Juan-Li Gu, Sanjay Kumar, George Poste, Robert R. Ruffolo, Jr and Giora Z. FeuersteinTian-Li Yue, Xin-Liang Ma, Xinkang Wang, Anne M. Romanic, Gao-lin Liu, Calvert Louden,

Apoptosis by CarvedilolReceptor Expression in Prevention of Ischemia/Reperfusion-Induced Cardiomyocyte Possible Involvement of Stress-Activated Protein Kinase Signaling Pathway and Fas

Print ISSN: 0009-7330. Online ISSN: 1524-4571 Copyright © 1998 American Heart Association, Inc. All rights reserved.is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231Circulation Research

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Possible Involvement of Stress-Activated Protein KinaseSignaling Pathway and Fas Receptor Expression in

Prevention of Ischemia/Reperfusion-InducedCardiomyocyte Apoptosis by Carvedilol

Tian-Li Yue, Xin-Liang Ma, Xinkang Wang, Anne M. Romanic, Gao-lin Liu, Calvert Louden,Juan-Li Gu, Sanjay Kumar, George Poste, Robert R. Ruffolo, Jr, Giora Z. Feuerstein

Abstract—Carvedilol, a new vasodilating b-adrenoceptor antagonist and a potent antioxidant, produces a high degree ofcardioprotection in a variety of experimental models of ischemic cardiac injury. Recent clinical studies in patients withheart failure have demonstrated that carvedilol reduces morbidity and mortality and inhibits cardiac remodeling. Thepresent study was designed to explore whether the protective effects of carvedilol on the ischemic myocardium includeinhibition of apoptosis of cardiomyocytes and, if so, to determine its mechanism of action. Anesthetized rabbits weresubjected to 30 minutes of coronary artery occlusion followed by 4 hours of reperfusion. Detection of apoptosis ofcardiomyocytes was based on the presence of nucleosomal DNA fragments on agarose gels (DNA ladder) and in situ nickend labeling. Carvedilol (1 mg/kg IV), administered 5 minutes before reperfusion, reduced the number of apoptoticmyocytes in the ischemic area from 14.760.4% to 3.461.8% (77% reduction, P,.001). Propranolol, administered atequipotent b-blocking dosage, reduced the number of apoptotic myocytes to 8.962.1% (39% reduction, P,.05). DNAladders were observed in the hearts of all six vehicle-treated rabbits but only one of six carvedilol-treated rabbits (P,.01).Immunocytochemical analysis of rabbit hearts demonstrated an upregulation of Fas protein in ischemic cardiomyocytes,and treatment with carvedilol reduced both the intensity of staining as well as the area stained. Myocardialischemia/reperfusion led to a rapid activation of stress-activated protein kinase (SAPK) in the ischemic area but not innonischemic regions. SAPK activity was increased from 2.160.3 mU/mg (basal) to 8.960.8 mU/mg after 30 minutes ofischemia followed by 20 minutes of reperfusion. Carvedilol inhibited the activation of SAPK by 53.466.5% (P,.05).Under the same conditions, propranolol (1 mg/kg) had no effect on SAPK activation. Taken together, these results suggestthat carvedilol prevents myocardial ischemia/reperfusion–induced apoptosis in cardiomyocytes possibly by downregulationof the SAPK signaling pathway, by inhibition of Fas receptor expression, and by b-adrenergic blockade. The former twoactions represent novel and important mechanisms that may contribute to the cardioprotective effects of carvedilol. (CircRes. 1998;82:166-174.)

Key Words: apoptosis n cardiomyocyte n ischemia n reperfusion n stress-activated protein kinase n Fas

Apoptosis (programmed cell death) is a process for disposingof senescent, injured, or redundant cells through self-

destruction. Apoptosis is an active gene-directed process of celldeath, characterized by fragmentation of chromosomal DNAthat can be recognized by electrophoresis on an agarose gel asa characteristic pattern of DNA-ladder formation.1 Apoptosishas been detected in cultured cardiomyocytes subjected tohypoxia or ischemia followed by reperfusion2,3 and in heartpapillary muscle exposed to overstretching.4 Ischemic injury–induced apoptosis in the myocardium has recently beendemonstrated in rabbit5 and rat6,7 models. The results con-firmed that cardiomyocytes are the major source of nucleoso-

mal DNA degradation when apoptosis occurs in the heart.5

Apoptosis in human cardiomyocytes has also been demon-strated in patients with end-stage congestive heart failureresulting from idiopathic dilated cardiomyopathy8 and inpatients with arrhythmogenic right ventricular dysplasia.9 Apo-ptotic myocytes have also been detected in myocardial samplesobtained from patients who died from acute myocardialinfarction.10 On the basis of these observations, it has beenproposed that apoptosis represents an important process in thecardiac remodeling and progression of myocardial dysfunctionthat occurs in patients with congestive heart failure.11 Accord-ingly, inhibition of apoptosis in the myocardium may represent

