chronic hypoxia induces apoptosis in cardiac myocytes: a possible role for bcl-2-like proteins

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Biochemical and Biophysical Research Communications 286, 419–425 (2001)

doi:10.1006/bbrc.2001.5406, available online at http://www.idealibrary.com on

hronic Hypoxia Induces Apoptosis in Cardiac Myocytes:Possible Role for Bcl-2-like Proteins

rank Jung,* Uli Weiland,* Roger A. Johns,† Christian Ihling,‡ and Stefanie Dimmeler*,1

Molecular Cardiology, Department of Internal Medicine IV, University of Frankfurt, Frankfurt, Germany;Department of Anesthesiology and Critical Care, Johns Hopkins University, School of Medicine, Baltimore,aryland 21287; and ‡Department of Pathology, University of Freiburg, Freiburg, Germany

eceived July 12, 2001

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The effect of prolonged hypoxia as well as the mo-ecular mechanisms on cardiac cell death is not wellstablished. A possible role of Bcl-2 and Bax inypoxia-induced apoptosis in different cell types haseen proposed. Here we demonstrate the effect of hyp-xia on the induction of apoptosis and the expressionf Bcl-2-like proteins in vivo and in vitro. Hearts fromats exposed to chronic hypoxia (n 5 4) showed anncreased rate of apoptosis compared to normoxicearts (n 5 4). The induction of apoptosis in hypoxicearts correlated with a significant decrease of Bcl-2rotein level, whereas Bax protein expression was in-reased. Exposure of isolated neonatal rat cardiacyocytes to hypoxia also resulted in a significant in-

rease in apoptosis. However, Bcl-2 and Bax proteinevels essentially remained unchanged. Our results

ay suggest a different molecular mechanism ofypoxia-induced apoptosis in vivo and in vitro. © 2001

cademic Press

Key Words: hypoxia; apoptosis; myocardium; Bcl-2;ax.

Apoptosis, or programmed cell death, has been de-cribed in many pathophysiological states leading toyocardial dysfunction, such as different forms of car-

iomyopathy, rejection after cardiac transplantationut also conditions of acute or prolonged hypoxia, suchs myocardial hibernation (1–5). To date controversytill exists as to whether hypoxia alone is sufficient tonduce apoptosis or if it requires a combination of hyp-xia with other cell damaging metabolic components,uch as acidosis or accumulation of cellular or extra-ellular metabolic waste products. Recently, Webster etl. have demonstrated that in an in vivo model acido-

1 To whom correspondence should be addressed at Department ofnternal Medicine IV, Molecular Cardiology, University of Frank-urt, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany. Fax: 149-69-301-7113. E-mail: [email protected].

419

lone are strong stimuli for the induction of apoptosis6). The diversity in different in vitro or in vivo models

ay result in a different cellular response to hypoxia.Controversy also exists about the precise initiating

timuli and signaling pathways leading to hypoxia-nduced apoptosis in cardiac myocytes. A long list of

ediators have been implicated in the process of apop-otic cell death. These include the cytokine TNFa, cer-mide, and high levels of the metabolic substrate glu-ose (7–9). It is a well accepted concept that intracel-ular proteins of the Bcl-2 family can be part of thepoptotic signaling cascade, of which Bcl-2 and Bcl-xLxhibit antiapoptotic properties and Bax and Bad ex-ibit proapoptotic actions (10, 11). These proteins canorm homo- or heterodimers, which modulate the in-uction of apoptosis. The ratio of the antiapoptoticrotein Bcl-2 and the proapoptotic protein Bax seemso be critical for cell survival (12–15). The activities ofhe Bcl-2 protein are also regulated by their subcellularocalization (cytosolic vs mitochondrial). A reduction ofcl-2 leading to an increase of Bax homodimers results

n translocation of Bax to the mitochondrial membranend subsequent mitochondrial dysfunction with loss ofhe membrane potential and release of cytochrome cnto the cytosol. Cytochrome c then forms a complexith apoptosis-activating-factor, activating caspase 9,hich triggers a proteolytic cascade leading to apopto-

ic cell death (16, 17). Moreover, it has been previouslyhown that hypoxia induces p53 and Fas-expression inardiac myocytes, although these studies have nothown a direct cause and effect relationship betweenncreased p53 expression and hypoxia-induced apopto-is (6, 18, 19). In contrast, other authors have demon-trated that hypoxia/acidosis mediated cell death wasndependent of p53, where equivalent levels of apopto-is occurred in cardiac myocytes from wild type and53 knock-out animals (6, 20).The goal of the present study was to evaluate the

eparate effects of prolonged hypoxia and the expres-

0006-291X/01 $35.00Copyright © 2001 by Academic PressAll rights of reproduction in any form reserved.

