrparew oy Wochemim玉.mwhc清Gw9,UGw灯Wd.,/wwy Rd位Augrd 3012.accepted40 cioher 2X位 Abstract Ohjerrive:Pro-intamaory cykines depress myocardial comtracrile funchon by enhancing peroxynimite producnon,ye the mochanism by which peruxynitrite does this is unknown.As malris mtalloproinases (MMPs)can be aclivalod by peruxynitrite and cun protoobtically clanve tropunin I in hearts,we dclermined whether this occurs in cylokine-inducod moucardial dysfunclion.Methods: Isolated workang rat hearts were pertused with butter contasng merteukin-1,imertern-y.and tumor necros tactor-u.Results: Cylukines induced a marked decline in mochanical findtion during 60-120 min of perfusiun.This decline wars aocompanicd hy incrcarad myocardil inducible NO syntlase aclivily and perfirale dilyroeine (a marker of perxynitrte),cumpared lo control hearts.Before the decline mecharical function there was echanced MMP.2 activity in the perfuate.This wis accompamed by decreased tismae levels of MMP.2.,tissue ichibitor of matrix metallopeoteinases-4 aed troponin I in cysolire-treaed hearts.The collazen cament of the heart was noc affected hy cylokine Ireaimenl.A ncilralizing,ani-MMP-2 anlibodly or the MMP inhihilors Ra31-9790 or PD166793 allenunkd the decline in myocardial function.Moreover.the MMP.2 antibody prevemed the decline in myocardial MMP.2 and troponin I levels. Cenclusious:Myocardial contractlle dysfunction caused by pro-icflammatory cytokines reats in MMP.2 activation and a decline in tissue inhihitor of matris mrtallopmteimeses-4 in the heant Tmoporin I is also a target for the proledlytie aetion of MMP-2 dunng acite heant fiilure triggered hy pro-infammaory cytokines Inhihion of MMPs may be a novel pharmarological segy for the tree of cue inflammaory heart disease. 2003 European Society of Cardiclogy.Published by Esevier Seience B.V.All nghts reserved Keyunbr.Couaclie funtlion Cykkine 1.Introduction wound healing.inflammation and tumor invasion.MMPs are synthesized in a latent form (zymogen cr pro-MMP) Matrix metalloproteinases (MMPs)are a family of zine- and are activated by proteolytie cleavage of an amino- dependent endopeptidases involved in remodeling the terminal domain or by coaformational changes induced by extracellular matrix during various physiological and denaturing agents or oxidative stress molecules such as pathological conditiors,such as embryonic develpment, peroxynitrile. NO nnd superoxide can renct a铺桑ditfur5io-limit mate to responding2h3.Te:+17452658;x+174B fomm peroxynitrite [1].Myocardial generation of perox- 973 -wa.dirers nichen时hulh区olberta ca (k Schuby Time for primary rnlew 22 days. 0008-5363/03/5-we fiont maer 2003 Euupean Socicty of Cadulgy.Pulsled bry Ebevicx Scicice B.V.All iigrs ieseived P11-90008-616310200719-8
Cardiovascular Research 57 (2003) 426–433 www.elsevier.com/locate/cardiores M atrix metalloproteinase-2 mediates cytokine-induced myocardial contractile dysfunction a b cb Cindy Qun Gao , Grzegorz Sawicki , Wilma L. Suarez-Pinzon , Tamas Csont , ´ d e a,b, Mieczyslaw Wozniak , Peter Ferdinandy , Richard Schulz ´ * a Department of Pediatrics, Cardiovascular Research Group, 4-62 Heritage Medical Research Center, University of Alberta, Edmonton, Alberta, Canada T6G 2S2 b Department of Pharmacology, Cardiovascular Research Group, 4-62 Heritage Medical Research Center, University of Alberta, Edmonton, Alberta, Canada T6G 2S2 c Department of Medicine, Cardiovascular Research Group, 4-62 Heritage Medical Research Center, University of Alberta, Edmonton, Alberta, Canada T6G 2S2 d Department of Clinical Chemistry, Medical University of Wroclaw, Wroclaw, Poland e Department of Biochemistry, Cardiovascular Research Group, University of Szeged, Szeged, Hungary Received 12 August 2002; accepted 4 October 2002 Abstract Objective: Pro-inflammatory cytokines depress myocardial contractile function by enhancing peroxynitrite production, yet the mechanism by which peroxynitrite does this is unknown. As matrix metalloproteinases (MMPs) can be activated by peroxynitrite and can proteolytically cleave troponin I in hearts, we determined whether this occurs in cytokine-induced myocardial dysfunction. Methods: Isolated working rat hearts were perfused with buffer containing interleukin-1b, interferon-g, and tumor necrosis factor-a. Results: Cytokines induced a marked decline in mechanical function during 60–120 min of perfusion. This decline was accompanied by increased myocardial inducible NO synthase activity and perfusate dityrosine (a marker of peroxynitrite), compared to control hearts. Before the decline in mechanical function there was enhanced MMP-2 activity in the perfusate. This was accompanied by decreased tissue levels of MMP-2, tissue inhibitor of matrix metalloproteinases-4 and troponin I in cytokine-treated hearts. The collagen content of the heart was not affected by cytokine treatment. A neutralizing anti-MMP-2 antibody or the MMP inhibitors Ro31-9790 or PD166793 attenuated the decline in myocardial function. Moreover, the MMP-2 antibody prevented the decline in myocardial MMP-2 and troponin I levels. Conclusions: Myocardial contractile dysfunction caused by pro-inflammatory cytokines results in MMP-2 activation and a decline in tissue inhibitor of matrix metalloproteinases-4 in the heart. Troponin I is also a target for the proteolytic action of MMP-2 during acute heart failure triggered by pro-inflammatory cytokines. Inhibition of MMPs may be a novel pharmacological strategy for the treatment of acute inflammatory heart disease. 2003 European Society of Cardiology. Published by Elsevier Science B.V. All rights reserved. Keywords: Contractile function; Cytokines 1. Introduction wound healing, inflammation and tumor invasion. MMPs are synthesized in a latent form (zymogen or pro-MMP), Matrix metalloproteinases (MMPs) are a family of zinc- and are activated by proteolytic cleavage of an aminodependent endopeptidases involved in remodeling the terminal domain or by conformational changes induced by extracellular matrix during various physiological and denaturing agents or oxidative stress molecules such as pathological conditions, such as embryonic development, peroxynitrite. NO and superoxide can react at a diffusion-limit rate to form peroxynitrite [1]. Myocardial generation of perox- *Corresponding author. Tel.: 11-780-492-6581; fax: 11-780-492- 9753. E-mail address: richard.schulz@ualberta.ca (R. Schulz). Time for primary review 22 days. 0008-6363/03/$ – see front matter 2003 European Society of Cardiology. Published by Elsevier Science B.V. All rights reserved. PII: S0008-6363(02)00719-8