Received May 13, 1997; accepted October 28, 1997.From the Departments of Pharmacology (T.-L.Y., X.W., A.M.R., J.-L.G., G.P., R.R.R., G.Z.F.), Cellular Biochemistry (S.K.), and Experimental

Pathology (C.L.), SmithKline Beecham Pharmaceuticals, King of Prussia, Pa, and the Division of Emergency Medicine (X.-L.M., G.L.), Thomas JeffersonUniversity, Philadelphia, Pa.

Correspondence to Tian-Li Yue, PhD, Department of Cardiovascular Pharmacology, Smith Kline Beecham Pharmaceuticals, UW-2510, PO Box 1539,King of Prussia, PA 19406.

E-mail Tian-Li—Yue@sbphrd.com© 1998 American Heart Association, Inc.

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a new therapeutic approach to inhibit the relentless progressionof left ventricular function that occurs in congestive heartfailure.12

Carvedilol is a new vasodilating b-adrenoceptor antagonistwith potent antioxidant activity.13,14 Carvedilol has been shownto reduce infarct size in a variety of experimental models ofacute myocardial infarction in several species.15 The degree ofinfarct size reduction produced by carvedilol was significantlygreater than that produced by other b-blockers at doses thatproduce equivalent degrees of b-adrenoceptor blockade, evenwhen vasodilators are administered with the other b-blockersto mimic the hemodynamic effects of carvedilol.15 Recentclinical studies in patients with congestive heart failure havedemonstrated that carvedilol significantly reduces morbidityand hospitalization and, more important, reduces mortality by65% and delays the progression of heart failure when admin-istered in addition to conventional therapy.16 The mechanismsresponsible for the high degree of cardiac protection producedby carvedilol in animals and in humans have not beenelucidated.

The objective of the present study was to determinewhether the cardioprotective effects of carvedilol includeinhibition of apoptosis in cardiomyocytes and, if so, to identifythe underlying mechanism(s). To examine these possibilities, astandard rabbit cardiac ischemia/reperfusion model was used inwhich ischemia/reperfusion-induced apoptosis of cardiomyo-cytes has been shown.5 The effects of carvedilol on theexpression of Fas and Bcl-2 in the myocardium were deter-mined because of the established death-promoting effect ofFas17 and the antiapoptotic effect of Bcl-2.18 Moreover, theeffect of carvedilol on the SAPK signaling pathway in myo-cardium was studied because the SAPKs, which represent afamily of novel kinases that activate the transcriptional activityof c-Jun,19,20 have recently been suggested to be involved in thesignaling pathway that leads to apoptosis.21

Materials and MethodsAnimal ModelMale New Zealand White rabbits (2.2 to 3.1 kg) were used for thepresent study, and a detailed description of the surgical procedure hasbeen previously described.22 Briefly, myocardial ischemia was pro-duced using a 3–0 silk suture placed around the major marginal branchof the left circumflex coronary artery located 10 to 12 mm from itsorigin. Arterial blood pressure and the ECG were continuouslyrecorded. After thoracotomy and a 30-minute stabilization period,myocardial ischemia was initiated (time 0) and maintained for 30minutes. After this period of ischemia, the ligature was removed, andthe ischemic myocardium was reperfused for 4 hours. Vehicle or testdrugs (carvedilol or propranolol, 1 mg/kg) were given as singleintravenous bolus injections 5 minutes before the initiation of reper-fusion. Samples of the myocardium from the normal area (ANAR)

and the ischemic area (AAR) were isolated using dye perfusion(Monastral dye). In part of the present study, the unstained portion ofmyocardium (ie, the AAR) was further separated into the necrotic andnonnecrotic areas using the method of nitroblue tetrazolium stainingas described previously.22

DNA Fragmentation (DNA Ladder)Hearts were removed from the perfusion device, and transmuralmyocardial samples were isolated as described above. Tissues werefrozen in liquid nitrogen and stored at 270°C for up to 1 week.Tissues were minced while thawing in lysis buffer (50 mmol/LTris-HCl, pH 8.0, 20 mmol/L EDTA, and 1% SDS) on ice for 5minutes, and proteinase K (100 mg/mL) was then added. Afterincubation at 55°C with shaking for 18 hours, DNA was extractedwith phenol/chloroform three times, precipitated in ethanol, treatedwith DNA-free RNase, reextracted, and precipitated again. DNAconcentration was determined, and 5 mg of DNA was used forelectrophoreses on a 1.8% agarose gel.23