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Vol. 286, No. 2, 2001 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

poptosis in myocardial cells in vivo and in vitro. Werovide evidence that an increased rate of apoptosisccurs in hearts from rats exposed to chronic hypoxiaompared to normoxia with a significant change in thecl-2/Bax ratio. On the other hand in vitro experimentssing neonatal cardiac myocytes also demonstrated an

ncreased rate of apoptosis with prolonged hypoxialone, however, with an essentially unchanged Bcl-2/ax ratio. Different molecular mechanisms of hypoxia-

nduced apoptosis in vivo or in vitro are discussed.

ATERIALS AND METHODS

Cell culture. Monolayer cultures of neonatal rat cardiac cellsere prepared by modifying the method of Simpson et al. (21).riefly, 1- to 2-day-old Sprague–Dawley rat pups were anesthetizednd sacrificed. The beating heart was extracted, washed in ice-coldBS and minced with a scissors. The tissue was trypsinized at 37°C

n a Hepes-buffered saline solution (Hepes–NaOH 20 mM [pH 7.6],aCl 130 mM, KCl 3 mM, NaH2PO4 1 mM, and glucose 4 mM and.15% trypsin). Dissociated cells were plated on 60 mm wells andllowed to seed for 24 h prior to exposure to hypoxia in the presencef 0.1 mM/L bromodeoxyuridine to prevent proliferation of nonmyo-ytes. For studies involving hypoxic conditions, cells were purgedith 95% N2, 5% CO2, for 20 min and then placed in a 1–2% O2, 5%O2 balanced N2 incubator for 6 to 36 h. Normoxia values were as

ollows: pH 7.31 6 0.1, pO2 119 6 2.3 mmHg, pCO2 44.3 6 4.8mHg; hypoxia values: pH 7.27 6 0.13, pO2 21.6 6 2.9 mmHg, pCO2

3.1 6 1.4 mmHg. FITC staining with a-actinin confirmed that morehan 95% of cells were cardiomyocytes.

In vivo animal protocol. Procedures followed in the care andacrifice of the animals were approved by the Animal Researchommittee of the University of Virginia (Dr. R. A. Johns and Dr. F.ung were previously employed at the University of Virginia). Therotocol for the exposure of rats to hypoxia has been previouslyescribed (22). Briefly, male Sprague–Dawley rats (250–300 g) werelaced in a Plexiglas chamber maintained at a 10% O2 atmospherehypoxic group) or in a chamber open to room air (normoxic group) forweeks, with a 12 h/12 h light/dark cycle. Hypoxia was maintainedsing a Pro:ox Model 350 unit (Reming Bioinstruments, Refield, NY),hich controlled fractional concentration of O2 in inspired gas by

olenoid controlled infusion of N2 (Roberts Oxygen, Rockville, MD)alanced against an inward leak of air through holes in the chamber.he hypoxic rats were exposed to room air for 10–15 min daily whileheir cages were changed. CO2, water vapor, and ammonia wereemoved by pumping the atmosphere of the hypoxia chamberhrough Bara Lyme (Barium hydroxide lime, USP; Chemetron Med-cal Division, Allied Healthcare Products, St. Louis, MO), Drieriteanhydrous calcium sulfate; Fisher Scientific, Atlanta, GA), and ac-ivated carbon (Fisher Scientific, Atlanta, GA).