C.Cu Cho el a.Carohurunealar Reatare 57(20031 426-433 427 ynitrite is acutely enhanced during reperfusion of the containing 1I mM glucose.5 mM pyruvate,100 uU/ml ichemic heart and coctrihutes to myocardial stunning insulin 1.75 mM Ca?',0.5 mM EDTA.0.1%bovine injury [2,3].More recently,we demonstrated that perox. serm albumin,and 0.3 mM L-tyrosine as described ynitrite is a major contributor to pro-inflammatory tytu (without pulmonary artery cannulatiun)[17].Cardiac work kine-induced myocardial dysfurction by concerted en (cardiac outputXpeak systolic nressure)was ued as an hancement in both superoxide and NO penerating setivities index of mechanical furetion After 20 min of oquilibra- in the heart 14].Peroxynitrite can react and modity vanous tion in the working mode,hearts were pertused for 120 bological molecules such as proteins,thiols,lipds,and min in the absence (Control.w-7)or presence of 5 ng/ml nuclic acids 5]Indeed,peroynitrite was shown to be a human IL-1B.9 ng/ml rat IFN-y.and 20 ng/ml human potent activator of'pro-MMPs without proccolytic removal TNF-a (Cytokines.-10)which was added as a bolus to of the propepeide domain,including pro-MMP-1 [6].pro the perfusion buffer at r=0 min.This mixturre of pro- MMP8I间po-MMP.9I间and pro-MMP.2IMe- infammatory cytokines mimics those measured in the while,peroxynitrite may also aler the structural and systemic inflammatory response and advanced heart filure binding characteristics of endogenous tisse inhibitors of syndromes,enhances myocardial peroxynitrite generation MMPs (TIMPs),thus inhibiting their ability to prevent and causes a rapid loss in myocurdial contractile function MMP activation [8].This imhalance between MMPs and within 1-2 h of perfusion [4]Perfusae samples were TIMPs favors the activation of MMPs and a proteclytie tsken al -1.1.30,60.90.and 120 min to determine MMP environmert within the cell.The upreguilation of MMP activities and dityrosie levels.The ventricles were frozen activity and downregulabon of TIMP action has been after 120 min of perfusion and processed as described [161 shown to occur in pathological alterations of myocardial Additional series of hearts were perfissed in the presence of ischemia [9].infarct 10]and dilated cardiomnyepathies cytokines for 120 min either with MMP inhibitors (Ro31- [11.12]The mechanism hy which these changes occur 9790,3μMPD166793.2M).DMS0 ehicle(03% during these processes are,however,not well known. v:v).a peptide generated.neutralizing MMP-2 nntibody Changes in MMP nctivity or expression that nffect (30 ag/ml)or unrelmed IgG (30 ug/ml)The specificity matrix remodeling are normally thought to occur on the and neutmlizing activity of the MMP.2 ancibody was time scale of hours to days However,there is increasing previously determined [18].PD166793 hns the following evidence that MMPs can also very rapidly regulate diverse reported nM ICso values (in parentheses)for MMP-I cellular functions within minutes,including platelet aggre (6100以MMP2(47.MP-3(I2xMP7(s100)ad gation [13].vascular tune [14]and modulation of the MMP-9 (9900)[191 whereas for Ro31-9790 they are inflammatory response [15],independent of their effects on MMP-I (3),MMP-2 (7)and MMP-9 (12)[20].Anocher the exctracellular matrix.Recently we discovered that in set of hearts was briedly perfused for 15 min with Krebs- cardine myocytes MMP-2 co-localizes with troponin I Herseleit buffer at a constant hydrostatic pressure of 7o (Inl)a regulatory clement of the actin-myosin controctile mmHg to allow for the washout of blood in order to apparatus.and that t is susceptible to Tnl proteolytie measure Inl content. degradation by MMP-2 1161 During neunte ischemin-re perfusion ingury of'the heart the proteolytic degradation of 22.P'ratione时tn路 Tnl by MMP.2 and mechanical dysfunctiun were pre vented by inhihiting MMP activity [16].Therefure,this For zymography and Western hlo experiments,the study was desigred to imvestigate whether there is an acute frozen vertricular tissue was powdered with a pestle and rule of MMPs in the heart in the setting of pro-inflamm mortar cooled to the temperature of liquid N,and then tory cytokine-induced myocardial dysfunction. hamogenized by sonicatian in 50 mM Tris-HCI (pH 7.4) containing3.I mM sucros线.ImMD1T,10μg/ml leupeptin,10 ug/ml soybean trypsin mhibitoe.2 ug/ml 2.Methods aprotimin,and 0.1%Triton X-100.The bomogerate was centrifiuged at 8500xg al 4C for 5 min and the superna- This investigaion conforms with The Gmide to the Care tamt was collected.For delermination of NO synthase and U'se of Experimentai Animais published by the actrvities Triton X-100 was omitted in the bomogenizstion Canadian Council on Animal Care (revised 1993) huffer and the samples were centrifiuged at 1000xg at 4C for 5 min 21.Lsolated heart prepuration 23ase程of NO syntha5ei Male Sprague-Dawley rats (250-330 g)were anes- thetized by intraperoreal injection of sodium penobarbi- NO synthase activities in the heart extracts (n-5 in each tal(60 mg/kg).The hearts were isolated and paced a 300 group)were determired frem the conversion of -[C]ar- beats per minule during perfusion at 37C as working ginine to LC]citrulline as deseribed [4,17].The protein hearts with 110 ml recirculating Krebs-Henseleet solutian concentration of heart extracts wes determined with the ue
C. Qun Gao et al. / Cardiovascular Research 57 (2003) 426–433 427 ynitrite is acutely enhanced during reperfusion of the containing 11 mM glucose, 5 mM pyruvate, 100 mU/ml 21 ischemic heart and contributes to myocardial stunning insulin, 1.75 mM Ca , 0.5 mM EDTA, 0.1% bovine injury [2,3]. More recently, we demonstrated that perox- serum albumin, and 0.3 mM L-tyrosine as described ynitrite is a major contributor to pro-inflammatory cyto- (without pulmonary artery cannulation) [17]. Cardiac work kine-induced myocardial dysfunction by concerted en- (cardiac output3peak systolic pressure) was used as an hancement in both superoxide and NO generating activities index of mechanical function. After 20 min of equilibrain the heart [4]. Peroxynitrite can react and modify various tion in the working mode, hearts were perfused for 120 biological molecules such as proteins, thiols, lipids, and min in the absence (Control, n57) or presence of 5 ng/ml nucleic acids [5]. Indeed, peroxynitrite was shown to be a human IL-1b, 9 ng/ml rat IFN-g, and 20 ng/ml human potent activator of pro-MMPs without proteolytic removal TNF-a (Cytokines, n510) which was added as a bolus to of the propeptide domain, including pro-MMP-1 [6], pro- the perfusion buffer at t50 min. This mixture of proMMP-8 [6], pro-MMP-9 [6] and pro-MMP-2 [7]. Mean- inflammatory cytokines mimics those measured in the while, peroxynitrite may also alter the structural and systemic inflammatory response and advanced heart failure binding characteristics of endogenous tissue inhibitors of syndromes, enhances myocardial peroxynitrite generation MMPs (TIMPs), thus inhibiting their ability to prevent and causes a rapid loss in myocardial contractile function MMP activation [8]. This imbalance between MMPs and within 1–2 h of perfusion [4]. Perfusate samples were TIMPs favors the activation of MMPs and a proteolytic taken at 21, 1, 30, 60, 90, and 120 min to determine MMP environment within the cell. The upregulation of MMP activities and dityrosine levels. The ventricles were frozen activity and downregulation of TIMP action has been after 120 min of perfusion and processed as described [16]. shown to occur in pathological alterations of myocardial Additional series of hearts