TUNELAfter ischemic intervention, the hearts were removed, mounted in aLangendorff perfusion apparatus, and perfused in a retrograde mannerwith lactated Ringer’s solution, which was changed to 4% parafor-maldehyde in PBS (pH 7.4, 4°C). Full-thickness slices of the nonis-chemic and ischemic left ventricular wall that includes the nonnecroticand necrotic areas were cut into four pieces each, fixed, dehydrated,and embedded. Paraffin-embedded myocardial sections (3 to 5 mm)were mounted on silanized slides and dried at 37°C overnight.Immunohistochemical procedures for detecting apoptotic cardiomyo-cytes were performed by direct immunoperoxidase detection ofdigoxigenin-labeled genomic DNA using ApopTag (Oncor) accord-ing to the manufacturer’s instructions. Cardiomyocytes from fourseparate sections that were picked randomly from each of the fourpieces of the tissue were analyzed per animal. The percentage ofpositively stained immunolabeled nuclei of myocytes was determinedby random counting 10 fields per section, except at the areas wheretypical signs of necrosis occurred, and the index of apoptosis wasdetermined (ie, number of apoptotic myocytes/total number ofmyocytes counted3100). Areas of extensive myocardial necrosis wereavoided and not included in the evaluation and analysis for apoptosis.As a positive control, sections of heart tissue were exposed to DNaseI for 20 minutes before nick end labeling. The numbers of myocytenuclei stained were easily identified (data not shown).

Immunohistochemical Analysis of Fas andBcl-2 ExpressionRepresentative sections of heart tissue were fixed in 10%neutral buffered formalin for 24 to 48 hours at 4°C and cutlongitudinally into sections 2 to 3 mm thick. After standardhistological processing and embedding in paraffin, 5-mm-thicksections were prepared for immunoperoxidase staining usingthe Vectastain ABC kit (Vector) according to the manufactur-er’s instructions. Briefly, endogenous peroxidase was quenchedwith 0.3% H2O2 in methanol for 30 minutes. Nonspecificimmunoglobulin binding sites were blocked with normal goatserum for 1 hour, and then the sections were incubated withthe primary antibody, mouse anti-human Fas (Upstate Bio-technology) or mouse anti-human Bcl-2 (DAKO) antibody,for 1 hour at room temperature. The sections were thenincubated for 30 minutes with a biotinylated goat anti-mouseIgM (Fas) secondary antibody (Vector) or a biotinylated goatanti-mouse IgG (Bcl-2) secondary antibody (Vector), followedby 30 minutes of incubation with ABC (Vector). Immuno-globulin complexes were visualized on incubation with 3,39-diaminobenzidine (Vector), then washed, counterstained with

Selected Abbreviations and Acronyms

AAR 5 area(s) at riskABC 5 avidin-biotin complex

ANAR 5 area(s) not at riskGST 5 glutathione S-transferase

SAPK 5 stress-activated protein kinaseTLV 5 total left ventricle

TUNEL 5 in situ nick end labeling

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Gill’s hematoxylin, cleared, mounted, and examined by lightmicroscopy.23

SAPK AssayRabbit hearts were quickly removed after 30 minutes of ischemiafollowed by a period of reperfusion. Full-thickness sides of theventricular wall were cut from the ischemic/reperfused left ventriclesand the nonischemic right ventricles (as a control) and frozen in liquidnitrogen. The ventricles were “freeze-clamped” using precooledaluminum tongs and pulverized under liquid nitrogen.24 The powderswere resuspended in ice-cold lysis buffer, and the protein content inthe detergent-soluble supernatant fraction was measured as describedpreviously.23

A fusion vector for GST–c-Jun(1–81) was constructed by cloning thec-Jun gene fragment (corresponding to the amino acid codons 1 to 81)into a pGEX 4T-3 vector, which contains a DNA sequence encodingGST. The fusion protein was expressed in Escherichia coli and purifiedwith glutathione-Sepharose chromatography.19,23 SAPK activity wasmeasured using GST–c-Jun(1–81) bound to glutathione-Sepharose 4Bas described by Verheij et al.25 Briefly, 100 mg of tissue protein extractwas incubated with anti-SAPK antibody–conjugated Sepharose beadsat 4°C for 3 hours. The immunoprecipitates were washed extensivelyand assayed for kinase activity at 30°C for 20 minutes using 4 mg

GST–c-Jun(1–81) fusion protein as a specific substrate. Phosphorylatedproteins were resolved by 10% SDS-polyacrylamide gel electrophore-sis followed by autoradiography. The incorporation of 32P intoGST–c-Jun(1–81) was determined by cutting the bands correspondingto GST–c-Jun(1–81) from the gel, and radioactivity was determined byscintillation counting. One unit of SAPK activity was defined as theincorporation of 1 pmol phosphate from ATP per minute into therespective substrate.24

Statistical AnalysisAll values in the text and figures are presented as mean6SEM of nindependent experiments. Statistical evaluation was performed byusing one-way ANOVA with subsequent post hoc paired compari-sons. The proportions of “DNA ladders” were assessed by x2 analysis.A value of P,.05 was accepted as statistically significant.

