IntroductionCardiac hypertrophy occurs in response to the bio-mechanical stress of pressure overload, which isoften caused by arterial hypertension. This adaptivegrowth is initially of a compensatory nature, but sus-tained hypertrophy later undergoes a transition toheart failure, a leading cause of mortality in devel-oped countries (1, 2).

Apoptosis, leading to myocyte loss, is thought to con-tribute to experimental and clinical heart failure (3–5).Activation of the Fas/APO-1 receptor by Fas ligand rep-resents a classical death signal causing apoptosis viaactivation of the caspase cascade in many cell types (6).

However, recent studies indicated that Fas signalingcan have apoptosis-unrelated biological effects such aspromotion of T cell proliferation (7). In cultured car-diomyocytes, Fas stimulation activates the prohyper-trophic transcription factor AP-1 rather than inducingapoptosis (8). The mechanical stress induced bystretching enhances cardiomyocyte Fas expression ofisolated papillary muscles (9). Upregulation of themyocardial Fas receptor was further described in bothexperimental volume-overload cardiac hypertrophy (8)

and failing human hearts (10). Finally, in transgenicmice, myocardial overexpression of Fas ligand wasshown to be sufficient to induce a hypertrophic phe-notype (11). Collectively, these data suggest that Fassignaling appears to be involved in various forms ofcardiac hypertrophy. However, the causative role of Fasreceptor activation following pressure overload as wellas the mechanism(s) of Fas-mediated hypertrophy incardiomyocytes is unknown.

Therefore, we investigated the signaling pathways ofFas receptor activation in cultured cardiomyocytes andthe cardiac phenotype of Fas signaling–defective lpr(lymphoproliferative disease) mice following pressureoverload induction. Our results demonstrate that Fasactivation–induced hypertrophy in cultured cardiomy-ocytes is dependent on phosphorylation and inactiva-tion of glycogen synthase kinase 3β (GSK3β), a nega-tive regulator of cardiomyocyte hypertrophy. Fassignaling is required for compensation of pressureoverload in vivo, and its absence leads to failure toinhibit GSK3β, rapid left ventricular dilatation,increased mortality, and absence of compensatoryhypertrophy following this biomechanical stress.

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Fas receptor signaling inhibits glycogen synthase kinase 3βand induces cardiac hypertrophy following pressure overload

Cornel Badorff,1 Hartmut Ruetten,2 Sven Mueller,1 Meike Stahmer,1 Doris Gehring,2

Frank Jung,1 Christian Ihling,3 Andreas M. Zeiher,1 and Stefanie Dimmeler1

1Molecular Cardiology, Department of Internal Medicine IV, University of Frankfurt, Frankfurt, Germany2Aventis Pharma, Frankfurt, Germany3Department of Pathology, University of Freiburg, Freiburg, Germany

Address for correspondence: Andreas M. Zeiher, Department of Internal Medicine IV, University of Frankfurt, Theodor Stern-Kai 7, 60590 Frankfurt, Germany. Phone: 49-69-6301-5789; Fax: 49-69-6301-7113; E-mail: Zeiher@em.uni-frankfurt.de.

Cornel Badorff and Hartmut Ruetten contributed equally to this work.

Received for publication July 17, 2001, and accepted in revised form December 17, 2001.

Congestive heart failure is a leading cause of mortality in developed countries. Myocardial hyper-trophy resulting from hypertension often precedes heart failure. Understanding the signaling under-lying cardiac hypertrophy and failure is of major interest. Here, we identified Fas receptor activation,a classical death signal causing apoptosis via activation of the caspase cascade in many cell types, asa novel pathway mediating cardiomyocyte hypertrophy in vitro and in vivo. Fas activation by Fas ligand induced a hypertrophic response in cultured cardiomyocytes, which was dependent on theinactivation of glycogen synthase kinase 3β (GSK3β) by phosphorylation. In vivo, lpr (lymphoprolif-erative disease) mice lacking a functional Fas receptor demonstrated rapid-onset left ventriculardilatation and failure, absence of compensatory hypertrophy, and significantly increased mortalityin response to pressure overload induction that was accompanied by a failure to inhibit GSK3β activ-ity. In contrast, Fas ligand was dispensable for the development of pressure overload hypertrophy invivo. In vitro, neonatal cardiomyocytes from lpr mice showed a completely abrogated or significant-ly blunted hypertrophic response after stimulation with Fas ligand or angiotensin II, respectively.These findings indicate that Fas receptor signaling inhibits GSK3β activity in cardiomyocytes and isrequired for compensation of pressure overload in vivo.

