hiromichi wada, teruhisa kawamura, tetsuhiko yanazume and

Hiromichi Wada, Teruhisa Kawamura, Tetsuhiko Yanazume and Shigetake SasayamaTsuyoshi Kakita, Koji Hasegawa, Eri Iwai-Kanai, Souichi Adachi, Tatsuya Morimoto,

Induced Apoptosis in Cardiac Myocytes−StressMediated Protection Against Oxidant−Calcineurin Pathway Is Required for Endothelin-1

Print ISSN: 0009-7330. Online ISSN: 1524-4571 Copyright © 2001 American Heart Association, Inc. All rights reserved.is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231Circulation Research

doi: 10.1161/hh1201.0917942001;88:1239-1246; originally published online June 7, 2001;Circ Res.

http://circres.ahajournals.org/content/88/12/1239World Wide Web at:

The online version of this article, along with updated information and services, is located on the

http://circres.ahajournals.org//subscriptions/

is online at: Circulation Research Information about subscribing to Subscriptions:

http://www.lww.com/reprints Information about reprints can be found online at: Reprints:

document. Permissions and Rights Question and Answer about this process is available in the

located, click Request Permissions in the middle column of the Web page under Services. Further informationEditorial Office. Once the online version of the published article for which permission is being requested is

can be obtained via RightsLink, a service of the Copyright Clearance Center, not theCirculation Researchin Requests for permissions to reproduce figures, tables, or portions of articles originally publishedPermissions:

by guest on February 21, 2013http://circres.ahajournals.org/Downloaded from

http://circres.ahajournals.org/content/88/12/1239

http://www.ahajournals.org/site/rights/

http://www.lww.com/reprints

http://circres.ahajournals.org//subscriptions/

http://circres.ahajournals.org/

Calcineurin Pathway Is Required for Endothelin-1–Mediated Protection Against Oxidant Stress–Induced

Apoptosis in Cardiac MyocytesTsuyoshi Kakita, Koji Hasegawa, Eri Iwai-Kanai, Souichi Adachi, Tatsuya Morimoto,

Hiromichi Wada, Teruhisa Kawamura, Tetsuhiko Yanazume, Shigetake Sasayama

Abstract—Endothelin-1 (ET-1) acts not only as a growth-promoting peptide but also as a potent survival factor againstmyocardial cell apoptosis. However, the signaling pathways leading to myocardial cell protection by ET-1 are poorlyunderstood. Using a culture system of primary cardiac myocytes derived from neonatal rats, we show in the presentstudy that ET-1 almost completely blocked the hydrogen peroxide–induced increase in the percentage of TdT-mediateddUTP-biotin nick-end labeling–positive myocytes. Apoptosis inhibition by ET-1 was confirmed by cytofluorometricanalysis as well as by examination of the ladder formation, morphological features, and caspase-3 cleavage. We havefound that ET-1 converts the nuclear factor of activated T lymphocytes (NFATc) in cardiac myocytes into high-mobilityforms and translocates cytoplasmic NFATc to the nuclei. In addition, ET-1 stimulates the interaction between NFATcand the cardiac-restricted zinc-finger protein GATA4 in these cells. The immunosuppressants cyclosporin A and FK506,which antagonize calcineurin, negated the inhibitory effect of ET-1 on apoptosis. Calcineurin activation de novo wassufficient to inhibit hydrogen peroxide–induced apoptosis. ET-1 induced the expression of an antiapoptotic protein bcl-2in cardiac myocytes in a cyclosporin A–dependent manner, but it did not alter the expression of bax. Cyclosporin A alsoattenuated the ET-1–stimulated transcription of the bcl-2 gene in these cells. These findings demonstrate that thecalcineurin pathway is required for the inhibitory effect of ET-1 on oxidant stress–induced apoptosis in cardiacmyocytes.(Circ Res. 2001;88:1239-1246.)

Key Words: apoptosisn endothelin-1n cardiac myocyten calcineurin

A poptosis, or programmed cell death, is a central featureof normal tissue development in the fetus and of cell

replacement in certain adult tissues (eg, the thymus). Apo-ptosis is most often associated with cells that are progressingthrough the cell cycle. However, accumulating evidencesuggests that terminally differentiated adult cardiac myocytesundergo apoptosis in various animal models of heart failure.These include models of rapid ventricular pacing1,2 andpressure overload caused by aortic constriction3 in agedspontaneously hypertensive rats.4 In addition, blockage of thesurvival pathway by cardiac-specific disruption of gp130results in massive myocardial cell apoptosis after pressureoverload, dilatation of the heart, and heart failure.5 Thus, theidentification of the signaling pathways that mediate cardiacmyocyte cell death and survival is critical to the ultimateelucidation of the molecular basis of cardiac muscle failure.

The control of programmed cell death is dependent on abalance between inhibitors and inducers of apoptosis. Anumber of humoral factors activated in congestive heartfailure6 may possibly play positive and negative roles in the

regulation of myocardial cell apoptosis. Endothelin-1 (ET-1),a 21-residue peptide originally isolated from vascular endo-thelium,7 is one such factor; the levels of ET-1 in plasma andin ventricular myocardium are markedly increased in humanand animal models of heart failure.8–10 ET-1 exerts diversephysiological effects, including vasoconstriction and growthpromotion. ET-1 is sufficient to induce the myocardial cellhypertrophy associated with the reactivation of the fetal geneprogram.11 It was recently reported that ET-1 is a survivalfactor in smooth muscle cells,12 fibroblasts,13 and cardiacmyocytes.14 However, the precise molecular mechanisms thatmediate these survival effects of ET-1 are unclear at present.