ResultsHemodynamic ChangesIn sham-operated rabbits, there were no significant changes inheart rates, left ventricular systolic pressure, and left ventricularend-diastolic pressure during the entire experimental proceed-ing. There was no significant difference among the three

Hemodynamic Effects of Carvedilol and Propranolol in Rabbits Subjected toIschemia and Reperfusion

Sham MI MI1Vehicle MI1CV MI1Pro

HR, bpm

Control 254611 25168 25468 25569

I-30 25668 25766 25267 25167

I-60 25368 24469 218610*† 22069*†

R-30 25469 24167 23466‡ 23367*‡

R-60 25169 248610 22968†‡ 22868†‡

R-120 24968 24768 22469*† 22566*†

R-180 24767 24067 22368 22467*

LVSP, mm Hg

Control 11764 11665 11865 11664

I-30 11865 9167* 9066* 8965*

I-60 11666 9565*† 8165*† 7865*†

R-30 11564 9366* 8366* 7766*

R-60 11565 9165* 8265* 8166*

R-120 11366 9065* 8566* 8166*

R-180 11365 9165* 9266* 8365*

LVEDP, mm Hg

Control 4.160.9 4.260.8 4.360.6 4.260.6

I-30 4.160.7 6.960.7‡ 6.860.6‡ 6.760.6‡

I-60 4.360.7 6.760.6‡ 6.660.6‡ 6.560.6‡

R-30 4.060.8 7.060.8‡ 6.860.8‡ 6.960.7‡

R-60 4.460.8 6.660.7‡ 6.360.6* ‡6.660.7‡

R-120 4.360.3 6.660.7‡ 5.860.7‡ 6.260.5‡

R-180 4.260.8 6.460.7 5.260.6939 5.760.6‡

MI indicates myocardial infarction; CV, carvedilol; Pro, propranolol; CV or Pro was administratedintravenously (over a period of 1 minute) at a dose of 1 mg/kg 5 minutes before reperfusion (55minutes after ischemia). Values are mean6SEM.

I-30 and I-60, 30- and 60-minute ischemia; and R-30, R-60, R-120, and R-180, 30-, 60-, 120-, and180-minute reperfusion.

HR, heart rate; LVSP, left ventricular systolic pressure; LVEDP, left ventricular end-diastolicpressure.

*P,.01 and ‡P,.05 vs control; †P,.05 vs MI1vehicle group.

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ischemia/reperfusion groups in hemodynamic parameters be-fore coronary occlusion. After coronary ligation, all animalsshowed a comparable blood pressure decrease. Administrationof carvedilol or propranolol 5 minutes before reperfusionsignificantly decreased heart rate and further decreased leftventricular systolic pressure. However, there was no significantdifference between carvedilol- and propranolol-treated rabbitsin heart rate, left ventricular systolic pressure, and left ventric-ular end-diastolic pressure either before coronary occlusion,before drug administration, after drug administration, or afterreperfusion (Table).

Protection of Myocardial Injury After Ischemiaand ReperfusionThere was no significant difference in the AAR expressed as apercentage of TLV among the three groups of animals,indicating that a comparable degree of ischemic risk existed inall three experimental groups. After 30 minutes of coronaryartery occlusion followed by 4 hours of reperfusion, thenecrotic area, expressed as a percentage of the AAR or apercentage of TLV, was 3766% and 13.563.0%, respectively.Both carvedilol and propranolol reduced the necrotic area,expressed as either a percentage of AAR or TLV, and theprotective effects of carvedilol treatment were more profoundthan those achieved with propranolol (Fig 1). Furthermore,there was a pronounced decrease in the severity of myocardialdegeneration and the heterophilic response in the drug-treatedrabbits that was consistent with previous observations in otherspecies.15

Detection of DNA Fragmentation (DNA Ladder)in Hearts Subjected to Ischemia and ReperfusionOf six vehicle-treated rabbits subjected to 30 minutes ischemiaand 4 hours of reperfusion, all showed a typical nucleosomalladder on DNA electrophoresis of tissue obtained from theischemic/reperfused left ventricles, indicating the occurrenceof apoptosis (Fig 2, V1 to V6, lanes marked with R). Nucleo-somal DNA ladders were not detected in nonischemic left

ventricles of either vehicle- or carvedilol-treated animals (lanesmarked with N). In the six animals treated with carvedilol (1mg/kg IV 5 minutes before reperfusion), DNA ladder forma-tion was observed in only one ischemic left ventricle (Fig 2, lastlane) (P,.01 versus vehicle).