were perfused in the presence of ischemia [9], infarct [10] and dilated cardiomyopathies cytokines for 120 min either with MMP inhibitors (Ro31- [11,12]. The mechanism by which these changes occur 9790, 3 mM; PD166793, 2 mM), DMSO vehicle (0.3% during these processes are, however, not well known. v:v), a peptide generated, neutralizing MMP-2 antibody Changes in MMP activity or expression that affect (30 mg/ml) or unrelated IgG (30 mg/ml). The specificity matrix remodeling are normally thought to occur on the and neutralizing activity of the MMP-2 antibody was time scale of hours to days. However, there is increasing previously determined [18]. PD166793 has the following evidence that MMPs can also very rapidly regulate diverse reported nM IC values (in parentheses) for MMP-1 50 cellular functions within minutes, including platelet aggre- (6100), MMP-2 (47), MMP-3 (12), MMP-7 (8100) and gation [13], vascular tone [14] and modulation of the MMP-9 (9900) [19], whereas for Ro31-9790 they are inflammatory response [15], independent of their effects on MMP-1 (3), MMP-2 (7) and MMP-9 (12) [20]. Another the extracellular matrix. Recently we discovered that in set of hearts was briefly perfused for 15 min with Krebs– cardiac myocytes MMP-2 co-localizes with troponin I Henseleit buffer at a constant hydrostatic pressure of 70 (TnI), a regulatory element of the actin–myosin contractile mmHg to allow for the washout of blood in order to apparatus, and that it is susceptible to TnI proteolytic measure TnI content. degradation by MMP-2 [16]. During acute ischemia–reperfusion injury of the heart the proteolytic degradation of 2 .2. Preparation of heart extracts TnI by MMP-2 and mechanical dysfunction were prevented by inhibiting MMP activity [16]. Therefore, this For zymography and Western blot experiments, the study was designed to investigate whether there is an acute frozen ventricular tissue was powdered with a pestle and role of MMPs in the heart in the setting of pro-inflamma- mortar cooled to the temperature of liquid N and then 2 tory cytokine-induced myocardial dysfunction. homogenized by sonication in 50 mM Tris–HCl (pH 7.4) containing 3.1 mM sucrose, 1 mM DTT, 10 mg/ml leupeptin, 10 mg/ml soybean trypsin inhibitor, 2 mg/ml 2. Methods aprotinin, and 0.1% Triton X-100. The homogenate was centrifuged at 85003g at 4 8C for 5 min and the supernaThis investigation conforms with The Guide to the Care tant was collected. For determination of NO synthase and Use of Experimental Animals published by the activities Triton X-100 was omitted in the homogenization Canadian Council on Animal Care (revised 1993). buffer and the samples were centrifuged at 10003g at 4 8C for 5 min. 2 .1. Isolated heart preparation 2 .3. Measurement of NO synthase activities Male Sprague–Dawley rats (250–330 g) were anesthetized by intraperitoneal injection of sodium pentobarbi- NO synthase activities in the heart extracts (n55 in each 14 tal (60 mg/kg). The hearts were isolated and paced at 300 group) were determined from the conversion of L-[ C]ar- 14 beats per minute during perfusion at 37 8C as working ginine to L-[ C]citrulline as described [4,17]. The protein hearts with 110 ml recirculating Krebs–Henseleit solution concentration of heart extracts was determined with the use
428 C.Dar GGau el ul.Carsluranular Rescarck 57(2003)426-433 of a bicinchoninic acid assay utilizing bovine serum drying the hydrolysale.Liquid chromstography (Eclipse al止min as a stardard. XDB-Cu column)/mass spectrometry analysis was per- formed on a Hewlett-Packard (series 1100.Allanta GA. 2.4.Mearrement of cardiac peroxynitrite gemeration USA)mass selectrve detector,montoring the ions with m上0fI88 We have previoushy detemmined that the measurement of dityrosine in heart perfusate,formed by the reaction of 2 8.Statsticoi aahsis peroxynitrite with tyrosine can he ued as an estimale of peroxynitrite generation [2,4].Perfusite samples taken at Results are expresed as mean+S.E M.for hearts As either I,30.60,90 or 120 min were assinyed by spec appropriate,Student's es,one-way ar two way (simple trodluorometry for dityrosine levels a described 21. or repented measures)ANOVA were used Differenoes were considered sipnificant at P<D05. 2.5 Mfeaswremen nf MMPs hy symugrophy Gelatin zymography was performed as described [18] 表民esl国 Briefly.perfsate samples or heart extracts (0 ug protein) were applied to 7.5%polyacrylamide gel copolymeriped Fig.IA shows the time course of changes in cardiac with 2 mg/ml gelatin.Afler electropboresis,gels were finction measured cardiac work.in control and cyto- rinsed three times for 20 min esch in 2.5%Triton X-100 in kine-treated hearts.Cardsae work in control hearts re- order to remove SDS.Then the gels were washed twice in mained stable for the 120 min of petfuson.while cyto- incubation bulfer (50 mM Trs-HCI.5 mM CaCl.150 kine-treated hearts showed a significant loss m cardiac mM NaCl and 0.05%NaN,for 20 min each at room work between 60 and 120 min of perfusion Neutralizing temperture and then incubated in incubation huffer at 37'C for either 18 or 48 h for the perfusate and heart A. extract samples,respectively.The gels were stained in 2% Coomasssie Brilliant blue G.25%methanul,10%acetic neid for 2 h and then destained for I h in 2%methanol- HI 4%ncetic seid.Zymograms were scanned and the band imensities were annlyzed using Sigmagel softwnre.MMP activities were expressed as a percentape of the MMP-2 standard (HT 1080 cell conditioned medium) 2.6.Western biot anolyses The heart extract was diluled with protein sample bufler (30%v/v glyoerol,2%w/v SDS.0.13 M Tns,0.1 mg/ml brumphenol blue,and 4%v/v mercaptoethanol,pH 6.8) boiled for 5 min and then stored at -20C until assay. Then,I or 20 ug protein wus applied to 12%poly- acrylamide gels and electrophcresed under reducing con ditions.After electrophoresis,samples were electroblotted onto polyvinylidene membranes (Bio-Rad Lnboratorics. Hereules,CA.USA)and probed for TIMP-4 (polyclonal nnti-mt TIMP-4 antibody.Chemicon.Temeculn.CA.USA) or for Tnl (anti-human Tnl nntibody.clone 81-7.Spectml Diagnosties.Tororto,Canada). 2.7 Collagen content 20 91 The collagen contert in heart tissue was determined by ThN Onn0 mass spectrometric analysis for 4-hydroxyproline [21].The Fig 1.Time course of chonges in cordiaz work in isoloed working rat frozen powdered heart tissue was freeze driod and an herts (A)Cadiac work of co山ula-)al cyiokin-beakod匀l imeral standard (N-methyl-a-proline)in 6 M HCL was n-I01 isalmed he及（月Specrhe MMI-2t办Ah.0nCl. added to the sample.Each sample was then hydralyzed n=4)山dlihol cyouki-udd年ewL,lbc2s山ul 04w=4 eserted an igniican✉◆Pc05r0mih山起 overnight at 115"C.The o-butyl ester derivatives were within the same group oneway repened measres ANDVAl:.0 prepared with 10%BF,butanol for 30 min at 120'C afier verua cunbol (Sladnt's il)