J. Clin. Invest. 109:373–381 (2002). DOI:10.1172/JCI200213779.

MethodsCell culture. Rat and mouse neonatal ventricular car-diomyocytes (12) and Jurkat T lymphocytes (13) wereisolated and cultured as described. Following serumstarvation for 18 hours, cells were stimulated withrecombinant Fas ligand (1–100 ng/ml; Upstate Biotech-nology Inc., Lake Placid, New York, USA), endothelin-1(100 nM; Alexis Corp., San Diego, California, USA),angiotensin II (1 µM; Calbiochem-Novabiochem Corp.,San Diego, California, USA), IGF-1 (40 ng/ml; Cell Con-cepts, Umkirch, Germany), or hydrogen peroxide (100µM). In some experiments, cells were preincubated for60 minutes with 20 µM Ly294002 or 200 nM wort-mannin (both from Calbiochem-Novabiochem Corp.).

Apoptosis assays. In cultured cells, apoptosis was detect-ed by DAPI and annexin V staining (13). In mouse hearts,DNA strand breaks were analyzed using TUNEL (14).

Protein synthesis. One microcurie per milliliter [3H]leucine (Amersham Pharmacia Biotech, Piscataway,New Jersey, USA) was added 1 hour before stimulationwith Fas ligand. Following lysis, proteins were precipi-tated using 5% trichloroacetic acid and [3H] leucine wasmeasured by scintillation counting.

Immunoblotting. Cells or tissue were homogenized inlysis buffer, and 25 µg was separated by SDS-PAGE,

transferred to PVDF membranes, and immunoblot-ted as described (15). Primary antibodies wereanti–phospho-Akt, anti-Akt, anti–phospho-GSK3β(all from Cell Signaling Technology, Beverly, Massa-chusetts, USA), anti-GSK3β (BD Transduction Labo-ratories, Los Angeles, California, USA), anti–phos-pho-JNK1, or anti-JNK1 (both from Santa CruzBiotechnology Inc., Santa Cruz, California, USA).Bound antibodies were detected by enhanced chemi-luminescence (Amersham Pharmacia Biotech).

Immunofluorescence staining and cell size measurement.Cardiomyocytes were stained with a rabbit anti-MLC2vantibody (a kind gift from Ju Chen, University of Cali-fornia–San Diego, San Diego, California, USA) followedby rhodamine-conjugated anti-rabbit IgG (Dianova,Hamburg, Germany). Nuclei were visualized by DAPI.Cells were imaged using an Axiovert 100M microscopeequipped with an AxioCam (Carl Zeiss Jena GmbH,Jena, Germany). Maximum cell length and cell surfacearea were measured using Axiovision software (CarlZeiss Jena GmbH).

Atrial natriuretic factor Northern blotting, RT-PCR, andRNase protection assays. Total RNA was isolated (QIAGEN,Hilden, Germany) and Northern blot analysis was per-formed using a radioactively labeled, PCR-amplified full-

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Figure 1Fas ligand induces cardiomyocyte hypertro-phy but not apoptosis. (a) Neonatal rat car-diomyocytes or Jurkat T lymphocytes weretreated with Fas ligand or H2O2, and apop-tosis was assessed by DAPI staining. Dataare mean ± SEM from four independentexperiments. *P < 0.05 versus control. (b)Annexin V staining of rat cardiomyocytes fol-lowing treatment with Fas ligand or H2O2.(c) [3H] leucine incorporation of rat car-diomyocytes following treatment with Fasligand, angiotensin II, or endothelin-1. Dataare mean ± SEM from four independentexperiments. *P < 0.05 versus control. (d)Myosin light chain 2v immunofluorescenceof rat cardiomyocytes following treatmentwith Fas ligand. (e) Increased ANF mRNAdetected by Northern blotting followingtreatment with Fas ligand for the times indi-cated. Representative images of three inde-pendent experiments are shown in c–e.

length rat atrial natriuretic factor (ANF) cDNA probe.ANF mRNA was quantitated by real-time RT-PCR (LightCycler; Roche Molecular Biochemicals, Indianapolis,Indiana, USA). RNase protection assays were fromPharMingen (San Diego, California, USA).