In Situ Determination of Apoptosis inIschemic/Reperfused MyocardiumHeart tissue from sham-operated rabbits and from nonischemicregions (ANAR) exhibited very low levels of staining forTUNEL (1.6060.47%, n56) (Figs 3A and 4). Because DNAdegradation can also occur nonspecifically in necrotic myocar-dium and because this might also be stained by TUNEL,apoptotic myocytes in areas where typical signs of necrosisoccurred (ie, loss of membrane integrity, cell lysis, or swelling)were not assessed. Apoptotic myocytes (stained positively)were localized to a greater degree in salvaged areas surroundingthe infarcted tissues than in other parts of the ischemic area,and the apoptotic myocytes were individually dispersed amongotherwise normal myocytes, as is characteristic of apoptosis.Significant numbers of myocyte nuclei from ischemic/reper-fused left ventricle were stained positively for TUNEL(14.761.4%, n56; Fig 3B, Fig 4) in vehicle-treated rabbits. Incontrast, there was a significant reduction in the numbers ofmyocyte nuclei staining positively in ischemic/reperfusedmyocardial tissue from rabbits treated with carvedilol(3.461.8%, n56, P,.001 versus vehicle; Fig 3C and Fig 4).The number of positively stained myocytes in the ischemic leftventricles was also reduced, but to a lesser degree, in animalstreated with propranolol (8.962.1%, n56, P,.05 versusvehicle; Fig 4).

Downregulation of Fas Expression in IschemicInjured Myocardium by CarvedilolIn nonischemic rabbit hearts, the basal level of Fas receptorexpression was not detectable by immunohistochemistry (Fig5A). However, Fas expression was significantly upregulated invehicle-treated ischemic/reperfused hearts (AAR) (Fig 5B).Immunostaining for Fas protein was more intense in thenonnecrotic zones compared with the necrotic zones. Whenmouse IgM was substituted for the primary antibody, positiveFas immunoreactivity was not detected (data not shown).Carvedilol treatment was associated with diminished Fas ex-

Figure 1. Protection by carvedilol and propranolol of myocardialinjury following ischemia and reperfusion in rabbits. Myocardialinjury was produced by occlusion of the left circumflex coronaryartery for 30 minutes, followed by reperfusion for 4 hours asdescribed in “Materials and Methods.” Carvedilol (1 mg/kg) orpropranolol (1 mg/kg) was given intravenously 5 minutes beforethe initiation of reperfusion. Samples of the myocardium fromthe normal (ANAR) and the ischemic (AAR) areas were isolatedusing dye perfusion (Monastral dye). The AAR was further sepa-rated into the necrotic (NEC) and nonnecrotic areas using themethod of nitroblue tetrazolium staining. The values in the figureare mean6SE (n56). *P,.05 and **P,.01 vs vehicle; #P,.05 vspropranolol.

Figure 2. Electrophoretic analysis of internucleosomal DNAextracted from rabbit hearts exposed to 30 minutes of ischemiaand 4 hours of reperfusion in the presence of vehicle (V1 to V6 )or carvedilol (Carv1 to Carv6 ) that was administered intrave-nously 5 minutes before reperfusion. Lanes labeled with N werefrom nonischemic ventricles (ANAR), and lanes labeled with Rwere from ischemic left ventricles (AAR). Each well was loadedwith 5 mg of DNA. The first lane is DNA size markers (1-kb DNAladder).

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pression in ischemic/reperfused hearts (Fig 5C). In contrast,Bcl-2 expression was detected only in intramyocardial arte-rioles, and no significant change was demonstrated after theischemia/reperfusion insult (Fig 5, inserts in bottom panels).