428 C. Qun Gao et al. / Cardiovascular Research 57 (2003) 426–433 of a bicinchoninic acid assay utilizing bovine serum drying the hydrolysate. Liquid chromatography (Eclipse albumin as a standard. XDB-C column)/mass spectrometry analysis was per- 18 formed on a Hewlett-Packard (series 1100, Atlanta, GA, 2 .4. Measurement of cardiac peroxynitrite generation USA) mass selective detector, monitoring the ions with m/z of 188. We have previously determined that the measurement of dityrosine in heart perfusate, formed by the reaction of 2 .8. Statistical analysis peroxynitrite with tyrosine can be used as an estimate of peroxynitrite generation [2,4]. Perfusate samples taken at Results are expressed as mean6S.E.M. for n hearts. As either 1, 30, 60, 90 or 120 min were assayed by spec- appropriate, Student’s t-test, one-way or two-way (simple trofluorometry for dityrosine levels as described [2]. or repeated measures) ANOVA were used. Differences were considered significant at P,0.05. 2 .5. Measurement of MMPs by zymography Gelatin zymography was performed as described [18]. 3. Results Briefly, perfusate samples or heart extracts (20 mg protein) were applied to 7.5% polyacrylamide gel copolymerized Fig. 1A shows the time course of changes in cardiac with 2 mg/ml gelatin. After electrophoresis, gels were function, measured as cardiac work, in control and cytorinsed three times for 20 min each in 2.5% Triton X-100 in kine-treated hearts. Cardiac work in control hearts reorder to remove SDS. Then the gels were washed twice in mained stable for the 120 min of perfusion, while cytoincubation buffer (50 mM Tris–HCl, 5 mM CaCl , 150 kine-treated hearts showed a significant loss in cardiac 2 mM NaCl and 0.05% NaN ) for 20 min each at room work between 60 and 120 min of perfusion. Neutralizing 3 temperature and then incubated in incubation buffer at 37 8C for either 18 or 48 h for the perfusate and heart extract samples, respectively. The gels were stained in 2% Coomassie Brilliant blue G, 25% methanol, 10% acetic acid for 2 h and then destained for 1 h in 2% methanol– 4% acetic acid. Zymograms were scanned and the band intensities were analyzed using Sigmagel software. MMP activities were expressed as a percentage of the MMP-2 standard (HT 1080 cell conditioned medium). 2 .6. Western blot analyses The heart extract was diluted with protein sample buffer (30% v/v glycerol, 2% w/v SDS, 0.13 M Tris, 0.1 mg/ml bromphenol blue, and 4% v/v mercaptoethanol, pH 6.8), boiled for 5 min and then stored at 220 8C until assay. Then, 1 or 20 mg protein was applied to 12% polyacrylamide gels and electrophoresed under reducing conditions. After electrophoresis, samples were electroblotted onto polyvinylidene membranes (Bio-Rad Laboratories, Hercules, CA, USA) and probed for TIMP-4 (polyclonal anti-rat TIMP-4 antibody, Chemicon, Temecula, CA, USA) or for TnI (anti-human TnI antibody, clone 81-7; Spectral Diagnostics, Toronto, Canada). 2 .7. Collagen content The collagen content in heart tissue was determined by mass spectrometric analysis for 4-hydroxyproline [21]. The Fig. 1. Time course of changes in cardiac work in isolated working rat hearts. (A) Cardiac work of control (n57) and cytokine-treated (cyt, frozen, powdered heart tissue was freeze dried and an n510) isolated hearts. (B) Specific MMP-2 antibody (Ab, 30 mg/ml, internal standard (N-methyl-L-proline) in 6 M HCL was n54) abolished cytokine-induced myocardial depression, whereas control added to the sample. Each sample was then hydrolyzed IgG (n54) exerted no significant effect. * P,0.05 versus 0 min value overnight at 115 [ 8C. The o-butyl ester derivatives were within the same group (one-way repeated measures ANOVA); P,0.05 prepared with 10% BF -butanol for 30 min at 120 8C after versus control (Student’s t-test). 3
C.Oar GGau cl ul.Curcliurarecnlar Rscarek 57(200)425-433 49 anti-MMP-2 antibedy [18]but not urrelated IgG prevented erhanced release of 72 kDa activity ino the coronary the cytokine-induced loss of cardiac work (Fig.IB). eflluent there was a significat decreasse in its content in Fig.2A shows that the isolated working rat beart heart tissue (by 44%)at the end of perfusion and this continuously releases MMP-2 into the recirculsting perfu- decrease was attenuated in hearts treated with ami MMP-2 sate.Gelstinolytic activities were detected in the zymog- antibody (Fig 3A and B). rams nt 75.72,and 62 kDa correspooding to a rodent- The MMP inhibitors Ro31-9790 (0.01-3 uM)and specific glycosylated MMP-2 (Chris Overall,University of PD166793 (02-20 HM)concentration-dependemly in- British Columbia,personal communication)pro-MMP.2 hihited the 72-kDa gelatinalytic activity of perfusate and MMP.2,respectively,by comparison with the human samples taken from aerohically perfused hearts (dta no HT1050 cell-derived standard.The 72-kDa activity consti- shown).We also tested their effects on cytakine-induced tutod the major gelatirase activity.In perfusate from changes in cardise work.Ro31-9790 (3 uM).PD166793 control hearts there ts a time-dependent aceumulation of 72 (2 uM).and anti-MMP-2 antibody (30 ug/ml)aterusted kDa sctivity within the first hour of perfusion.Addition of the cytokine-induced loss in cardiac work as measured at cytokines markedly inereasod pertusate 72 kDa nctivity 120 min of perfision (Fie.4).The vehicle for both MMP especinlly in the first 60 min of'pertusion (Fig.2B and C) inhibitors was withour effect (Fig.4). This preceded the onset of the delayed depression in Since the hnlance between MMPs and TIMPs mipht be cardise work.which was first significantly impaired in the altered in an inflammatory envirurment.and TIMP-4 is a cytokine group at 90 min perfusion (Fig.IA).There was a potent inhibitor of MMP-2 [22]and abundantly expressed similar increase in 61 kDa activity in the perfusate from in the beart [23].we then determined myocardial TIMP-4 cytokine trealed hearts,however.this was not within a level.TIMP-4 was detected in control hearts and its level quantiliable range (data not shown).Concomitant wih the was significantly decreased by 54%in cytokine-tresed eart本(Fig.5) To evaluate whether the extracellular matrix of the heart control cyt wus affected in resporse to cytokines we measured total collagen by determining 4.hydroxyproline content.In control,cyokine and cytokine plus anti-MMP.2 antibody groups the amount of 4-hydroxyproline was not signfi- c0 ntly diffcrent10.58±0.10.0.66±0070nd06500g ug/me dry tissue weight,respeetively). St·Ab 0 3000010 72 4081 室0 00 B 10 30 120 Iig (A)Reprertative zymegram rwals zresencn nd 74.72-.and 52-kDa zclatiadlyuc aztivities in perfusone fort tsoloced rat heorts afler control Ab 0 in uf ac比e perfesion.Sd inictex stmdard (hurren H打103Oal sondmtoned medium)(Represemacme togramts of pertisare tirte 3.(AI Represenatve zymogram of MMIP-2 acnvry in heart nsae couse sumsles fium ourtrul anal t ryoulin-ealod feyt)heL (C) urps ufler 2 h pefipion Saripes fiun twu differonr heeta in cuch Dersionetric arolyuis of speciir 2 kDh erlinelytic artivity in gst导me shown图Detsinnetric ara小is of sperife拉k perfasne from como (n)and cytokmetreaed (cyt10)hears gelnnolytie Ectivity in all heort tissue sempkes.+PeD.05 versus cortrol 4P505(wEdE日Eu ANOAL (use-way ANOVA,n-7-10 pr goupl