Stimulation with ET-1 results in an increase in intracellularcalcium levels.15 Calcium-activated phosphatase calcineurinis necessary for the nuclear import of the nuclear factor ofactivated T lymphocytes (NFAT) transcription factors, whichmediate changes in gene expression in response to calciumsignaling from the T-cell receptor (reviewed by Rao et al16).NFAT3, a member of the NFAT family, interacts with highaffinity and specificity with the cardiac-restricted zinc-finger

Original received November 13, 2000; resubmission received March 7, 2001; revised resubmission received April 20, 2001; accepted April 20, 2001.From the Department of Cardiovascular Medicine (T. Kakita, K.H., E.I.-K., T.M., H.W., T. Kawamura, T.Y., S.S.), Graduate School of Medicine,

Kyoto University, Kyoto, and Department of Pediatrics (S.A.), Graduate School of Medicine, Kyoto University, Kyoto, Japan.Correspondence to Koji Hasegawa, Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, 54 Kawara-cho,

Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan. E-mail [email protected]© 2001 American Heart Association, Inc.

Circulation Researchis available at http://www.circresaha.org

1239 by guest on February 21, 2013http://circres.ahajournals.org/Downloaded from


protein GATA4.17 Although calcineurin-GATA4 is involvedin the transcriptional pathways that modulate cardiac hyper-trophy,17 a role for this pathway in ET-1–mediated protectionagainst myocardial cell survival is unknown. The presentstudy was conducted to determine whether the calcineurinpathway is involved in the inhibitory effects by ET-1 onmyocardial cell apoptosis induced by oxidant stress.

Materials and MethodsDetection of DNA FragmentationPrimary ventricular cardiac myocytes were prepared fromneonatal rats as previously described.14,18 The cells weresubjected to terminal deoxynucleotidyl transfer–mediated endlabeling of fragmented nuclei (TdT-mediated dUTP-biotinnick-end labeling [TUNEL] assay) and nucleosomal ladderassay as previously described.14,18

Cytofluorometric AnalysisCells were incubated for 10 minutes at 37°C in a culturemedium containing 2.5 mmol/L 5,59,6,69-tetrachloro-1,19,3,37-tetra-ethylbenzimidazolocarbocyanine iodide(JC-1) and 5mg/mL propidium iodide (both from MolecularProbes), followed by analysis within 30 minutes of theaddition of fluorochrome in a Becton Dickinson FAC-Scalibur cytofluorometer. After suitable compensation, fluo-rescence was recorded at different wavelengths: JC-1 at 525nm and propidium iodide at 600 nm.

Immunoprecipitation/Western BlottingImmunoprecipitation and Western blotting were performed aspreviously described.19 Aliquots of the lysates containing 100mg were immunoprecipitated by using an anti-mouse GATA4polyclonal antiserum (Santa Cruz Biotechnology) or normalgoat IgG in low-stringency buffer for 16 hours at 4°C andincubated with protein G (Sigma) beads for 1 hour at 4°C.The precipitate was washed 4 times in the same buffer andsubjected to Western blots by using a monoclonal antibodyagainst murine NFATc (Santa Cruz Biotechnology).

As a primary antibody in Western blots for caspase-3, weused anti–caspase-3 polyclonal antibody (Santa Cruz Bio-technology). This antibody detects both the full-length andthe cleaved fragment of caspase-3.

For the detection of bcl-2 and bax, we have used an anti-ratbcl-2 monoclonal antibody (Medical & Biological Laborato-ries) and an anti-mouse bax monoclonal antibody (Santa CruzBiotechnology). The images were analyzed with computer-assisted densitometry (NIH Image software).

ImmunocytochemistryThe cardiac myocytes were grown in a flask-style chamberwith glass slides. The cells were then fixed with 3% formal-dehyde in PBS for 15 minutes at room temperature. Immu-nocytochemical staining for NFATc was performed by usingthe indirect immunofluorescence method. Cells were incu-bated with anti-NFATc monoclonal antibody (Santa CruzBiotechnology) at a dilution of 1:100. Signals of NFATc weredetected with anti-mouse FITC-conjugated secondary anti-body at a dilution of 1:500 for 45 minutes.

Plasmid ConstructsAn expression vector encoding a constitutively active form ofcalcineurin A subunit (CaNDA) and encoding a wild-typecalcineurin B subunit (CaNB) were provided by Dr StephenJ. O’Keefe, Merck Research Laboratories, Rahway, NJ. Theplasmid pCMVb-gal is a b-gal expression vector and hasbeen described prevously.19 The plasmid construct pbcl-2luc,20 a firefly luciferase–reporter plasmid driven by a DNAfragment containing sequences from21796 to2999 relativeto the translation start site of the human bcl-2 gene, waskindly donated by Dr Linda M. Boxter, Stanford UniversitySchool of Medicine, Stanford, Calif. pRSVCAT contains thebacterial chloramphenicol acetyl transferase (CAT) genedriven by Rous sarcoma virus (RSV) long-terminal repeatsequences.19

Transfection and Luciferase/CAT AssaysCardiac myocytes were cotransfected with 2mg of pbcl-2 and0.1 mg of pRSVCAT by using Lipofectamine Plus (GIBCOBRL) and were subjected to assays for luciferase and CATactivities as described previously.19

Statistical AnalysisData are presented as mean6SE. Statistical comparisonswere performed by using unpaired two-tailed Student’st testsor ANOVA with Scheffe’s test when appropriate, with aprobability value less than 0.05 taken to indicate significance.