Plasmid cloning, mutagenesis, transfection, and luciferaseassay. The full-length human GSK3β cDNA was PCR-amplified and cloned into pcDNA3.1-mycHis vector(Invitrogen Inc., Carlsbad, California, USA). Site-direct-ed mutagenesis was performed with the Quick ChangeMutagenesis Kit (Stratagene, La Jolla, California, USA).The entire 3.7-kb rat ANF promoter was PCR-amplifiedand cloned into pGL3-enhancer vector (PromegaCorp., Madison, Wisconsin, USA). Plasmids were trans-fected into cardiomyocytes using Effectene (QIAGEN).Six hours after transfection, cells were serum-starvedfor 18 hours prior to stimulation with Fas ligand (50ng/ml), endothelin-1 (10 nM), or LiCl (10 mM) for 24hours. Luciferase activity was measured using a com-mercial assay (Promega Corp.). Each data point isderived from an independent experiment and repre-sents the mean of triplicate wells.

GSK3β and c-jun N-terminal kinase 1 immunocomplexkinase assay. Total GSK3β or c-jun N-terminal kinase 1(JNK1), respectively, was immunoprecipitated frommyocardial homogenates using monoclonal antibodies,and the in vitro kinase assay was performed as described(16) with protein phosphatase inhibitor-2 (Calbiochem-Novabiochem Corp.) as GSK3β substrate or GST-c-jun(Santa Cruz Biotechnology Inc.) as JNK1 substrate.

Animal experiments. C57BL/6+/+, Fas-deficient C57BL/6lpr/lpr, and Fas ligand–deficient C57BL/6gld/gld mice wereobtained from The Jackson Laboratories (Bar Harbor,Maine, USA). Animal experiments were performed inaccordance with the German animal protection law.

Pressure overload hypertrophy was induced byabdominal aortic constriction between the diaphragmand the renal arteries as described (17). Echocardio-grams were performed in anesthetized mice, and, for

the assessment of pressure-volume relationships, theleft ventricle was catheterized retrogradely using animpedance-pressure catheter (17).

Statistical analysis. Unpaired, two-tailed t test was usedfor the comparison of two groups. For multiple groups,one-way ANOVA analysis (least significant differencetest) was used. Mortality was analyzed by χ2 test.

ResultsFas receptor activation induces hypertrophy of cultured car-diomyocytes. As previously reported (6), recombinant Fasligand caused apoptosis in Jurkat T lymphocytes after6 hours with significantly increased nuclear fragmen-tation (Figure 1a). In contrast, Fas ligand did not resultin increased apoptosis in neonatal rat ventricular car-diomyocytes, even after 18 hours, as measured bynuclear morphology (Figure 1a) and annexin V stain-ing (Figure 1b). Importantly, the functional integrity ofthe apoptosis execution program in neonatal car-diomyocytes was intact as evidenced by H2O2-induced

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Figure 2Fas ligand induces Akt and GSK3β phosphorylation in cardiomy-ocytes. (a) Rat cardiomyocytes were stimulated with IGF-1, endothe-lin-1, or Fas ligand for the times indicated. Lysates were immuno-probed for phospho-Akt or phospho-GSK3β, followed by reprobesfor total Akt or total GSK3β, respectively. A representative blot fromthree independent experiments is shown. (b) Quantitative densito-metric analyses of the phospho-Akt and phospho-GSK3β bands nor-malized for total Akt or GSK3. Data are mean ± SEM from three inde-pendent experiments for each data point. *P < 0.05 versus control.(c) Cardiomyocytes were pretreated with Ly294002 or wortmanninfollowed by stimulation with Fas ligand or IGF-1, immunoblottingwith phospho-GSK3β antibody, and a reprobe for total GSK3β. (d)Cardiomyocytes were cotransfected with an ANF promoter-drivenluciferase reporter together with either empty vector (left) or a non-phosphorylatable GSK3β construct (GSK3βS9A) (right) followed bystimulation with Fas ligand. Data are mean ± SEM from three to fourindependent experiments. *P < 0.05 for stimulation versus control;#P < 0.05 for GSK3βS9A versus empty vector. FasL, Fas ligand.

apoptosis (Figure 1, a and b). RNase protection assaysconfirmed that the Fas receptor and downstream apop-totic signaling molecules such as Fas-associated deathdomain and caspase-8 were expressed in the neonatalcardiomyocytes used (data not shown).