C. Qun Gao et al. / Cardiovascular Research 57 (2003) 426–433 429 anti-MMP-2 antibody [18] but not unrelated IgG prevented enhanced release of 72 kDa activity into the coronary the cytokine-induced loss of cardiac work (Fig. 1B). effluent there was a significant decrease in its content in Fig. 2A shows that the isolated working rat heart heart tissue (by 44%) at the end of perfusion and this continuously releases MMP-2 into the recirculating perfu- decrease was attenuated in hearts treated with anti MMP-2 sate. Gelatinolytic activities were detected in the zymog- antibody (Fig. 3A and B). rams at 75, 72, and 62 kDa corresponding to a rodent- The MMP inhibitors Ro31-9790 (0.01–3 mM) and specific glycosylated MMP-2 (Chris Overall, University of PD166793 (0.2–20 mM) concentration-dependently inBritish Columbia, personal communication), pro-MMP-2 hibited the 72-kDa gelatinolytic activity of perfusate and MMP-2, respectively, by comparison with the human samples taken from aerobically perfused hearts (data not HT1080 cell-derived standard. The 72-kDa activity consti- shown). We also tested their effects on cytokine-induced tuted the major gelatinase activity. In perfusate from changes in cardiac work. Ro31-9790 (3 mM), PD166793 control hearts there is a time-dependent accumulation of 72 (2 mM), and anti-MMP-2 antibody (30 mg/ml) attenuated kDa activity within the first hour of perfusion. Addition of the cytokine-induced loss in cardiac work as measured at cytokines markedly increased perfusate 72 kDa activity 120 min of perfusion (Fig. 4). The vehicle for both MMP especially in the first 60 min of perfusion (Fig. 2B and C). inhibitors was without effect (Fig. 4). This preceded the onset of the delayed depression in Since the balance between MMPs and TIMPs might be cardiac work, which was first significantly impaired in the altered in an inflammatory environment, and TIMP-4 is a cytokine group at 90 min perfusion (Fig. 1A). There was a potent inhibitor of MMP-2 [22] and abundantly expressed similar increase in 64 kDa activity in the perfusate from in the heart [23], we then determined myocardial TIMP-4 cytokine treated hearts, however, this was not within a levels. TIMP-4 was detected in control hearts and its level quantifiable range (data not shown). Concomitant with the was significantly decreased by 54% in cytokine-treated hearts (Fig. 5). To evaluate whether the extracellular matrix of the heart was affected in response to cytokines we measured total collagen by determining 4-hydroxyproline content. In control, cytokine and cytokine plus anti-MMP-2 antibody groups the amount of 4-hydroxyproline was not signifi- cantly different (0.5860.10, 0.6660.07 and 0.6560.08 mg/mg dry tissue weight, respectively). Fig. 2. (A) Representative zymogram reveals presence of 75-, 72-, and 62-kDa gelatinolytic activities in perfusate from isolated rat hearts after 60 min of aerobic perfusion. Std indicates standard (human HT 1080 cell conditioned medium). (B) Representative zymograms of perfusate time Fig. 3. (A) Representative zymogram of MMP-2 activity in heart tissue course samples from a control and a cytokine-treated (cyt) heart. (C) samples after 2 h perfusion. Samples from two different hearts in each Densitometric analysis of specific 72 kDa gelatinolytic activity in group are shown. (B) Densitometric analysis of specific 72 kDa perfusate from control (n57) and cytokine-treated (cyt, n510) hearts. gelatinolytic activity in all heart tissue samples. * P,0.05 versus control * P,0.05 (two-way repeated measures ANOVA). (one-way ANOVA, n57–10 per group)
430 C.Par Gav er ul.Cursliuraneular Reacarck 57(2003)435-433 50 the mechanical dysfurctiun of the heart fullwing is chemia-reperfusion injury [16].We therefure measured the level of Tnl as a target for the action of MMP-2 in the 2-h perfused heart groups (Fig.6).In cytokine treated hearts there was a dramstic reduction in the level of 31 kDa Tnl compared to control Addition of either ami-MMP-2 antibody or the MMP inhihitor PD 166793 to the cviokine treated hearts prevented the loss in Tnl coment.Indeed the level of Tnl in cytokine plus anti-MMP-2 antibody treated hearts was significantly higher than in cortrol hearts.As a 2-h period of'isolsted workme heart perfusion per se is a mild oxidative stress 171.we therefore analyzed an 卡中中 additional set of hearts perfused acrobieally for only 15 ++州 min in vitro to wash them free of blood.Indeed the level of contral vohiclg PD Ro Ab Tnl in these hearts was comparable to the 2-h isolaned Fg.4.Cardo work remaiine atter 2 h pertusion measared in control working hearts perfused with cytokines plus the anti- andl ryuukite-rod fyt)ral lst b und cfb uf MMP.2 inkibilpex MMP-2 antibudy ()166791.2 uM.Pl.Ro 1-9791.3 uM Rol.their wzhide (DMsOI ar To confirm our previous results that mvocardial induc- UMMP2 ar.bedy(30Hg/El,A味.”Pc00 s versus comrd (one4w ible NO synthase activity and peroxynitrite production ANOVA,n-4-10 per goupl were upregulated in the heart by pro-inflammatory cyto- kines [4].we measured NO synthase activities in some We recently showed that there is intrapellular co-locali- hearts and perfusnte dityrosine levels In ventricular tissue zation of MMP.2 with troponin I (Tnl),the regulatory from henrts perfused for 120 min,the Cadependent NO element of contractile proteins within the surcomere of the candisc myocyte [16].We also found that Tnl is suxeptible synthase activity (between 1-2 pmol/min/mg protein)was to proeolytic degradation by MMP-2 which contributes to 15 min Std control cyt cyt+PD cyl+Ab pectuxiun Std control g 31 RDa 23 120 100 80 40 6 20 20 16min PO 山 t Fig 6.(A)Represerlative Westem blots foc woporin I is heuts (e=2 htpg四p以Hter回fuod fi eiher上h in uorking mode as Fg.5.(A]Represemacve westem blts r TIMP-4 mn heores thor comtrol h3飞mn●e presence of cy3 ines Icytl,oTa动adaition orPD corol and cylikins-livuled (oyl)nul lae b flt 120 ttin pefipion (-4 166753 (eyt+PD]ut uli-MMP.2 alibuly (yi+All.Aiube suup of rt线Sid denores TIMI-eded nbtaired fmom rt myncardial tio网n ht水ug理hnefhy perfuord onhy fre I5 mn fer comgarison个nh时 (B)Densitomerie anelysis of TIXP-4 wesra blts *Pe0D5 versus (B)Derosikomemrie ardlysis of 31 kDa troponin I band irtersity =4-7 comrol (Stadem:'x i-ev) hFgs甲以◆Pc05 venis cutnd (n-uDAO