ResultsET-1 Inhibits H 2O2-Induced Apoptosis inCardiac MyocytesTo determine the effects of ET-1 on hydrogen peroxide(H2O2)–induced myocardial cell apoptosis, neonatal rat car-diac myocytes were treated with saline, H2O2 (1025 mol/L)alone or H2O2 plus ET-1 (1027 mol/L) in serum-free mediumfor 48 hours. We provide representative photographs ofTUNEL staining in Figure 1A and quantitative data in Figure1B. Under our experimental conditions, in which cells wereplated at a high density, serum deprivation alone did notincrease the number of TUNEL-positive cells (,10%, panelA in Figure 1A and bar 1 in Figure 1B). However, thestimulation with H2O2 markedly increased the number ofTUNEL-positive cells (.35%, panel B in Figure 1A and bar2 in Figure 1B). These positive cells may specifically indicatethe presence of internucleosomal DNA fragmentation, be-cause no positive cells were found when we omitted theterminal deoxytransferase treatment (panel C in Figure 1A).The cells stimulated with H2O2 displayed small condensednuclei, cell shrinkage, and nuclear fragmentation, consis-tent with the morphological features of apoptosis. Fewermyocardial cells treated with ET-1 in addition to H2O2

were positive for internucleosomal cleavage by TUNELstaining (panel D in Figure 1A and bar 3 in Figure 1B)compared with the cells treated with H2O2 alone.Figure 1C(lane 2) shows the H2O2-induced typical ladder formationof fragmented internucleosomal DNA in agarose gels, ahallmark of apoptosis. As shown in lane 3 of Figure 1C,ET-1 completely inhibited the internucleosomal cleavageof genomic DNA in H2O2-stimulated cardiac myocytes. To

1240 Circulation Research June 22, 2001



confirm the inhibitory effect of ET-1 on myocardial cellapoptosis, we examined whether ET-1 will inhibit theactivation of the caspase cascade by Western blot analysisof the active p17 subunit of caspase-3. As illustrated inFigure 1D, stimulation of cardiac myocytes with H2O2

induced an increase in p17 of caspase-3, which indicatesthe proteolytic activation of caspase-3. In addition to H2O2,ET-1 inhibited this increase. These findings suggest thatET-1 has an antiapoptotic effect on oxidant stress–inducedapoptosis in cardiac myocytes.

ET-1 Inhibits H 2O2-Mediated Reduction ofMitochondrial Transmembrane Potential inCardiac MyocytesThe reduction of mitochondrial transmembrane potentialprecedes DNA fragmentation in apoptosis. To further confirmthe inhibitory effect of myocardial cell apoptosis by ET-1, weexamined mitochondrial membrane potential and cell mem-brane permeability by cytofluorometric analysis at 24 hoursafter the stimulation with H2O2. Stimulation of cardiac myo-cytes with H2O2 did not alter cell membrane permeability as

Figure 1. ET-1 inhibits H2O2-inducedapoptosis in cardiac myocytes. Neonatalrat cardiac myocytes were treated withsaline as a control or with H2O2 (1025

mol/L) in the presence or the absence ofET-1 (1027 mol/L) for 48 hours. A, Repre-sentative photographs of TUNEL-staining. Cardiac myocytes were sub-jected to TUNEL staining in the absenceof H2O2 or ET-1 (panel A), in the pres-ence of H2O2 (1025 mol/L) (panels B andC), in the presence of H2O2 plus ET-1(1027 mol/L) (panel D). Terminaldeoxytransferase (TdT) was omitted inpanel C. Arrowheads show cells withevidence of apoptosis, including chro-matin condensation. B, Quantitativeanalysis of TUNEL staining. TUNEL-positive nuclei were counted andexpressed as the percentage of totalnuclei. An average of 400 to 500 nucleiwere counted in random fields on eachslide. The results are the mean6SE of 3independent experiments. C, GenomicDNA fragmentation. Genomic DNA wasisolated from myocytes and loaded on a1.5% agarose gel. M indicates molecularmarkers. D, Caspase-3 cleavage.Lysates from cardiac myocytes weresubjected to Western blotting with anantibody that recognized both the full-length and the p17 subunit ofcaspase-3. E, Mitochondrial transmeme-brane potential. Mitochondrial trans-membrane potential and cell membranepermeability were assayed by incubatingthese cells for 10 minutes at 37°C in aculture medium containing 2.5 mmol/Lof JC-1 and 5 mg/mL propidium iodide.The x axis is JC-1 fluorescence and they axis is propidium iodide fluorescence.

Kakita et al Inhibition of Cardiac Apoptosis by Calcineurin 1241



shown by no increase in propidium iodide binding to DNA.However, H2O2 stimulation increased the number of cellswith low JC-1 fluorescence, indicating that H2O2 reducedmitochondrial transmembrane potential. It was found that3064.2% of the H2O2-treated cells and 4.661.3% of thesaline-stimulated cells had low JC-1 fluorescence (lower leftquadrant in Figure 1E). However, ET-1 reversed this effect ofH2O2 and reduced the number of cells with low JC-1fluorescence to 8.262.1%. These findings suggest that H2O2

stimulation specifically reduces mitochondrial membrane po-tential and provide further evidence for the inhibitory effectof ET-1 against H2O2-induced apoptosis.