Protein-isolation and Western blot analysis from cardiac myocytesnd rat heart tissue. Isolated cardiac myocytes were washed withce-cold PBS and lysed (20 mM Tris (pH 7.4), 150 mM NaCl, 1 mMDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1M b-glycerolphosphate, 1 mM Na3VO4, 1 mg/ml leupeptin, 1 mMMSF) for 5 min on ice. Then, cells were scraped off the plates,onicated and lysed on ice for 20 min. Cells were then centrifuged for0 min at 20,000g at 4°C, and protein concentration was determinedn the supernatants using the Bio-Rad reagent (Bio-Rad, Munchen,ermany).For isolation of whole protein extracts from rat hearts, tissuesere homogenized in liquid nitrogen and lysed in RIPA buffer (50M Tris, pH 8, 1% Nonidet P-40, 150 mM NaCL, 0.1% SDS, 0.5%

eoxycholic acid) for 30 min on ice. After centrifugation at 20,000g

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Bio-Rad, Munchen, Germany) using BSA as standard.Proteins (50 mg/lane) were loaded onto 8% SDS–polyacrylamide

els and blotted onto PVDF membranes. After blocking with 5% milkowder at room temperature for 2 h, the antibodies were incubateds followed: anti-Bcl-2 (Biolabs, Schwalbach, Germany) 1:500, anti-ax (Biolabs, Schwalbach, Germany) 1:1000 or anti-PARP (Cell Sig-aling, Frankfurt, Germany) 1:1000 at 4°C over night in TBS (50M Tris/HCl, pH 8; 150 mM NaCl, 2.5 mM KCl), 0.1% Tween 20, 3%

ovine serum albumin (BSA). After incubation with the second an-ibody (anti-rabbit: 1:4000) for 1 h, enhanced chemiluminescenceas performed according to the instructions of the manufacturer

Amersham, Germany). Blots were reprobed with actin (Boehringerannheim, Germany) (1:2000). Blots were scanned and semiquan-

itatively analyzed.

Immunohistochemistry. We used polyclonal rabbit IgG as pri-ary antibodies against Bcl 2 (1:250, Santa Cruz Biotechnology) anddirectly biotinylated rabbit IgG against Bax (1:20; Bax; Oncogene).

mmunohistochemistry was performed using a one step (Bax) as wells a two-step (Bcl 2) avidin–biotin complex technique as previouslyescribed (23–25). Antigens were unmasked by pressure cooking in0 mM citric acid, pH 6, for 5 min. Peroxidase activity was visualizedy 3-amino-9-ethycarbazol (AEC, Sigma) to yield a brown reactionroduct. The nuclei were slightly counterstained with hematoxylin.

Detection of apoptosis. For morphological staining of nuclei, cellsere centrifuged for 10 min at 700g, then fixed in 4% formaldehydend stained with DAPI (0.2 mg/ml in 10 mM Tris/HCl, pH 7, 10 mMDTA, 100 mM NaCl) for 20 min. Three visual fields were counted by

wo independent blinded investigators and the percentage of apopto-ic cells per total number of cells was determined.

FACS analysis. Cells were grown as described above and starvedn serumfree medium over night prior to exposure to normoxia orypoxia. The adherent cells were detached with trypsin, washed inBS and were incubated with 2.5 ml Annexin-PE for 15 min at RTccording to the manufacturer (Pharmingen, Annexin V-Pe Apopto-is Kit) and analysed by FACS using a FACS SCAN flow cytometerBD) and Cell Quest software (BD).

DNA fragmentation. DNA was extracted by phenol-chloroformxtraction as described previously (26). The DNA samples werereated with 5 U of Klenow polymerase using 0.5 mCi of [a-32P]dCTPn the presence of 10 mM Tris–HCl (pH 7.5) and 5 mM MgCl2. Theeaction was terminated after addition of 10 mM EDTA and thenincorporated nucleotides were removed by Sephadex G-50 col-mns. The radioactive DNA was separated by gel electrophoresis1.8% agarose in 13 TBE (90 mM Tris–borate, 2 mM EDTA, 2.5 h at0 V)], transferred to nitrocellulose Hybond N1 (Amersham) andxposed to X-ray film.

Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-iotin nick end labeling (TUNEL). The detection of DNA strandreaks in situ by TUNEL was performed according to the instructionsf the manufacturer (Boehringer Mannheim). In brief frozen sectionsere fixed in formalin for 30 min, pretreated with 3% citric acid androteinase K (20 mg/ml) and subsequently permeabilized on ice. ThenUNEL reaction mixture (terminal deoxynucleotidyl transferase (TdT)nd fluorescein-dUTP) was added and sections were incubated for 60in at 37°C. Labeled nuclei were detected by peroxidase immunostain-

ng and sections counterstained with hematoxylin.