Next, we investigated total protein synthesis, sar-comere organization, and gene induction of ANF ashallmarks of the hypertrophic response. Fas liganddose-dependently (1–100 ng/ml) increased protein syn-thesis in neonatal cardiomyocytes after 24 hours. Theincrease in protein synthesis achieved by Fas ligand wascomparable to the effects of well-established hyper-trophic stimuli such as angiotensin II or endothelin-1(Figure 1c). Fas ligand treatment profoundly increasedcell size and sarcomeric organization in neonatal car-diomyocytes after 48 hours (Figure 1d). Furthermore,Fas ligand caused a time-dependent increase of ANFmRNA in cultured cardiomyocytes (Figure 1d). Thus,Fas signaling induces a hypertrophic response ratherthan apoptosis in neonatal cardiomyocytes.

Fas ligand–induced hypertrophy in cardiomyocytes is GSK3β-dependent. Recent studies have shown that inactivationof GSK3β via phosphorylation, e.g., by the upstreamkinase Akt, is both necessary and sufficient for car-diomyocyte hypertrophy in vitro (18, 19). As a result ofGSK3β inactivation, the downstream effector nuclearfactor of activated T cells (NF-AT) translocates to thenucleus to initiate hypertrophic gene expression (18).

In neonatal cardiomyocytes, Fas activation by Fas lig-and (10 ng/ml) transiently increased Akt and GSK3β

phosphorylation, as did endothelin-1 (100 nM) andIGF-1 (40 ng/ml), two established inducers of GSK3βphosphorylation (18, 19) (Figure 2a). Quantitative den-sitometric analysis revealed that Akt and GSK3β phos-phorylation peaked 40 minutes and 60 minutes afteraddition of Fas ligand, respectively, consistent with theconcept that Akt is upstream of GSK3β (Figure 2b).Downstream of GSK3β, we observed a prominentnuclear translocation of NF-AT at 60 minutes after Fasreceptor activation (data not shown). GSK3β phos-phorylation induced by Fas ligand and IGF-1 was sig-nificantly inhibited by pretreatment (60 minutes) withLy294002 or wortmannin, confirming a PI 3-kinase–dependent (PI3K-dependent) pathway (Figure 2c).

To assess the causative role of GSK3β phosphorylationin Fas ligand–induced hypertrophy, neonatal cardiomy-ocytes were cotransfected with an ANF promoter-drivenluciferase reporter and a nonphosphorylatable, domi-nant-active construct of GSK3β (GSK3βS9A). In contrastto cotransfection with empty vector, the GSK3βS9A con-struct significantly suppressed Fas ligand–induced ANFpromoter activation. Fas ligand– and endothelin-1–induced ANF transcription was equally sensitive to thepresence of GSK3βS9A. In contrast, ANF induction byLiCl was unaffected by GSK3βS9A because LiCl inhibitsGSK3β independent of its phosphorylation status (Fig-ure 2d). Thus, Fas ligand–induced cardiomyocyte hyper-trophy is GSK3β-dependent.

Fas-deficient lpr mice develop congestive heart failure andenhanced mortality in response to pressure overload. Havingdemonstrated that Fas activation induces cardiomy-ocyte hypertrophy in vitro, we investigated the necessi-ty of this pathway in Fas-deficient lpr mice after induc-tion of pressure overload by abdominal aortic banding(17, 20). As control, wild-type strain (C57BL/6) sex- andage-matched mice were used.

Pressure-volume loops were recorded 2 days aftersham-operation or abdominal aortic banding. Fol-lowing sham-operation, wild-type and lpr mice bothhad a normal cardiac phenotype with similar pres-sure-volume loops (Figure 3a) and hemodynamics(Table 1), ruling out a major physiological defect in lprhearts at base line.

The trans-stenotic pressure gradients were compara-ble in wild-type (39.1 ± 5.3 mmHg) and lpr mice (37.6 ± 6.4 mmHg), resulting in a significantly

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Figure 3Heart failure of lpr mice in response to pressure overload. (a and b)Representative, superimposed, steady-state pressure-volume loopsof a wild-type (black) or lpr mouse (gray) 2 days after sham-opera-tion (a) or abdominal aortic banding (b). (c and d) Representativepressure-volume loops during transient reduction of cardiac preload2 days after sham-operation (c) or abdominal aortic banding (d). (e and f) Echocardiographic measurements of surviving wild-type(WT) and lpr mice 14 days after induction of pressure overload. Themean ± SEM from six mice per group is shown. *P < 0.05 for aorticbanding versus control. LVEDD, left ventricular end-diastolic diam-eter; LVESD, left ventricular end-systolic diameter.