Inhibition by Carvedilol of Ischemia/Reperfusion-Induced Activation of SAPKin MyocardiumThe basal SAPK activity in right ventricles (2.060.5 mU/mg,n56) was similar to the basal level of SAPK in nonischemic leftventricles (2.160.3 mU/mg, n56) and was not activatedwhen the left ventricles were subjected to ischemia/reperfu-sion (2.060.3 mU/mg, n56) (Figs 6 and 7). On the basis ofthis fact, the SAPK activity of the right ventricle was used asthe control, and the ratio of SAPK activity in the ischemic leftventricle to SAPK activity in the nonischemic right ventriclewas determined (IR/C in Figs 6 and 7) to assess the fold

increase in SAPK activity induced by ischemia and reperfusion.Ischemia alone for 30 minutes had no effect on SAPK activityin rabbit myocardium (Fig 6). However, SAPK was remark-ably activated by ischemia/reperfusion. A significant increasein SAPK activity was detected by 10 minutes, peaked at 20minutes, and then returned to basal levels 60 minutes afterreperfusion. Administration of carvedilol (1 mg/kg IV) 5minutes before reperfusion significantly diminished ischemia/reperfusion-induced activation of SAPK in myocardium from8.960.8 to 4.160.6 mU/mg (53.466.5% reduction, P,.05).Accordingly, the fold change of SAPK activity (IR/C in Figs6 and 7) induced by ischemia/reperfusion was reduced from5.360.6 to 2.360.4 (56.666.2% reduction, P,.05; Fig 7).Under the same condition, propranolol had no effect onischemia/reperfusion-induced activation of SAPK (Fig 7).

DiscussionThe two most widely used techniques to identify apoptosis,DNA ladder formation on agarose gel and histochemicalvisualization of nuclear DNA fragments by TUNEL, wereused in the present study. It is widely assumed that anoligonucleosomal ladder-type fragmentation into monomersand oligomers of 180 bp affecting nuclear but not mitochon-drial DNA is indicative of apoptotic cell death, and accord-ingly, DNA ladder formation is one of the hallmarks ofapoptosis. The TUNEL method was also used to identify thecell type undergoing DNA fragmentation in which the free39-OH end of DNA can be labeled with modified nucleotidesin an enzymatic reaction. The specificity of TUNEL todistinguish between apoptotic versus necrotic cells has beencontroversial. In order to avoid possible errors caused bycounting necrotic myocytes that localize in necrotic zones asapoptotic cells, we counted only those apoptotic myocytes thatoccurred in the AAR but not within the infarcted tissue.Apoptotic myocytes having an intact plasma membrane werelocalized primarily within salvaged areas of myocardium sur-rounding the infarcted tissues in the AAR (in the border zoneof histologically infarcted myocardium), and the apoptotic

Figure 3. Photomicrographs of in situ detection of DNA frag-ments in rabbit heart tissue. Left ventricles from normal rabbits(A) or rabbits subjected to 30 minutes of ischemia followed by 4hours of reperfusion in the absence (B) or presence (C) of carve-dilol, which was administered 5 minutes before reperfusion,were sectioned and stained using the TUNEL method, asdescribed in “Materials and Methods.” Magnification 3200.

Figure 4. Percentage of nuclei staining positive for TUNEL inheart tissues exposed to 30 minutes of ischemia and 4 hours ofreperfusion from rabbits treated with vehicle, carvedilol (1mg/kg), or propranolol (1 mg/kg) intravenously 5 minutes beforereperfusion. The AAR and ANAR of the ischemic/reperfused leftventricles were cut into four pieces each, fixed, dehydrated,embedded, and sectioned (3 to 5 mm). Four sections randomlypicked from each of these four pieces were analyzed per ani-mal. The stained immunolabeled nuclei of myocytes was deter-mined by random counting of 10 fields per section except theareas where typical signs of necrosis occurred. Each value rep-resents the mean6SE of six animals. *P,.05 and **P,.001 vsvehicle.

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myocytes were detected as individual cells dispersed amongotherwise normal myocytes. Apoptotic myocytes were rarelydetected in ANAR. DNA ladder formation was detected in allleft ventricles from six rabbits subjected to ischemia/reperfu-sion. Carvedilol, when administered immediately before reper-fusion, significantly reduced the number of apoptotic myocytesby 77% (P,.001) as well as the occurrence of DNA ladders inischemic/reperfused hearts (P,.01). Propranolol, at equipo-tent b-blocking dosage, also reduced, but to a lesser degree, thenumber of apoptotic myocytes in ischemic/reperfused leftventricles. To our knowledge, this represents the first study todemonstrate the effectiveness of a cardiovascular drug ap-proved for congestive heart failure to protect against acute

ischemia/reperfusion injury–induced cardiomyocyte apoptosisin vivo.