ET-1 Converts NFATc Into High-Mobility Formsand Stimulates the Interaction With GATA4To determine whether ET-1 dephosphorylates NFATc incardiac myocytes, we performed Western blotting. Neonatalcardiac myocytes were stimulated with ET-1 or saline as acontrol for 3 hours, and then whole lysates derived from thesecells were subjected to Western blotting with the anti-NFATcantibody. As shown in Figure 2A, the expression level ofNFATc did not differ between saline- and ET-1–stimulatedcardiac myocytes. However, all 3 forms of NFATc inET-1–stimulated cardiac myocytes (lane 2) migrated fasterthan NFATc in saline-stimulated cells (lane 1). Becausedephosphorylated NFATc migrates faster than phosphorylat-ed NFATc, this might indicate dephosphorylation of NFATcby ET-1 stimulation. To further confirm this hypothesis, wehave used the immunosuppressants cyclosporin A (Cys A),which inhibits the ability of calcineurin to activate NFATtranscription factors.21,22As shown in Figure 2A, a therapeu-tic concentration of Cys A (431027 mol/L) reversed the effectof ET-1 on the migration of NFATc.

To determine whether NFATc interacts with GATA4 incardiac myocytes by ET-1 stimulation, we performed immu-noprecipitations followed by Western blotting. Neonatal car-diac myocytes were stimulated with ET-1 or saline as acontrol for 3 hours, and then whole lysates derived from thesecells were subjected to immunoprecipitation with an anti-NFATc antibody as a positive control (Figure 2B, lanes 1 and4), IgG as a negative control (Figure 2B, lanes 2 and 5), or ananti-GATA4 antibody (Figure 2B, lanes 3 and 6). Westernblotting with the anti-NFATc antibody showed that theanti-NFATc antibody immunoprecipitated all 3 forms ofNFATc in saline- and ET-1–stimulated cardiac myocytes(lanes 1 and 4). The anti-GATA4 antibody (lane 6), but notIgG (lane 5), coprecipitated NFATc protein in lysates fromcardiac myocytes stimulated with ET-1, even after extensivewashing. The main form of NFATc coprecipitated was theone with the highest mobility (compare lanes 6 and 8). Theanti-GATA4 antibody did not coprecipitate NFATc in lysatesfrom saline-stimulated myocytes (lane 3). However, theexpression level of GATA4 did not differ between saline- andET-1–stimulated cells (data not shown). Thus, ET-1 stimu-lated the interaction between NFATc and GATA4 in cardiacmyocytes.

ET-1 Translocates NFATc Into the Nucleus in aCalcineurin-Dependent MannerTo examine nuclear translocation of endogenous NFATc inresponse to ET-1 treatment in cardiac myocytes, we per-

Figure 2. ET-1 converts NFATc into high-mobility forms andstimulates the interaction between NFATc and GATA4. A, Neo-natal cardiac myocytes were cultured in serum-free medium inthe presence or absence of ET-1 (1027 mol/L) and Cys A(431027 mol/L) as indicated for 3 hours. Lysates from thesecells were subjected to Western blotting with an anti-NFATcantibody. B, Neonatal cardiac myocytes were stimulated withsaline or 1027 mol/L ET-1 for 3 hours. Lysates derived fromthese cells were immunoprecipitated with an anti-NFATc anti-body (lanes 1 and 4), IgG (lanes 2 and 5), or an anti-GATA4antibody (lanes 3 and 6). After electrophoresis and electroblot-ting, the membranes containing immobilized immunocomplexeswere subjected to Western blotting with an anti-NFATc anti-body. Lysates were subjected to Western blotting withoutimmunoprecipitation in lanes 7 and 8.

Figure 3. ET-1 translocates NFATc intothe nucleus in a calcineurin-dependentmanner. A through C, Cultured cardiacmyocytes were treated with ET-1 (1027

mol/L, B), ET-1 plus Cys A (431027

mol/L, C), or saline as a control (A) for3 hours in serum-free conditions, andwere then subjected to immunofluores-cence with an anti-NFATc antibody. Dand E, Cultured cardiac myocytes were

transfected with 0.2 mg of CaNDA and 0.2 mg of CaNB together in E or with 0.4 mg of pCMVb-gal alone in panel D. Forty-eighthours later, these cells were subjected to immunofluorescence with an anti-NFATc antibody.




formed immunofluorescence. As shown in Figure 3A,NFATc was detected in cytoplasm of nearly all saline-stimulated cardiac myocytes. However, the stimulation ofcardiac myocytes with ET-1 markedly changed this localiza-tion and caused the nuclear translocation of NFATc (Figure3B). ET-1–mediated translocation was reversed by Cys A(Figure 3C), suggesting a requirement of calcineurin activa-tion for this translocation.

To examine the effects of calcineurin activation de novo oncardiac myocytes, we cotransfected an expression vectorencoding a constitutively active form of CaNDA and CaNB.As a control, we transfected the corresponding amount ofpCMVb-gal. Then, we examined the localization of endoge-nous NFATc in cardiac myocytes by immunofluorescence.The transfection of pCMVb-gal did not change the locationof NFATc in cardiac myocytes (Figure 3D). However,cotransfecting CaNDA and CaNB resulted in the nucleartranslocation of endogenous NFATc in almost all cells(Figure 3E).