Statistical analysis. Data are expressed as means 6 SEM from ateast 3 independent experiments. Statistical analysis was performedith ANOVA for multiple variable and t test for comparison of tworoups with SPSS software.

ESULTS

Prolonged hypoxia induces apoptosis in vivo. It istill not clear if prolonged or chronic hypoxia causes

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poptosis in myocardial cells in vivo. Thereforeprague–Dawley rats (250–300 g) were placed in alexiglas chamber maintained at a 10% O2 atmosphere

hypoxic group) or in a chamber open to room air (nor-oxic group) for 3 weeks. Hearts were harvested and

issues were processed for DNA fragments, a hallmarkf apoptotic cell death. Typical DNA ladder demon-trated increased DNA fragmentation in heart tissuerom rats exposed to hypoxia compared to rats exposedo normoxia (Fig. 1A). TUNEL staining confirmed thatn hypoxic hearts there was a significant increase inhe rate of apoptosis compared to normoxic hearts6.3 6 1% vs 1.2 6 0.7%, respectively) (Fig. 1B). Theistribution of apoptotic cells was not localized to apecific region. It was equally distributed between theight and left ventricle.

Bcl-2 and Bax protein expression under hypoxia inivo. To elucidate the effect of hypoxia on the apopto-is regulatory proteins Bcl-2 and Bax, immunohisto-hemistry was performed in sections from normoxic

FIG. 1. Prolonged hypoxia induces apoptosis in vivo. Hearts fromprague–Dawley rats exposed to prolonged hypoxia (n 5 4) orormoxia (n 5 4) for 3 weeks were harvested and tissues wererocessed for DNA-fragmentation assays. (A) TUNEL staining fromormoxic and hypoxic hearts was performed as described underaterials and Methods. Five visual fields were counted to determine

he percentage of apoptotic cells per total number of cells (loweranel). Data are mean 6 SEM (n 5 5), *P , 0.05 (B).

421

xpressed mainly in cardiac myocytes (Fig. 2A). After 3eeks of hypoxia, Bcl-2 immunostaining was reduceds representatively illustrated in Fig. 2A. In contrast,he expression of the proapoptotic protein Bax wasncreased upon hypoxia (Fig. 2B). Bax immunostainingas also mainly localized to cardiac myocytes (Fig. 2B).Furthermore, we quantified Bcl-2 and Bax protein

xpression in protein homogenates from normoxic andypoxic hearts. Western blot analysis revealed thatcl-2 protein levels were significantly down-regulatedfter hypoxia (Fig. 3A), whereas Bax protein expres-ion was significantly increased (Fig. 3B). Overall, thecl-2/Bax ratio was changed from 2.44 to 0.52 suggest-

ng a potential regulatory role of Bcl-2-like proteins inypoxia-induced cardiac myocyte apoptosis in vivo.

Prolonged hypoxia induces apoptosis in vitro. Toest the direct effect of hypoxia on cardiac myocytes,rimary neonatal cardiac myocytes were exposed toypoxia and normoxia for 18, 24, or 36 h. Under hyp-xic conditions the pO2 was reduced to 21.6 6 2.9mHg and the pCO2 was 33.1 6 1.4 mmHg with a pH

ot significantly changed (pH 7.31 6 0.1 vs 7.27 6

FIG. 2. Bcl-2 and Bax protein is expressed in cardiac myocytesnd regulated under hypoxic conditions in vivo. To determine theocalization of Bcl-2 (A) and Bax (B) protein is expressed in normoxicnd hypoxic hearts, immunohistochemistry was performed on tissueections from normoxic and hypoxic rat hearts. Immunohistochem-stry was performed using a one step (Bax) as well as a two-step (Bcl) avidin–biotin complex technique as described under Materialsnd Methods. Bcl-2 protein expression was present in cardiac myo-ytes under normoxia and significantly reduced under hypoxia. Ex-ression of Bax protein levels in cardiac myocytes was low underormoxia, whereas it was significantly increased under hypoxia.issue sections shown are representative of sections from 4 differentnimals.