Apoptosis is an active gene-directed process of cell suicidecontrolled by proapoptotic and antiapoptotic genes. Apopto-sis-related genes in cardiomyocytes have been the subject ofintense investigation, yet limited information is currentlyavailable.26,27 Because carvedilol significantly inhibited theoccurrence of apoptosis induced by ischemia/reperfusionwhen assessed by two different techniques, we investigated theeffects of carvedilol on the expression of Fas (proapoptotic)17

and Bcl-2 (antiapoptotic)18 proteins in the rabbit heart. Fas is amember of the tumor necrosis factor receptor family, Fas-related apoptosis has been demonstrated in a variety of cell

Figure 5. Immunocytochemical detection of Fas (top panels) and Bcl-2 (bottom panels) protein in ischemic left ventricles from rabbitsexposed to 30 minutes of ischemia followed by 4 hours of reperfusion and treated with vehicle (B and E) or carvedilol (1 mg/kg IV) 5minutes before reperfusion (C and F). Panels A and D were from nonischemic left ventricles. Inserts in the bottom panels are intramyo-cardial arterioles.

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types,17 and tumor necrosis factor-a–induced cardiomyocyteapoptosis has been demonstrated in vitro.28 The basal level ofFas in nonischemic ventricular tissue was low or below adetectable level, which is consistent with observations in otherspecies.29 However, Fas expression was markedly upregulated

in areas at risk in left ventricles of the rabbits subjected toischemia followed by reperfusion. In carvedilol-treated ani-mals, the expression of Fas was reduced significantly, both interms of staining intensity and the size of area stained positivelyfor Fas. Conversely, expression of Bcl-2 was not detected incardiomyocytes either within the AAR or ANAR, and wasonly detected in the intramyocardial arterioles; this expressionwas not affected by either ischemia/reperfusion or by carve-dilol. This finding is in accord with a recent report by Ohno etal.30 Our data suggest, therefore, that Fas, but not Bcl-2, isinvolved in ischemia/reperfusion-induced apoptosis in rabbitmyocytes. The results of the present study also indicate thatoverexpression of Fas in rabbit myocytes plays an importantrole in the acceleration of cellular damage after ischemic injuryand that downregulation of Fas expression by carvedilol maybe critically involved in the protection of myocytes againstapoptosis.

Recent evidence has suggested that the induction of apo-ptosis involves activation of a signaling system.31 However,many elements of the signaling pathway leading to apoptosis,especially in cardiomyocytes, remain unknown.32 SAPK hasrecently been implicated as an important signaling pathwaymediating programmed cell death.20 In contrast to mitogen-activated protein kinase, SAPK is weakly activated by growthfactors but is strongly activated by cellular stresses. Overex-pression of SAPK, or activation of its upstream kinases, inducesapoptosis in cultured PC-12, U937, and bovine aortic endo-thelial cells. In contrast, blockade of its downstream effect byexpression of a dominant-negative c-Jun mutant prevented celldeath.21,25 The apparent relationship between the blockade ofthe SAPK signaling pathway and the resistance to cell death hassuggested that SAPK may be a mediator of cell death. Thesefindings are of particular relevance to hearts that have beenexposed to pathological stress. Recently, activation of SAPKhas been observed in vitro in perfused rat hearts exposed toischemia/reperfusion injury,24 and it has been proposed thatSAPK may modulate an apoptotic response in myocytes.33

However, activation of the SAPK pathway in the heart in vivohas not yet been demonstrated. Reperfusion is associated witha dramatic increase in SAPK activity, which was significantlyinhibited by carvedilol administered before reperfusion. Therapid activation of SAPK is consistent with a role for this kinasein the activation of transcription factors and the stress-activatedsignaling cascades that follow cellular stress. This represents thefirst in vivo study to demonstrate quantitatively the activationof SAPK in the heart by ischemia/reperfusion-induced injuryand the ability to inhibit this activation by a clinically relevanttherapeutic agent.

Carvedilol is a multiple action cardiovascular agent withb-adrenergic blocking, vasodilating, and antioxidant prop-erties. The former two actions can produce a significantreduction in myocardial oxygen demand and thereforereduce ischemic tissue injury; this mechanism of actioncould well account for its protection of cardiomyocytesfrom apoptosis. The effectiveness of propranolol, a nonse-lective b-blocker, in reduction of myocyte apoptosis ob-served in the present study supports this hypothesis. How-ever, at equipotent b-blocking dosage, propranolol showedsignificantly less cardiac protection and antiapoptotic activ-

Figure 6. Time course of ischemia/reperfusion-induced activa-tion of SAPK in rabbit hearts. Rabbit hearts were quicklyremoved after 30 minutes of ischemia alone or after ischemiafollowed by a period of reperfusion indicated in the figure. Sam-ples of the myocardium from the ischemic/reperfused left ventri-cles (IR) and the nonischemic right ventricles (control, C) wereisolated. The tissue extraction and SAPK assay were performedas described in “Materials and Methods.” The ratio of SAPKactivity in ischemic left ventricle vs that in right ventricle of eachanimal (IR/C) was calculated. Each point in the top panel repre-sents mean6SE of at least three animals. *P,.05 and **P,.01vs time 0. The bottom panel is a representative autoradiogram.Each time point contains two lanes: the left lane is the right ven-tricle (control), and the right lane is the ischemic left ventricle.