Cys A and FK506 Neutralize the AntiapoptoticEffect of ET-1We examined whether the calcium-activated phosphatasecalcineurin is required for the inhibition of myocardial cellapoptosis by ET-1. We exposed neonatal cardiac myocytes toH2O2 and ET-1 in the presence or the absence of the Cys Aand FK506, which inhibit the ability of calcineurin to activateNFAT transcription factors.23 As shown in Figure 4, ET-1inhibits the H2O2-mediated increase in the number ofTUNEL-positive myocytes (compare bars 1, 4, and 7). Atherapeutic concentration of Cys A (431027 mol/L) or FK506(131029 mol/L) in addition to H2O2 and ET-1 increased thenumber of TUNEL-positive myocytes (compare bars 7, 8,and 9). However, the same concentration of Cys A andFK506 did not increase the number of TUNEL-positive

myocytes in saline-stimulated cardiac myocytes (comparebars 1, 2, and 3). Thus, the effect of Cys A and FK506 onET-1 may not be explained by the cytotoxicity of theseagents. To exclude the possibility that Cys A and FK506augment proapoptotic effects of H2O2, we treated cardiacmyocytes with Cys A and FK506 in addition to H2O2.However, Cys A and FK506 did not increase the number ofTUNEL-positive cells in H2O2-stimulated states (comparebars 4, 5, and 6). These results indicate that the apoptosis-in-hibitory effect of ET-1 is Cys A– and FK506-sensitive andtherefore involves calcineurin activation.

To examine the effects of calcineurin/NFATc activation denovo on myocardial cell apoptosis, we cotransfected anexpression vector encoding a constitutively active form ofCaNDA and CaNB. As a control, we transfected the corre-sponding amount of pCMVb-gal. Transfection of CaNDAand CaNB but not that of pCMVb-gal results in the nucleartranslocation of NFATc in cardiac myocytes as describedearlier (Figures 3D and 3E). As shown in Figure 5, thetreatment of pCMVb-gal–transfected cardiac myocytes withH2O2 markedly increased the number of TUNEL-positivenuclei. In contrast, H2O2 did not increase the TUNEL-positivity in cells cotransfected with CaNDA and CaNB.These findings demonstrate that the activation of calcineurin/NFATc is sufficient to inhibit H2O2-induced myocardial cellapoptosis.

Cys A Inhibits ET-1–Induced Transcription ofbcl-2 in Cardiac MyocytesBcl-2 is a proto-oncogene–encoded protein that preventsapoptosis induced by various stimuli.24 To determine whetherstimulation of cardiac myocytes with ET-1 induces theexpression of bcl-2, and if so, whether a calcineurin pathwayis involved in this process, neonatal cardiac myocytes weretreated with ET-1 in the presence or absence of Cys A

Figure 4. Cys A and FK506 neutralize ET-1–mediated inhibitionof apoptosis. Cultured cardiac myocytes were treated for 48hours in serum-free medium in the presence or absence of H2O2

(1025 mol/L), ET-1 (1027 mol/L), Cys A (431027 mol/L), andFK506 (131029 mol/L). TUNEL-positive nuclei were counted andexpressed as the percentage of total nuclei. An average of 400to 500 nuclei were counted in random fields on each slide. Theresults are the mean6SE of 3 independent experiments.

Figure 5. Calcineurin activation de novo inhibits H2O2-inducedapoptosis. Cultured cardiac myocytes were transfected with 0.2mg of CaNDA and 0.2 mg of CaNB together (bars 3 and 4) orwith 0.4 mg of pCMVb-gal alone (bars 1 and 2). These cellswere treated for 48 hours in a serum-free medium in the pres-ence or absence of H2O2 (1025 mol/L) as indicated and werethen subjected to TUNEL staining. The results are the mean6SEof 3 independent experiments.




(431027 mol/L). Forty-eight hours later, lysates from thesecells were subjected to Western blotting with anti–bcl-2antibody. As shown in Figures 6A and 6B, ET-1 stimulationinduced the expression of bcl-2 protein in cardiac myocytes(lane 2) by 3.660.3-fold compared with saline simulation(lane 1). Blockage of calcineurin activation by Cys A inhib-ited the induction of expression of bcl-2 by ET-1 in these cells(lane 3). However, neither ET-1 nor Cys A altered theexpression of the proapoptotic molecule bax. These resultsindicate that calcineurin activation is required for the induc-tion of the expression of an antiapoptotic molecule, bcl-2, byET-1. To determine whether ET-1 stimulates the transcrip-tional activity of the upstream regulatory sequences of thebcl-2 gene, we evaluated the expression of a luciferase-reporter gene driven by DNA sequences from21796 to2999 relative to the translation start site of the human bcl-2gene in saline- and ET-1–stimulated cardiac myocytes. Thesesequences contain the major transcriptional promoter P1.20

Neonatal cardiac myocytes were transfected with pbcl-2lucand a small quantity of pRSVCAT as an internal control tonormalize for transfection efficiency and were then stimu-lated with saline or ET-1 in the presence or the absence ofCys A (431027 mol/L). Forty-eight hours later, luciferase andCAT activities were assessed in lysates from these cells. Asshown in Figure 7, ET-1 increased the relative luciferase

activity of pbcl-2luc by 3.7-fold. Cys A inhibited the ET-1–stimulated increase in the bcl-2 promoter activity by 34%(P,0.01), suggesting that calcineurin activation is, in part,involved in ET-1–responsive bcl-2 transcription.

DiscussionAccumulating evidence suggests that myocyte apoptosis oc-curs in failing hearts.1–4 Because adult cardiac muscle cellsare terminally differentiated and have lost their proliferativecapacity, the maintenance of cardiac muscle cell survival iscritical for the maintenance of systolic cardiac function.Recently, it has been shown that ET-1 is not only a growth-promoting peptide, but it is also a potent protective factoragainst apoptosis in various cell types, including cardiacmyocytes.12–14However, the precise signaling pathways lead-ing to antiapoptotic effects of ET-1 are poorly understood.The present study has demonstrated that the calcineurinpathway is required for the inhibitory effect of ET-1 onoxidant stress–induced apoptosis in cardiac myocytes. Thesefindings provide further insights into the role of calcineurin inheart failure in vivo.