increased end-systolic pressure (ESP) and arterial elas-tance (Ea) in both groups (Table 1). Following 2 days ofpressure overload, both wild-type and lpr mice main-tained their cardiac output (CO) with enhanced con-tractility during the isovolumetric phase (dp/dtmax) aswell as the ejection phase, end-systolic pressure-volumerelationship (Ees) and maximumchamber elasticity (Emax), of the sys-tole (Figure 3, c and d, and Table 1).However, already 2 days after induc-tion of pressure overload, lpr micedisplayed significantly enlarged leftventricular end-diastolic volumes(Table 1), resulting in pressure-vol-ume loops extended to the right(Figure 3, a and b) and a significant-ly increased preload recruitablestroke work (PRSW) (Table 1) asearly indicators of impaired left ven-tricular hemodynamics.

Most importantly, 7 days after aor-tic banding, lethality rates were 55%(51/92 mice) in lpr mice comparedwith 17% (14/81 mice) in wild-typemice (P < 0.001).

Fourteen days after induction ofpressure overload, surviving lpr miceechocardiographically exhibited asignificant increase of their left ven-tricular end-systolic and end-dias-tolic diameter (Figure 3e) as well as asignificant decrease in fractionalshortening (Figure 3f), demonstrat-ing overt left ventricular failure.

Thus, Fas signaling is required for compensation ofpressure-overload; its absence leads to rapid-onsetleft ventricular dilatation and failure as well asincreased mortality.

Failure to develop pressure overload–compensating hyper-trophy in mice lacking a functional Fas receptor. In order tomorphologically assess the cardiac hypertrophicresponse to the induction of pressure overload, wedetermined left ventricular wall diameters by echocar-diography. Following sham-operation, wild-type andlpr mice had identical left ventricular wall thickness-es. Importantly, only wild-type but not lpr miceresponded to the induction of pressure overload with

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Table 1Left ventricular mechanics as measured by pressure-volume loops of wild-type and lpr mice2 days after induction of pressure overload by aortic constriction

Wild-type lprParameter Sham Aortic banding Sham Aortic banding

(n = 7) (n = 7) (n = 7) (n = 7)Hemodynamics

BW (g) 26.5 ± 0.7 26.0 ± 0.9 25.6 ± 0.8 26.11 ± 1.2HR (min–1) 505 ± 23 562 ± 24 492 ± 33 532 ± 20ESP (mmHg) 85.3 ± 5 126 ± 7A 81.5 ± 8 116.8 ± 7A

EDP (mmHg) 3.4 ± 0.6 4.6 ± 0.9 3.7 ± 0.7 5.6 ± 1ESV (µl) 19.2 ± 5 23.5 ± 4 16.1 ± 3 22.2 ± 2A

EDV (µl) 35.1 ± 5 36.6 ± 5 32.8 ± 4 41.8 ± 4A

CO (µl/min) 9862 ± 822 10544 ± 1022 10160 ± 843 14009 ± 1165

Systolic function

dp/dtmax (mmHg/s) 9412 ± 664 14222 ± 1230A 8550 ± 946 12030 ± 1520A

Ea (mmHg/µl) 3.1 ± 0.4 7.1 ± 0.6A 3 ± 0.3 4.6 ± 0.4A,B

Ees (mmHg/µl) 3.0 ± 0.4 4.4 ± 0.3A 3.4 ± 0.6 5.1 ± 1.3A

PRSW 65 ± 6 62 ± 5 55 ± 7 75 ± 8A

Emax (mmHg/µl) 6.37 ± 0.4 13.6 ± 3.8A 4.3 ± 0.8 12.5 ± 2.6A

Diastolic function

dp/dtmin (mmHg/s) 9128 ± 592 11457 ± 1230 8102 ± 798 10020 ± 724τ (Glantz) (ms) 6.1 ± 0.2 10.2 ± 1.8A 7.1 ± 0.2 10.1 ± 1.5A

Shown are means ± SEM. BW, body weight; HR, heart rate; ESP, end-systolic pressure; EDP, end-dias-tolic pressure; ESV, end-systolic volume; EDV, end-diastolic volume; CO, cardiac output; dp/dtmax,maximal rate of pressure development; dp/dtmin, maximal rate of pressure decay; Ea, arterial elas-tance; Ees, end-systolic pressure-volume relationship; PRSW, preload recruitable stroke work; Emax,maximum chamber elasticity; τ, monoexponential time constant of relaxation. AP < 0.05 aortic band-ing versus sham-operation; BP < 0.05 lpr versus wild-type after aortic banding.