430 C. Qun Gao et al. / Cardiovascular Research 57 (2003) 426–433 the mechanical dysfunction of the heart following ischemia–reperfusion injury [16]. We therefore measured the level of TnI as a target for the action of MMP-2 in the 2-h perfused heart groups (Fig. 6). In cytokine treated hearts there was a dramatic reduction in the level of 31 kDa TnI compared to control. Addition of either anti-MMP-2 antibody or the MMP inhibitor PD 166793 to the cytokine treated hearts prevented the loss in TnI content. Indeed the level of TnI in cytokine plus anti-MMP-2 antibody treated hearts was significantly higher than in control hearts. As a 2-h period of isolated working heart perfusion per se is a mild oxidative stress [17], we therefore analyzed an additional set of hearts perfused aerobically for only 15 min in vitro to wash them free of blood. Indeed the level of TnI in these hearts was comparable to the 2-h isolated working hearts perfused with cytokines plus the anti- Fig. 4. Cardiac work remaining after 2 h perfusion measured in control and cytokine-treated (cyt) rat hearts and effects of MMP-2 inhibitors MMP-2 antibody. (PD166793, 2 mM, PD; Ro31-9790, 3 mM, Ro), their vehicle (DMSO) or To confirm our previous results that myocardial inducanti-MMP-2 antibody (30 mg/ml, Ab). * P,0.05 versus control (one-way ible NO synthase activity and peroxynitrite production ANOVA, n54–10 per group). were upregulated in the heart by pro-inflammatory cytokines [4], we measured NO synthase activities in some We recently showed that there is intracellular co-locali- hearts and perfusate dityrosine levels. In ventricular tissue zation of MMP-2 with troponin I (TnI), the regulatory 21 from hearts perfused for 120 min, the Ca -dependent NO element of contractile proteins within the sarcomere of the synthase activity (between 1–2 pmol/min/mg protein) was cardiac myocyte [16]. We also found that TnI is susceptible to proteolytic degradation by MMP-2 which contributes to Fig. 6. (A) Representative Western blots for troponin I in hearts (n52 hearts per group). Hearts were perfused for either 2 h in working mode as Fig. 5. (A) Representative western blots for TIMP-4 in hearts from control hearts or in the presence of cytokines (cyt), or with addition of PD control and cytokine-treated (cyt) rat hearts after 120 min perfusion (n54 166793 (cyt1PD) or anti-MMP-2 antibody (cyt1Ab). Another group of each). Std denotes TIMP-4 standard obtained from rat myocardial tissue. hearts were briefly perfused only for 15 min for comparison (open bar). (B) Densitometric analysis of TIMP-4 western blots. * P,0.05 versus (B) Densitometric analysis of 31 kDa troponin I band intensity (n54–7 control (Student’s t-test). hearts per group). * P,0.05 versus control (one-way ANOVA)
C.Oaw Gav el ul.Carsiurancalar Rescarek 57(2003)436-433 431 MMP-2 activity and the level of its endogenous irhibitor TIMP-4 in heart tissue were reduced in cytokine-tresed hearts.The collagen content of the heart was unaltered by cytokine trealmert,however.the level of Tnl was di- minished.Pharmacological inhibitors of MMP oc a neutral- izing MMP.2 antibody procected the heart from the lss in Tnl as well as the myocardial dysfunction caused by cytokines. We have recently shown using this model of acute myocardial contractile dysfunction that pro-inflammatory cytokines enhance the myocardal generation of the pro- oxidant spocics peroxyntrite through an enhancement in both superoxide and NO genernting enzyme activities, Ca?+dependent Cu2+.independent which are topether responsible for the depression in contractile function [4]This wns confirmed in the present study with the enhanced inducihle NO synthase nctivity in 129 heart tissue and perfusale dityrosine level.an indicator of peroxynitrite generation [2.4].We also recenly found that 10 the activation and rekase of MMP-2 from the heart contributes to another oxidant stress induced injury to the heart caused by ischemsa and reperfusion [18].The oxidant stress of acute myocardial ischemia-reperfusion injury is also mediaed by the generation of peroocynitrite [2,3].As MMPs can be activated by oxidant stress,we determined here whether tytokine-induced cardiac dysfunction is also mediated through MMPs In accordance with our previous report 18].the main eclatinolyte activity in eororary ettluent from the nerobi- 30 0 120 Timo (min) enlly perfised heart is 72 kDn.corresponding to pro- MMP-2.A rapid and significant inerease in 72 kDa activitv Fw.ZCd”小endeat wd Cu'”i:peieal aiti:uide c was ohserved within the first hour in the perfusmte from activitiex in wertricube honopmakes fom cortml and cytokine-tealed hearts expesed to cytokines along with some enhanced cyti working rat hears at 120 min perfusion and effect of MMP-2 release of 64 kDa activity corresponding to MMP-2.albeit artiluly (Ah,n-5sguu◆Pcn05aald.(B影Tinc course of chanpes in perfiale ditymeine level n comtml (o-7)and at a reduced leveL Cytokine treatment caused a clear-cut eyukise-treuted hexts [o=10 PE0.05 versis I mit vlue lone-wry depletion of MMP-2 actrvity in the myocardium.Pro- ANOVA MMPs may be actrvaed by breaking a zinc-cysteine bond in the catalytic centre [24],which then exposes the catalytic site followed by proceolytic activation,or through not significanatly different between control and cytokins. oxidant-induced conformational changes [6 Previous treated hearts (Fig.7A).In cuntrast,cytokine treatment studies suggested oxygen free radicals may he capable af resulted in a near seven-fold increase in Ca*'independent activating matrix metallopruteinases in perfused rat hearts ndivity.which was not abolishod by anti-MMP-2 anti- 125]Indeed.the powerful oxidant peroxynitrite ean acti- body.In accordnnce with the inerease in Ca-independent vare pro-MMP-2 in human smooth muscle cells 7]and NOS activity.the level of perfusme dityrosine (n mnrker 1-20 uM peroynitrite activmed purifiod pro-MMPs with- for peroxynitnte)in eytokine treated henrts showed a out a change in molecular weight 6 Recently we hnve time-dependent incrense which wns statistically significnnt shown that the acute mechanical dysfunction cnused by the at 90 and 120 min (Fig 7B) infusion of peroxynitrite imo the heart is mediated through MMP-2 261.It is therefore more accurate to specify the MMP-2 activities reported here by molecular weight than 4.Discussion by the use of the desagnations 'pro-MMP-2'and 'MMP-2 which imply inactrve and active forms of the erzyme, We show here for the first time that pro-mtlammalory respectively,when this indeed may not be the case cytokines caurse a rapid and enhanced release of MMP.2 Endogenous tissue inhibitors of metalloproteinases acivity from the heart imo the perfusae.This preceded the (TIMPs)also cantrol MMP activity.Interestingly,perax. oneet of a marked depressiomn in cardic mechanical ynitrite was shown to inactivale TIMP.I in vitro [8].We function.At the end of perfusion both the remaining chose to imvestigate TIMP.4 as it is the most abundantly