Using primary neonatal rat cardiac myocytes, we showedthat ET-1 blocked an H2O2-induced increase in the TUNEL-positive cells. We cannot rule out the possibility that TUNEL-positive cells contain a subset of false-positive cells. How-

Figure 6. Cys A inhibits ET-1–induced expression of bcl-2. Cardiac myocytes were cultured in serum-free medium in the presence orabsence of ET-1 (1027 mol/L) and Cys A (431027 mol/L) as indicated for 48 hours. Cell lysates were subjected to Western blotting withan anti–bcl-2 antibody, anti-bax antibody, or anti–b-actin antibody.




ever, inhibition of apoptosis in rat cardiac myocytes wasdemonstrated by another 4 lines of evidence: (1) the inhibi-tion of nucleosomal ladder formation detected by agarose gelelectrophoresis, (2) the decrease in the number of cellsshowing nuclear condensation, a morphological feature ofapoptosis, (3) the inhibition of cleavage of caspase-3, and (4)the increase of mitochondrial transmembrane potential shownby cytofluorometric analysis. These findings are consistentwith the idea that ET-1 is a potent protective factor againstmyocardial cell apoptosis.

The levels of ET-1 in plasma and in ventricular myocar-dium markedly increase in close association with systolicdysfunction.8–10 Blockade of the myocardial ET-1 pathwayby ET-1 receptor antagonists has been shown to improvecardiac function and survival in experimental animal modelsof heart failure.8–10 These data indicate that activation ofmyocardial ET-1 pathway worsens cardiac function in thedevelopment of heart failure. Our data that ET-1 protectscardiac myocytes from apoptosis are paradoxical to data ofthese previous studies, assuming that myocardial cell apopto-sis is the only factor that determines cardiac function in thedevelopment of heart failure. However, an apoptosis-independent model of heart failure has been reported,25 andwe speculate that ET-1 could worsen cardiac functionthrough a mechanism other than apoptosis. For example,ET-1 receptors bind with Gi as well as Gq and decreasecAMP levels in cardiac myocytes.26 Thus, an increase ofmyocardial ET-1 may impair cardiac pump function bydecreasing intracellular cAMP levels in the development ofheart failure. Although the precise mechanisms by whichET-1 impairs systolic function should be further investigated,endogenous ET-1 may provide self-protection against myo-cardial cell apoptosis as well as function as a harmful factorunder the conditions of heart failure.

ET-1 signaling is coupled with an increase in intracellularcalcium levels.15 Increased calcium activates calcineurin, aubiquitously expressed serine/threonine phosphatase, by

binding the 19-kDa regulatory subunit of calcineurin.27 Theactivated form of calcineurin dephosphorylates NFAT tran-scription factors, which enables them to translocate to thenucleus.16,17 NFAT transcription factors interact with highaffinity and specificity with the cardiac-restricted zinc fingerprotein GATA4.17 Cys A and FK506 bind the immunophilincyclophilin and FK506-binding protein (FKBP12), respec-tively, forming complexes that bind the calcineurin catalyticsubunit and inhibit the ability of calcineurin to activate NFATtranscription factors.23 The present study demonstrated thatET-1 translocated NFATc into the nucleus and stimulated theinteraction between NFATc and GATA4. These findingsclearly indicate that ET-1 activates calcineurin. Cys A andFK506 antagonized the protective effects of ET-1. However,the same concentration of Cys A and FK506 alone increasedthe number of apoptotic cells in neither saline-stimulatedcells nor H2O2-stimulated cells. This indicates that theseagents might block the downstream signaling pathway bywhich ET-1 prevents apoptosis. These results demonstratethat calcineurin activation requires the antiapoptotic effect ofET-1. Several possibilities should be taken into account whenthe data of this study are applied to the in vivo setting in theadult. First, because myocardial development is not completeat birth, differences may exist between neonatal and adultcardiac myocytes. Second, the biological properties of disas-sociated myocytes in culture and myocytes in the organizedheart in vivo may differ. However, a recent study showed thattransgenic mice overexpressing an activated form of cal-cineurin exhibited less myocardial cell apoptosis after myo-cardial ischemia than wild-type mice.28 Thus, the inhibitionof cardiomyocyte apoptosis through calcineurin-dependentsignaling pathways is not confined to in vitro assays inneonatal cells but may also occur in adult cardiac myocytes invivo.

Recently, it has been reported that Cys A and FK506significantly inhibit myocardial cell apoptosis induced by theb-adrenergic agonist isoproterenol.29 Calcineurin has alsobeen implicated in proapoptotic signaling in thymocytesthrough mechanisms that are thought to involve the activationof the nur77 gene30; thus, the calcineurin pathway is involvedin the induction of apoptosis in some situations. One possibleexplanation of the opposing effects of calcineurin on apopto-sis is the crosstalk with other signaling pathways. Theactivation of calcineurin by isoproterenol stimulation resultsin the dephosphorylation of Bad, which may be involved inthe induction of apoptosis.29 In contrast, because ET-1 isfunctionally related to the activation of Akt/Bad, calcineurinactivation by ET-1 does not result in dephosphorylation ofBad. Despite the opposing effects of calcineurin activation onapoptosis, our findings demonstrate that calcineurin activa-tion de novo inhibits H2O2-induced myocardial cell apoptosis,in agreement with a previous report.25 However, precisemechanisms by which calcineurin could have diametricallyopposing effects in different contexts should be furtherinvestigated.