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.13). DAPI staining of nuclei from cardiac myocytesevealed an approximately 3-fold increase in the rate ofpoptosis in cells exposed to hypoxia compared to cellsxposed to normoxia for 18 and 36 h (Fig. 4A). Theseesults were confirmed by FACS analysis of neonatalardiomyocytes using Annexin-staining, showing alsosignificant increase in the rate of apoptotic nuclei inypoxic cells compared to normoxic cells (normoxia—.8 6 0.2% vs hypoxia—3.9 6 1.2% at 18 h and nor-oxia—2.3 6 1% vs hypoxia—5.0 6 1.1% at 24 h) (Fig.

B). Moreover, induction of apoptosis was also exam-ned by proteolytic cleavage of poly(ADP-ribose)-poly-

erase (PARP). Immunoblot analysis using a monoclo-al antibody demonstrated a significant reduction ofhe full length PARP protein in cells exposed to hyp-xia for 24 h compared to cells exposed to normoxia100% vs 57.6%) (Fig. 4C).

Bcl-2 and Bax protein expression in cardiac myocytesn vitro under hypoxia. To elucidate the involvementf Bcl-2-like proteins in hypoxia-induced apoptosis inardiac myocytes in vitro, Western blot analysis waserformed after 6, 18, and 36 h of hypoxia. Bcl-2 andax protein expression was not significantly affected

Figs. 5A and 5B). The Bcl-2/Bax protein ratio essen-

FIG. 3. Bcl-2 and Bax protein expression in cardiac myocytes inxpression Western blot analysis was performed using protein extraith anti-Bcl-2, anti-Bax, or anti-p53. Blots are representative of 3

n the right panel (mean 6 SEM, *P , 0.05).

422

ially remained unchanged (2.31 vs 2.41). Moreover53 expression also was unchanged (data not shown).

ISCUSSION

To date controversy exists if hypoxia alone is suffi-ient to induce apoptosis in cardiac myocytes or if ad-itional factors are required, such as acidosis or accu-ulation of cellular or extracellular metabolic waste

roducts. Moreover, the precise initiating stimuli andhe signaling pathways leading to hypoxia-inducedpoptosis are not well established. We have shown thatn increased rate of apoptosis occurs in hearts fromats exposed to chronic hypoxia compared to normoxiaith a significant change in the Bcl-2/Bax ratio. In

solated cardiac myocytes an increased rate of apopto-is with prolonged hypoxia was also observed, however,ith an essentially unchanged Bcl-2/Bax ratio.Two important points can be made from our study

esults: (1) Hypoxia alone can induce apoptosis of car-iac myocytes as demonstrated in our isolated in vitroystem. (2) Modulation of Bcl-2 or Bax-expression inardiac myocytes may depend on the different experi-ental model used and the type of hypoxic exposure.

o under hypoxia. For determination of Bcl-2 (A) and Bax (B) proteinom rat hearts exposed to normoxia and hypoxia. Blots were probed

ependent experiments. Semiquantitative analysis (n 5 4) is shown

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It is still not clear if prolonged hypoxia alone cannitiate apoptosis in cardiac myocytes or if other stim-li, such as acidosis, are required. Recently, Webstert al. have proposed that acidosis, reoxygenation, oreperfusion, but not hypoxia alone are strong stimula-ors for programmed cell death (6). In contrast, ourtudies demonstrate that hypoxia is sufficient to in-uce apoptosis in vitro. The discrepancy between thesetudies may be explained by different experimentalonditions. Webster and co-workers used insulin-sup-lemented medium. Insulin is well established to pre-ent apoptosis by activation of Akt (27). Moreover, ourndings that hypoxia is sufficient to induce cardiacyocyte apoptosis in vitro are in accordance with other

tudies (28). Our in vivo data also demonstrate thatxposure to prolonged hypoxia can cause myocardialell loss. Clearly one could speculate that in our animal

FIG. 4. Prolonged hypoxia induces apoptosis in vitro. Primary nawley rat pups and exposed to hypoxia and normoxia for 18 and 3ypoxia for 18 and 36 h were fixed in 4% formaldehyde and stained w

nvestigators and the percentage of apoptotic cells per total numbernd **P , 0.05. FACS analysis was performed using Annexin-5 stauantified and data are expressed as mean 6 SEM (n 5 4) #P , 0.ormoxia and hypoxia for 24 h and Western blot analysis was perfo