C. Qun Gao et al. / Cardiovascular Research 57 (2003) 426–433 431 MMP-2 activity and the level of its endogenous inhibitor TIMP-4 in heart tissue were reduced in cytokine-treated hearts. The collagen content of the heart was unaltered by cytokine treatment, however, the level of TnI was diminished. Pharmacological inhibitors of MMP or a neutralizing MMP-2 antibody protected the heart from the loss in TnI as well as the myocardial dysfunction caused by cytokines. We have recently shown using this model of acute myocardial contractile dysfunction that pro-inflammatory cytokines enhance the myocardial generation of the prooxidant species peroxynitrite through an enhancement in both superoxide and NO generating enzyme activities, which are together responsible for the depression in contractile function [4]. This was confirmed in the present study with the enhanced inducible NO synthase activity in heart tissue and perfusate dityrosine level, an indicator of peroxynitrite generation [2,4]. We also recently found that the activation and release of MMP-2 from the heart contributes to another oxidant stress induced injury to the heart caused by ischemia and reperfusion [18]. The oxidant stress of acute myocardial ischemia–reperfusion injury is also mediated by the generation of peroxynitrite [2,3]. As MMPs can be activated by oxidant stress, we determined here whether cytokine-induced cardiac dysfunction is also mediated through MMPs. In accordance with our previous report [18], the main gelatinolytic activity in coronary effluent from the aerobically perfused heart is 72 kDa, corresponding to proMMP-2. A rapid and significant increase in 72 kDa activity 21 21 Fig. 7. (A) Ca -dependent and Ca -independent nitric oxide synthase was observed within the first hour in the perfusate from activities in ventricular homogenates from control and cytokine-treated hearts exposed to cytokines along with some enhanced (cyt) working rat hearts at 120 min perfusion and effect of MMP-2 release of 64 kDa activity corresponding to MMP-2, albeit antibody (Ab, n55 hearts per group; * P,0.05 versus control). (B) Time at a reduced level. Cytokine treatment caused a clear-cut course of changes in perfusate dityrosine level in control (n57) and depletion of MMP-2 activity in the myocardium. Pro- cytokine-treated hearts (n510), * P,0.05 versus 1 min value (one-way ANOVA). MMPs may be activated by breaking a zinc–cysteine bond in the catalytic centre [24], which then exposes the catalytic site followed by proteolytic activation, or through not significantly different between control and cytokine- oxidant-induced conformational changes [6]. Previous treated hearts (Fig. 7A). In contrast, cytokine treatment studies suggested oxygen free radicals may be capable of 21 resulted in a near seven-fold increase in Ca -independent activating matrix metalloproteinases in perfused rat hearts activity, which was not abolished by anti-MMP-2 anti- [25]. Indeed, the powerful oxidant peroxynitrite can acti- 21 body. In accordance with the increase in Ca -independent vate pro-MMP-2 in human smooth muscle cells [7] and NOS activity, the level of perfusate dityrosine (a marker 1–20 mM peroxynitrite activated purified pro-MMPs withfor peroxynitrite) in cytokine treated hearts showed a out a change in molecular weight [6]. Recently we have time-dependent increase which was statistically significant shown that the acute mechanical dysfunction caused by the at 90 and 120 min (Fig. 7B). infusion of peroxynitrite into the heart is mediated through MMP-2 [26]. It is therefore more accurate to specify the MMP-2 activities reported here by molecular weight than 4. Discussion by the use of the designations ‘pro-MMP-2’ and ‘MMP-2’ which imply inactive and active forms of the enzyme, We show here for the first time that pro-inflammatory respectively, when this indeed may not be the case. cytokines cause a rapid and enhanced release of MMP-2 Endogenous tissue inhibitors of metalloproteinases activity from the heart into the perfusate. This preceded the (TIMPs) also control MMP activity. Interestingly, peroxonset of a marked depression in cardiac mechanical ynitrite was shown to inactivate TIMP-1 in vitro [8]. We function. At the end of perfusion both the remaining chose to investigate TIMP-4 as it is the most abundantly
432 C.Oa Gau el ul.Curolurancalar Rscarek 57(2003)415-433 expressed TIMP in heart tissue compared with other matrix.An intracellular associstion of MMP-2 with the organs [23]and is foumnd in both human [23]and murin sarcolemma was found in hearts from patients with dilsted hearts [271.Moreover,it has been demoostraled that cardiomyopauthy [This indicsles that elemerts of the TIMP-4.unlike TIMP-2.does not promote but actually contractile apparatus may represent a molecular target for inhibas the activalion of MMP-2 221 There was also a the detrimental actions of MMP-2.We recently sbowed decrense in TIMP4 levels resulting from the exposure of that the contractile protein regulatory element Tnl is the heart to cytokines.It is interesting to speculate that this susceptible to rapid proceolytic cleavage hy MMP.2 and loss in TIMP-4 protein also contributed to enhanced MMP that inhibition of MMP activity preverts Tnl degradation activity and the depression in myocardial function. while improving the recovery of mechanical function in Our results provide compelling evidence that MMP.2 ischemic-reperfused hearts [16].Mareover several lines of octs as a modiator to cyokine-induced myocardial evdenoe showed the co-localization of MMP-2 with 'Tnl in dysfunction.Several observations support this:(1)cyto- cardiac myocytes 116].Indeed.results from the present kine-treated hesrts showed a signficant loss in cordine experiments show thet cytokine treatment caused a marked work between 60 and 120 mnin of'perfusion.(2)this was lass in myocardial Tnl coment which was prevented by the preceded by a rapid and marked inerease of MMP-2 ncutralizing ati-MMP-2 antibody.This provides further nctivity in perfusare.(3)this occared in conjunction with evidence for the intrncellular action of MMP-2 and that a decrease of MMP-2 and TIMP-4 in heart tissue following Tnl is a target for MMP-2 in the setting of cytokine- cytokine treatment.and (4)Ro31-9790 and PD166793. induced myoc山al depression.Rent山y.eral other inhibeors of MMP activity.prevented and a neutralizing novel substrates and resulting biological activites of MMP-2 antibody abolished the depression in mechanical MMP-2.independert of its well-described activities on the funcbon.The MMP-2 antbody.however.did not anlect extracellulsr matr,have been described.This includes the cviokine-induced increase in inducible NO synthase roles for MMP-2 in the comtrol of vascular tone [14] activity,excluding the possihility that it nonspecifically platelet aggregitian [13]and inflammation [15]suggesting inctivated cytokines or interfered with the expression of that this protease can have effects on a more rapid time inducible NO synchase. scale and with hoth intracellular ard extracellular loci of In accordance with our findings,activation of MMPs is action. mplcated in cardine inury resulting from drverse In sumiary.we Fave demonstraed thst pro-intlamma- pothologsc insults auch as ischemia.infaretion,and inflan- tory cytokines induced depression of mechanical function mation Cheung ct nl.[18]found in isolated rt hearts that of'the heart is medintod by MMP-2.Inhibition of MMP MMP-2 was rapidly relensed irto the perfusate duning the actrvity protects the heart from cytokine-induced myocar- first minute of reperfusion after ischemia.Inhibitors of dinl injury,thus MMP-2 may be a viable target for the MMP or a neutralizing anti-MMP-2 antibody impeoved the therapeutic inervention of inflammslory beart disesse recovery of post-ischemic mechanical functon [18].Li et al.