The bcl-2 gene product is a 25-kDa membrane protein thatfunctions to prevent apoptosis by various stimuli.24 Preven-tion of apoptosis by increased bcl-2 expression has also beenshown in adult cardiac myocytes.21 The present study dem-

Figure 7. Cys A inhibits ET-1–mediated activation of the bcl-2promoter. Two micrograms of pbcl-2 and 0.1 mg of pRSVCATwere cotransfected into primary cultures of neonatal rat cardiacmyocytes, which were subsequently stimulated with saline or1027 mol/L ET-1 in the presence or absence of Cys A (431027

mol/L) as indicated for 48 hours. The relative luciferase activities(luc/CAT) of pbcl-2 in the saline-stimulated state was set at 1.0in each experiment. Values are mean6SE of 2 independentexperiments, each carried out in duplicate.




onstrated that stimulation of cardiac myocytes with ET-1increased the expression of bcl-2 protein. ET-1 signaling isfunctionally linked to phospholipase C to induce phospho-inositide breakdown.22 It is also becoming clear that an ET-1pathway cross-talks with Ras and MAP kinase cascades.31

Because ET-1 affects multiple signaling pathways, the up-regulated bcl-2 expression may not be the only mechanismfor the antiapoptotic effects of ET-1. However, the potentability of bcl-2 to block apoptosis in many cell types suggeststhat this upregulation is, at least in part, involved in theprotective effects of ET-1.

Calcineurin-GATA4 is involved in the transcriptional path-ways that modulate cardiac hypertrophy.17 Calcineurin inhibi-tion by Cys A is sufficient to block cardiac hypertrophy in atransgenic animal model of hypertrophic cardiomyopathy.32

Although calcineurin seems to be involved in the developmentof compensating hypertrophy evoked by pressure overload, CysA instead worsens systolic function.28 Whether the deteriorationof systolic function caused by Cys A is attributable to an increaseof myocardial cell apoptosis should be further investigated.Further elucidation of signaling pathways leading to myocardialcell hypertrophy and survival by calcineurin activation will beuseful for understanding the role of this pathway in the devel-opment of heart failure in vivo.

AcknowledgmentsThis work was supported in part by grants to K.H. from the Ministryof Education, Science and Culture of Japan. We thank N. Sowa forhis excellent technical assistance.

References1. Liu Y, Cigola E, Cheng W, Kajstura J, Olivetti G, Hintze TH, Anversa P.

Myocyte nuclear mitotic division and programmed myocyte cell deathcharacterize the cardiac myopathy induced by rapid ventricular pacing indogs.Lab Invest. 1995;73:771–787.

2. Sharov VG, Sabbah HN, Shimoyama H, Goussev AV, Lesch M,Goldstein S. Evidence of cardiocyte apoptosis in myocardium of dogswith chronic heart failure.Am J Pathol. 1996;148:141–149.

3. Teiger E, Than VD, Richard L, Wisnewsky C, Tea BS, Gadoury L,Tremblay J, Schwartz K, Hamet P. Apoptosis in pressure overload-induced heart hypertrophy in the rat.J Clin Invest. 1996;97:2891–2897.

4. Li Z, Bing OH, Long X, Robinson KG, Lakatta EG. Increased cardio-myocyte apoptosis during the transition to heart failure in the sponta-neously hypertensive rat.Am J Physiol. 1997;272:H2313–2319.

5. Hirota H, Chen J, Betz UA, Rajewsky K, Gu Y, Ross J Jr, Muller W,Chien KR. Loss of a gp130 cardiac muscle cell survival pathway is acritical event in the onset of heart failure during biomechanical stress.Cell. 1999;97:189–198.

6. Packer M. The neurohormonal hypothesis: a theory to explain themechanism of disease progression in heart failure.J Am Coll Cardiol.1992;20:248–254.

7. Yanagisawa M, Kurihara H, Kimura S, Tomobe Y, Kobayashi M, MitsuiY, Yazaki Y, Goto K, Masaki T. A novel potent vasoconstrictor peptideproduced by vascular endothelial cells.Nature. 1988;332:411–415.

8. Wei CM, Lerman A, Rodeheffer RJ, McGregor CGA, Brandt RR, WrightS, Heublein DM, Kao PC, Edwards WD, Burnet JC Jr. Endothelin inhuman congestive heart failure.Circulation. 1994;89:1580–1586.

9. Sakai S, Miyauchi T, Kobayashi M, Yamaguchi I, Goto K, Sugishita K.Inhibition of myocardial endothelin pathway improves long-term survivalin heart failure.Nature. 1996;384:353–355.

10. Iwanaga Y, Kihara Y, Hasegawa K, Inagaki K, Kaburagi S, Araki M,Sasayama S. Cardiac endothelin-1 plays a critical role in the functionaldeterioration of left ventricles during the transition from compensatoryhypertrophy to congestive heart failure in salt-sensitive hypertensive rats.Circulation. 1998;98:2065–2073.

11. Shubeita HE, McDonough PM, Harris AN, Knowlton KU, GlembotskiCC, Brouwn JH, Chien KR. Endothelin induction of inositol phospholipid

hydrolysis, sarcomere assembly, and cardiac gene expression in ventric-ular myocytes: a paracrine mechanism for myocardial cell hypertrophy.J Biol Chem. 1990;265:20555–20562.