ndependent experiments. Semiquantitative analysis is shown in th

423

odel the development of pulmonary hypertensionith right ventricular hypertrophy associated with ex-osure to a 3-week period of hypoxia may be responsi-le for apoptotic cell death in the pressure overloadedight ventricle. However, apoptosis was equally ob-erved in both, right and left ventricles. Other groupsave reported that hypoxia-induced apoptotic celleath in vivo may require reoxygenation or reperfusion2, 6). We cannot completely exclude the possibilityhat a daily 10-min period of reoxygenation of the ratso room air for feeding and cage-change may haveontributed to apoptosis of cardiac myocytes.Different animal models and the type of hypoxic

xposure may involve other signaling mechanism me-iating apoptotic cell death. The ratio between thentiapoptotic Bcl-2 and the proapoptotic protein Baxeems to be critical for cell survival in different cell

atal cardiac myocytes were isolated from 1- to 2-day-old Sprague–. For morphological staining of nuclei, cells exposed to normoxia orDAPI. Three visual fields were counted by two independent blindedells was determined (A). Data are mean 6 SEM (n 5 3) *P , 0.05ng as described under Materials and Methods. Apoptotic cells were(B). For the PARP cleavage assay cardiac myocytes were exposed tod using monoclonal PARP antibodies. Blots are representative of 3ght panel (mean 6 SEM, ##P , 0.05) (C).

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ypes possibly via regulation of mitochondrial perme-bility and caspase-3 activity (29–31). In models ofcute hypoxia, increased Bcl-2 expression was ob-erved in the salvaged regions surrounding infarctedissue, whereas Bax was predominantly expressed inater infarct-stages (15). Apoptosis has also been linkedo the translocation of the proapoptotic protein Bax tohe mitochondria, with subsequent release of cyto-hrome c and caspase activation (17). However, in theetting of chronic myocardial hypoxia, such as in hiber-ating or stunned myocardium, the role of the Bcl-2amily proteins is not clear. In our studies the Bcl-2nd Bax protein expression in an isolated in vitro hyp-xia model was essentially unchanged whereas it wasncreased in a chronic hypoxia animal model. In vitro,

echanisms independent from the Bcl-2/Bax ratioeem to be responsible for hypoxia-induced apoptosis.hese may include an up-regulation of the inducibleO-Synthase, which is known to be induced by hypoxiand promotes apoptosis (32), although inhibition ofO-synthesis by N-mono-methyl-L-arginine did not af-

ect hypoxia-induced apoptosis (data not shown). Inivo, hypoxia-induced apoptosis is associated with ahange in the Bcl-2/bax ratio, which is in line with thending, that there was a trend of p53 upregulation inhe vivo model (data not shown). Modulation of thecl-2/Bax expression in the in vivo model may there-

ore depend on the influence of other co-factors, such asnflammatory mediators, NO or the influence of non-

yocardial cells, whereas in the isolated in vitro modelther effector pathways may be responsible for theirect proapoptotic effects of hypoxia on isolated car-iac myocytes.

FIG. 5. Bcl-2 and Bax protein expression in cardiac myocytes initro under hypoxia. Neonatal cardiac myocytes were exposed toormoxia (N) or hypoxia (H) for 6, 18, or 36 h and Bcl-2 (A) and BaxB) protein expression was determined by Western blot analysis.lots were probed with anti-Bcl-2 and anti-Bax and subsequently

eprobed with anti-actin as a loading control. Blots are representa-ive of 3 different experiments. Bcl-2 or Bax protein expression wasot statistically different between normoxia and hypoxia (P 5 ns).

424

xia is a sufficient stimulator to induce apoptosis inardiac myocytes in vivo and in vitro. However, theignaling pathways mediating this effect may be dis-inct. The mechanisms of hypoxia-induced cell deathay vary depending on the experimental conditions

sed and different factors involved, such as length ofypoxia, reoxygenation, preconditioning of cells, or the

nfluence of cofactors. Further studies are required tolucidate the complex mechanisms leading to hypoxia-nduced apoptosis of cardiac myocytes.

CKNOWLEDGMENTS

We thank Barbara Lafferton for the excellent technical assistance.his work was supported by grant support from the Deutsche For-chungsgemeinschaft (Ju241/2-1) and from the NIH (ROI HL 39706nd GM 49111-RAJ).

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chronic hypoxia induces apoptosis in cardiac myocytes: a possible role for bcl-2-like proteins

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