Figure 4Cardiac hypertrophy in wild-type but not lpr mice after pressure over-load. (a) Echocardiographic septal (white bars) and posterior wall(black bars) thicknesses of wild-type and lpr mice 14 days after induc-tion of pressure overload. The mean ± SEM from six mice per groupis shown. *P < 0.05 for aortic banding versus control. (b) Heart-to-body-weight ratios of wild-type (white bars) versus lpr mice (blackbars) 14 days after induction of pressure overload. The mean ± SEMfrom six mice per group is shown. *P < 0.05 for aortic banding ver-sus control. (c and d) ANF mRNA expression 2 days after inductionof pressure overload in wild-type and lpr mice compared with sham-operated animals. (c) A representative ANF Northern blot (upperpanel) is shown. 18S RNA (lower panel) demonstrates equal load-ing. (d) ANF mRNA group data derived from real-time RT-PCR ofwild-type (white bars) and lpr mice (black bars) at the times indicat-ed following abdominal aortic banding. The mean ± SEM from threemice per group is shown. *P < 0.05 for aortic banding versus control.

significant increases of the septal as well as posteriorwall thickness (Figure 4a). This hypertrophic responsein the wild-type mice was confirmed by a significant-ly increased heart-to-body-weight ratio (Figure 4b). Incontrast, wall thicknesses and the heart-to-body-weight ratio remained unchanged in lpr mice, indi-cating a failure to develop pressure overload–inducedhypertrophy (Figure 4, a and b).

As a molecular marker of cardiomyocyte hypertrophy,we analyzed ANF mRNA induction at various timesafter aortic banding. Wild-type but not lpr miceresponded to pressure overload with a robust induc-

tion of ANF transcription (Figure 4c). Real-time RT-PCR revealed a significant fivefold increase of theANF mRNA in wild-type but not lpr mice 2 days afterinduction of pressure overload (Figure 4d). These mor-phological and molecular data demonstrate that Fas-deficient lpr mice fail to develop adaptive cardiac hyper-trophy in response to pressure overload.

Inhibition of GSK3β following pressure overload is abro-gated in lpr mice. Next, we sought to identify the sig-naling pathway(s) defective in lpr mice in response topressure overload.

In order to determine whether altered phosphoryla-tion and inactivation of GSK3β is involved in the fail-ure of lpr mice to develop compensatory left ventricu-lar hypertrophy in response to the induction ofpressure overload, GSK3β immunocomplex kinaseassays of myocardial homogenates were performed atvarious time points following aortic banding. GSK3βactivity was transiently but significantly reduced inwild-type mice 4 and 24 hours after induction of pres-sure overload. In contrast, lpr mice exhibited an entire-ly different regulation of GSK3β activity, with a signif-icant increase in GSK3β activity 24 hours after aorticconstriction (Figure 5, a and b). Moreover, base-lineGSK3β activity in the absence of pressure overload waslower in lpr mice (66.3% ± 6.5% of wild-type mice, P < 0.05). Thus, in mice lacking a functional Fas recep-tor, pressure overload–induced inhibition of GSK3βactivity is completely abrogated.

Because our cell culture studies as well as previousreports suggested that GSK3β inactivation involvesphosphorylation by the kinase Akt (18, 19), we investi-gated the effects of pressure overload induction on Aktphosphorylation. Indeed, aortic banding induced a sus-tained phosphorylation of Akt up to 48 hours, whichoccurred in lpr mice to a lesser degree and more tran-siently (Figure 5c).

JNK1 activity and apoptosis in lpr mice following pressureoverload. Since pressure overload activates multiple sig-naling pathways, and JNKs are known regulators of car-diac hypertrophy in vivo (16, 21), we determined thephosphorylation of JNK1, the predominant JNK iso-form in the heart, as an index of kinase activity. Where-as wild-type mice showed a potent cardiac induction ofJNK1 activity in response to pressure overload, theincrease of JNK1 phosphorylation in lpr mouse heartswas significantly attenuated after 24 and 48 hours (Fig-ure 5d). Consistent with these results, Fas receptor acti-vation in cultured cardiomyocytes induced JNK1 activ-ity about threefold after 40 minutes (data not shown).

Because Fas signaling causes apoptosis in variouscell types, we assessed cardiomyocyte apoptosis by insitu TUNEL staining (n = 6 mice per group). In sham-operated animals, the percentage of TUNEL+ cellswas similar in lpr (0.39% ± 0.2%) and wild-type mice(0.4% ± 0.1%). Although the number of apoptoticnuclei slightly increased 2 days after induction ofpressure overload, there were no differences betweenwild-type (0.76% ± 0.4%) and lpr mice (1.1% ± 1.5%).