[12]demonstraed a selectrve downregulation of both TIMP-I and-3 transcripts and proteins along with upregu Acknewledgeme维t国 lation of myocardial MMP-9 gelatinolylie activity in cardiomyopathic ventricles.Much evidence supports that We thank Dr Damon Meyes (University of Alherta)for MMPs directly mediate crucial steps during the patho. advice on statistical nnalysis This project was funded by n genesis of myocardial infarction and also determine the gmnt from the Heart and Stroke Foundarion of Alberta clinical outcome.MMP activity rapidly increases in the NWI and NunavuL Tamas Csont is a Fellow and Richard myocardium during infarction and remairs elevated Schulz is a Senior Scholar of the Alberta Heritage Founda- through the healing phase 110].Also infarcted hearts from tion for Medical Research. MMP-9 deficient mice show a reduction in both carly mvocardal rupture 28]as well as progressive ventneular dilotion 29].Experiments trom several laboratories indi- cae that in both humans and experimental nnimal models References of henrt failure that there is incrensed nctivation of myocardisl MMPs and that inhibition of MMPs can block 1]Beckmr JS,Becknan TW.Chen Mrdull PA,Freenan BA ventriculsr dilation [30-321.Furthermore.Kim et al.[33] Apporent hydrovy rical productin ty peroynitrte:mglicanons fie eluhelid inuy fom nik wide a superuside.Poe Nall demonstraled that transgenic overexpression of the human c时sa1841971-ln4 MMP-I gere in the mouse ventricle leads to myocyte 21 Yesrin,Saynodka KD.Sch业R.Gee山on of pe@miie hypertrophy and ventricular dyslunction. We shawed here that the total collagen pontent of the d3Res19w7.33:22-2 heart was not changed following cytokine treatment, 3]Wang P,Zweicr JL..Measaciaad uf nii wile usd perunynikil neratin in the potizhemic heort Evience far pemeynirte- suggesting that other actions of MMP.2 may have occurred ned aed reperfusion inuny.J Hidl Chen 1996,27129223-29230. apart from degradstion or remodeling of the extracellular Ferdireny P,Dunil H,Arhta 1,Ruthery RA.Schuz R.Perox-
432 C. Qun Gao et al. / Cardiovascular Research 57 (2003) 426–433 expressed TIMP in heart tissue compared with other matrix. An intracellular association of MMP-2 with the organs [23] and is found in both human [23] and murine sarcolemma was found in hearts from patients with dilated hearts [27]. Moreover, it has been demonstrated that cardiomyopathy [34]. This indicates that elements of the TIMP-4, unlike TIMP-2, does not promote but actually contractile apparatus may represent a molecular target for inhibits the activation of MMP-2 [22]. There was also a the detrimental actions of MMP-2. We recently showed decrease in TIMP-4 levels resulting from the exposure of that the contractile protein regulatory element TnI is the heart to cytokines. It is interesting to speculate that this susceptible to rapid proteolytic cleavage by MMP-2 and loss in TIMP-4 protein also contributed to enhanced MMP that inhibition of MMP activity prevents TnI degradation activity and the depression in myocardial function. while improving the recovery of mechanical function in Our results provide compelling evidence that MMP-2 ischemic-reperfused hearts [16]. Moreover several lines of acts as a mediator to cytokine-induced myocardial evidence showed the co-localization of MMP-2 with TnI in dysfunction. Several observations support this: (1) cyto- cardiac myocytes [16]. Indeed, results from the present kine-treated hearts showed a significant loss in cardiac experiments show that cytokine treatment caused a marked work between 60 and 120 min of perfusion, (2) this was loss in myocardial TnI content which was prevented by the preceded by a rapid and marked increase of MMP-2 neutralizing anti-MMP-2 antibody. This provides further activity in perfusate, (3) this occurred in conjunction with evidence for the intracellular action of MMP-2 and that a decrease of MMP-2 and TIMP-4 in heart tissue following TnI is a target for MMP-2 in the setting of cytokinecytokine treatment, and (4) Ro31-9790 and PD166793, induced myocardial depression. Recently, several other inhibitors of MMP activity, prevented and a neutralizing novel substrates and resulting biological activities of MMP-2 antibody abolished the depression in mechanical MMP-2, independent of its well-described activities on the function. The MMP-2 antibody, however, did not affect extracellular matrix, have been described. This includes the cytokine-induced increase in inducible NO synthase roles for MMP-2 in the control of vascular tone [14], activity, excluding the possibility that it nonspecifically platelet aggregation [13], and inflammation [15] suggesting inactivated cytokines or interfered with the expression of that this protease can have effects on a more rapid time inducible NO synthase. scale and with both intracellular and extracellular loci of In accordance with our findings, activation of MMPs is action. implicated in cardiac injury resulting from diverse In summary, we have demonstrated that pro-inflammapathologic insults such as ischemia, infarction, and inflam- tory cytokines induced depression of mechanical function mation. Cheung et al. [18] found in isolated rat hearts that of the heart is mediated by MMP-2. Inhibition of MMP MMP-2 was rapidly released into the perfusate during the activity protects the heart from cytokine-induced myocar- first minute of reperfusion after ischemia. Inhibitors of dial injury, thus MMP-2 may be a viable target for the MMP or a neutralizing anti-MMP-2 antibody improved the therapeutic intervention of inflammatory heart disease. recovery of post-ischemic mechanical function [18]. Li et al. [12] demonstrated a selective downregulation of both TIMP-1 and -3 transcripts and proteins along with upregu- Acknowledgements lation of myocardial MMP-9 gelatinolytic activity in cardiomyopathic ventricles. Much evidence supports that We thank Dr Damon Meyes (University of Alberta) for MMPs directly mediate crucial steps during the patho- advice on statistical analysis. This project was funded by a genesis of myocardial infarction and also determine the grant from the Heart and Stroke Foundation of Alberta, clinical outcome. MMP activity rapidly increases in the NWT and Nunavut. Tamas Csont is a Fellow and Richard ´ myocardium during infarction and remains elevated Schulz is a Senior Scholar of the Alberta Heritage Founda- through the healing phase [10]. Also infarcted hearts from tion for Medical Research. MMP-9 deficient mice show a reduction in both early myocardial rupture [28] as well as progressive ventricular dilation [29]. Experiments from several laboratories indi- References cate that in both humans and experimental animal models of heart failure that there is increased activation of [1] Beckman JS, Beckman TW, Chen J, Marshall PA, Freeman BA. myocardial MMPs and that inhibition of MMPs can block Apparent hydroxyl radical production by peroxynitrite: implications ventricular dilation [30–32]. Furthermore, Kim et al. [33] for endothelial injury from nitric oxide and superoxide. Proc Natl demonstrated that transgenic overexpression of the human Acad Sci USA 1990;87:1620–1624. 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