12. Wu-Wong JR, Chiou WJ, Dickinson R, Opgenorth TJ. Endothelinattenuates apoptosis in human smooth muscle cells.Biochem J. 1997;328:733–737.

13. Shichiri M, Sedivy JM, Marumo F, Hirata Y. Endothelin-1 is a potentsurvival factor for c-Myc-dependent apoptosis.Mol Endocrinol. 1998;12:172–180.

14. Araki M, Hasegawa K, Iwai-Kanai E, Fujita M, Sawamura T, KaburagiS, Sasayama S. Endothelin Type A receptor-dependent signalingpathways inhibitb-adrenergic agonist-induced apoptosis in cardiacmyocytes.J Am Coll Cardiol. 2000;36:1411–1418.

15. Touyz RM, Fareh J, Thibault G, Tolloczko B, Lariviere R, Schiffrin EL.Modulation of Ca21 transients in neonatal and adult rat cardiomyocytes byangiotensin II and endothelin-1.Am J Physiol. 1996;270:H857–H868.

16. Rao A, Luo C, Hogan PG. Transcription factors of the NF-AT family:regulation and function.Annu Rev Immunol. 1997;15:707–747.

17. Molkentin JD, Lu JR, Antos CL, Markham B, Richardson J, Robbins J,Grant SR, Olson EN. A calcineurin-dependent transcriptional pathway forcardiac hypertrophy.Cell. 1998;93:215–228.

18. Iwai-Kanai E, Hasegawa K, Araki M, Kakita T, Morimoto T, SasayamaS.a- andb-Adrenergic pathways differentially regulate cell type-specificapoptosis in rat cardiac myocytes.Circulation. 1999;100:305–311.

19. Kakita T, Hasegawa K, Morimoto T, Kaburagi S, Wada H, Sasayama S.p300 protein as a coactivator of GATA-5 in the transcription of cardiac-restricted atrial natriuretic factor gene.J Biol Chem. 1999;274:34096–34102.

20. Chen HM, Boxer LM. Pi 1 binding sites are negative regulators of bcl-2expression in pre-B cells.Mol Cell Biol. 1995;15:3840–3847.

21. Kirshenbaum LA, de-Moissac D. The bcl-2 gene product prevents pro-grammed cell death of ventricular myocytes.Circulation. 1997;96:1580–1585.

22. Sugawara F, Ninomiya H, Okamoto Y, Miwa S, Mazda O, Katsura Y,Masaki T. Endothelin-1-induced mitogenic responses of Chinese hamsterovary cells expressing human endothelin A: the role of a wortmannin-sensitive signaling pathway.Mol Pharmacol. 1996;49:447–457.

23. Shaw KTY, Ho AM, Raghavan A, Kim J, Jian J, Park J, Sharma S, RaoA, Hogan PG. Immunosuppressive drugs prevent a rapid dephosphory-lation of transcription factor NFAT1 in stimulated immune cells.ProcNatl Acad Sci U S A. 1995;92:11205–11209.

24. Hockenberry D, Nunez G, Milliman C, Schreiber RD, Korsmeyer SJ.Bcl-2 is an inner mitochondorial membrane protein that blocks pro-grammed cell death.Nature. 1990;348:334–336.

25. De Windt LJ, Lim HW, Taigen T, Wencker D, Condorelli G, Dorn GWII, Kitsis RN, Molkentin JD. Calcineurin-mediated hypertrophy protectscardiomyocytes from apoptosis in vitro and in vivo.Circ Res. 2000;86:255–263.

26. Jones LG. Inhibition of cyclic AMP accumulation by endothelin is per-tussis toxin sensitive and calcium independent in isolated adult felinecardiac myocytes.Life Sci. 1996;58:115–123.

27. Stemmer PM, Klee CB. Dual calcium ion regulation of calcineurin bycalmodulin and calcineurin B.Biochemistry. 1994;33:6859–6866.

28. Meguro T, Hong C, Asai K, Takagi G, McKinsey TA, Olson EN, VatnerSF. Cyclosporin attenuates pressure-overload hypertrophy in mice whileenhancing susceptibility to decompensation and heart failure.Circ Res.1999;84:735–740.

29. Saito S, Hiroi Y, Zou Y, Aikawa R, Toko H, Shibasaki F, Yazaki Y,Nagai R, Komuro I.b-Adrenergic pathway induces apoptosis throughcalcineurin activation in cardiac myocytes.J Biol Chem. 2000;275:34528–34533.

30. Youn HD, Liu JO. Cabin1 represses MEF2-dependent Nur77 expressionand T cell apoptosis by controlling association of histone deacetylasesand acetylases with MEF2.Immunity. 2000;13:85–94.

31. Bogoyevitch MA, Glennon PE, Andersson MB, Clerk A, Lazou A,Marshall CJ, Parker PJ, Sugden PH. Endothelin-1 and fibroblast growthfactors stimulate the mitogen-activated protein kinase signaling cascadein cardiac myocytes: the potential role of the cascade in the integration oftwo signaling pathways leading to myocyte hypertrophy.J Biol Chem.1994;269:1110–1119.

32. Sussman MA, Lim HW, Gude N, Taigen T, Olson EN, Robbins J, ColbertMC, Gualberto A, Wieczorek DF, Molkentin JD. Prevention of cardiachypertrophy in mice by calcineurin inhibition.Science. 1998;281:1690–1693.




hiromichi wada, teruhisa kawamura, tetsuhiko yanazume and

Documents