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Figure 5Lpr mice fail to inhibit GSK3β in response to pressure overload. (a)Representative GSK3β immunocomplex kinase assay dot blots ofmyocardial homogenates at the times indicated following aorticbanding. (b) Group data derived from densitometric quantitation.Shown is the relative change (mean ± SEM) in GSK3β activity com-pared with control in wild-type (white bars) and lpr hearts (blackbars); n = 3 per group. *P < 0.05 for aortic banding versus control. (c)Immunoblotting for phospho-Akt (top panel) followed by a reprobefor total Akt (bottom panel) of myocardial homogenates at the timesindicated following induction of pressure overload. Shown is a repre-sentative blot from three animals in each group. (d) JNK1 activity inmouse heart homogenates following induction of pressure overload.Shown is the mean ± SEM in wild-type (white bars) and lpr hearts(black bars); n = 3 per group. *P < 0.05 for wild-type versus lpr.

Given that lpr mice develop a generalized lympho-proliferative disease, albeit at a considerably older agethan the mice used for the present study, we immuno-histochemically analyzed serial myocardial sections oflpr mice subjected to pressure overload for inflamma-tory infiltrates. Lpr mice did not reveal any myocardialinflammatory cells following aortic banding (data notshown). This suggests that the distinct cardiac responseof lpr mice to pressure overload is not due to a myocar-dial inflammatory process.

Fas ligand is dispensable for pressure overload–induced hyper-trophy in vivo. Cardiac overexpression of Fas ligand, theprototypic binding partner of the Fas receptor, is suffi-cient to induce hypertrophy in transgenic mice (11). Totest whether Fas ligand is necessary for pressure over-load hypertrophy in a manner similar to the Fas recep-tor, we performed aortic constriction of homozygous gld(generalized lymphoproliferative disease) mice, whichharbor a carboxyl-terminal Fas ligand point mutationrendering Fas ligand nonfunctional (20).

At base line, gld mice had ventricular dimensions andwall thicknesses similar to those of wild-type and lprmice (Figures 6, a and b). Following induction of pres-sure overload, gld mice maintained their ventriculardimensions without enlargement (Figure 6a), as well astheir fractional shortening (data not shown). Gld miceresponded to aortic banding with significant increasesof the septal as well as posterior left ventricular wall(Figure 6b). This corresponded to a significantlyincreased left-ventricular-to-body-weight ratio 14 daysafter aortic banding (4.02 ± 0.31 vs. 3.44 ± 0.23 mg/g, n = 8, P ≤ 0.05). Most importantly, 7 days after aorticbanding, the lethality rate of gld mice was 29% (4/14mice, P = not significant vs. wild-type, P ≤ 0.05 vs. lpr).Thus, gld mice behave similarly to wild-type mice fol-lowing the induction of pressure overload, indicatingthat Fas ligand is dispensable for the development ofcompensatory hypertrophy.

The Fas receptor mediates hypertrophy signaling beyond Fasligand in vitro. The striking phenotypic disparitybetween lpr and gld mice following aortic banding sug-gested that the Fas receptor may mediate hypertrophicsignaling beyond Fas ligand. Therefore, we investigat-ed this possibility in vitro using neonatal cardiomy-ocytes from wild-type and lpr mice.

Without stimulation, wild-type and lpr cardiomy-ocytes had identical cell lengths and surface areas (Fig-ure 6d). In conjunction with the results shown in Fig-ure 1, wild-type cardiomyocytes showed a significantincrease in both cell length and cell surface when treat-ed with either Fas ligand or angiotensin II. Fas ligandinduced a more elongated phenotype, whereasangiotensin II caused a greater increase in cell surfacethan in cell length. Expectedly, lpr cardiomyocytes wereunable to respond to Fas ligand treatment. Important-ly, the response of lpr cardiomyocytes to angiotensin IIwas significantly blunted with no detectable increase incell length and an increase of cell surface area, whichwas significantly lower than the one observed with

wild-type cardiomyocytes (Figure 6d). These resultsindicate that the Fas receptor mediates hypertrophysignaling beyond Fas ligand in vitro, thus supportingour in vivo findings obtained with lpr and gld mice.