Figure 7. Effects of carvedilol and propranolol on ischemia/reperfusion-induced activation of SAPK in rabbit hearts sub-jected to 30 minutes of ischemia followed by 20 minutes ofreperfusion. Carvedilol (1 mg/kg), propranolol (1 mg/kg), or vehi-cle was administered intravenously 5 minutes before reperfusionthat continued for 20 minutes. The ventricular walls were cutfrom the ischemic/reperfused left ventricles (IR) and the nonis-chemic right ventricles (control, C), and the SAPK activity wasdetermined (top panel). The left bar graph shows the SAPKactivity in control ventricles of three groups. The middle bargraph shows the SAPK activity in the IR ventricles of threegroups. The ratio of SAPK activity in ischemic left ventricle vsthat in right ventricle of each animal (IR/C) was calculated. Theright bar graph shows the mean6SE of the ratios of threegroups (n56). *P,.05 and **P,.01 vs vehicle-treated group. Thebottom panel is a representative autoradiogram.

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ity, indicating the involvement of other mechanisms. Therewas no significant difference in hemodynamic changesbetween carvedilol- and propranolol-treated rabbits, sug-gesting that the vasodilating activity of carvedilol may notbe the mechanism that makes the difference in the cardio-protection. Carvedilol has been demonstrated to be a potentantioxidant in a variety of experimental animal models (forreview see Reference 15) and in humans.34 The higherdegree of cardioprotection produced by carvedilol com-pared with other b-blockers has been attributed, at least inpart, to the antioxidant activity of the drug.15 Accumulatingdata have now provided strong evidence that oxygen-derived free radicals play an important role in cell apoptosis(for review see Reference 35). Addition of oxygen-derivedfree radicals, the induction of free radical formation, and thedepletion of cellular antioxidants (such as glutathione) allhave the capacity to induce apoptosis.36,37 Moreover, Fas-related apoptosis can be blocked by antioxidants.38 Further-more, oxidative stress has been demonstrated to activateSAPK, and an increase in intracellular antioxidants preventsthe activation of SAPK in cultured cells.39,40 The activationof SAPK, which was observed only during reperfusion inthe present study, strongly suggests an involvement of freeradicals in ischemia/reperfusion-induced SAPK activation.The absence of effect by propranolol on SAPK activityfurther supported the role of free radicals in activation ofSAPK since propranolol has no antioxidant activity.14 Inas-much as oxygen-derived free radicals are potent inducers ofSAPK as well as apoptosis and the growing body of evidenceindicates that the SAPK signaling pathway may play animportant role in apoptosis, it is conceivable that thesuperior antiapoptotic effect of carvedilol demonstrated inthe present study may be, at least partially, attributed to itsunique antioxidant activity. Carvedilol, by virtue of itsantioxidant activity, may directly reduce oxygen-derivedfree radical–induced apoptosis in myocytes and/or inhibitfree radical–induced SAPK activation and Fas upregulation,thereby indirectly reducing cardiomyocyte apoptosis.Moreover, we cannot rule out the possibility that a syner-gistic interaction between b-blocking activity and antioxi-dant potential is operating as well. Because the real role ofSAPK signaling pathway in cell apoptosis is still underinvestigation, the mechanisms of carvedilol for inhibition ofapoptosis in cardiomyocytes remain to be further clarified.

In summary, we have demonstrated that cardiac ischemiafollowed by reperfusion results in cardiomyocyte apoptosis inthe rabbit heart in vivo. Yin et al recently reported theactivation of JNK2 (SAPK2, 55 kD) in canine heart in vivoafter ischemia/reperfusion.41 They observed a strong correla-tion between stress kinase activation and initiation of apoptoticcell death and provided evidence to support a role for JNK inapoptosis in vivo. The SAPK signaling pathway is activated inthe heart during this process, and expression of Fas in myocytesis significantly upregulated. Carvedilol, when administeredbefore reperfusion, inhibits SAPK activation, attenuates Fasexpression, and reduces apoptosis in cardiomyocytes. Ourresults suggest that inhibition of cardiomyocyte apoptosis,possibly through suppression of the SAPK signaling pathway,downregulation of Fas expression, and b-adrenergic blockade,

may represent an important mechanism for therapeuticcardioprotection.

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