DiscussionThe results of the present study establish Fas receptorsignaling as a novel pathway required for adaptive,

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Figure 6Fas ligand is dispensable for pressure overload hypertrophy in vivo,and Fas receptor mediates Fas ligand–independent signaling. (a)Echocardiographic left ventricular end-diastolic diameter (LVEDD)and end-systolic diameter (LVESD) of gld mice at base line (left, n = 4)and 14 days after induction of pressure overload (right, n = 8). Themean ± SEM is shown. *P ≤ 0.05 for aortic banding versus control.(b) Echocardiographic septal (white bars) and posterior wall (blackbars) thicknesses of gld mice at base line (left, n = 4) and 14 days afterinduction of pressure overload (right, n = 8). The mean ± SEM isshown. *P ≤ 0.05 for aortic banding versus control. (c) Representa-tive photomicrographs (×400) of wild-type (top) or Fas-deficient lpr(bottom) mouse neonatal cardiomyocytes unstimulated (left) or stim-ulated with either Fas ligand (middle) or angiotensin II (Ang II; right)at the indicated concentrations for 48 hours. A typical experiment ispresented. (d) Maximal cell length (left panel) and cell surface area(right panel) of wild-type (white bars) and lpr cardiomyocytes (blackbars). n = 10 randomly chosen cells per group. *P ≤ 0.01 for stimula-tion versus control; #P ≤ 0.01 for wild-type versus lpr.

compensatory hypertrophy following the biomechani-cal stress of pressure overload. In vitro, Fas receptoractivation caused cardiomyocyte hypertrophy depend-ing on the inhibition of GSK3β activity by phosphory-lation. In vivo, lpr mice lacking a functional Fas recep-tor demonstrated abrogated GSK3β inhibition,rapid-onset left ventricular dilatation and failure,absence of compensatory hypertrophy, and signifi-cantly increased mortality following the induction ofpressure overload. Results obtained with Fasligand–defective gld mice and with lpr cardiomyocytesin vitro indicate that the Fas receptor mediates signal-ing beyond its classical binding partner.

Although the Fas/Fas ligand system is a well-recog-nized and potent activator of apoptosis in many celltypes (6), a growing body of evidence suggests car-diomyocyte-specific downstream signaling of the Fasreceptor leading to survival and hypertrophy instead ofapoptotic cell death. In cultured cardiomyocytes, Fasreceptor activation was shown to activate the prohy-pertrophic transcription factor AP-1 (8). Transgenicmice overexpressing Fas ligand in the heart exhibit noenhanced cardiomyocyte apoptosis, but mild cardiachypertrophy in a transgene dose-dependent manner(11). Our results with cultured rat and mouse neonatalcardiomyocytes indicate that Fas receptor activationwith up to 100 ng/ml Fas ligand results in a specificelongated form of cardiomyocyte hypertrophy similarto the one observed with gp130 receptor stimulationand different from the phenotype evoked byangiotensin II (22). The concentrations of Fas ligandused did not cause cardiomyocyte apoptosis. However,much higher concentrations of Fas ligand (5000ng/ml) can principally cause cardiomyocyte apoptosis,in particular when combined with doxorubicin (23).

Consistent with a prominent role for Fas signaling tomediate hypertrophic responses in cardiomyocytes, thefailure of lpr mice lacking a functional Fas receptor toactivate a compensatory hypertrophic response follow-ing induction of pressure overload indicates that Fasreceptor signaling is required for at least this form ofcardiac hypertrophy in vivo. The phenotype of rapid-onset left ventricular dilatation and heart failure fol-lowing induction of pressure overload accompanied bya substantially increased mortality observed in lpr miceis similar to the one of mice with cardiac-restricted genetargeting of gp130, a cell-surface receptor for car-diotrophin-1 (5). However, the underlying mechanismsappear to be different: In contrast to mice with cardiac-restricted loss of gp130, lpr mice do not developincreased cardiomyocyte apoptosis following pressureoverload. In addition, lpr mice but not cardiac-restrict-ed gp130–/– mice have lost the ability to activate ahypertrophic response on a molecular level (5).

Strikingly, Fas ligand–deficient gld mice behaved sim-ilarly to wild-type mice when challenged with pressureoverload. First, this suggests that the phenotypeobserved in lpr mice did not occur secondarily due toautoimmunity disorders, since both lpr and gld mice are

prone to lymphoproliferative disorders (20). Second,the fact that Fas ligand is dispensable for pressure over-load hypertrophy in vivo suggested that the Fas recep-tor may mediate signaling beyond Fas ligand. In sup-port of these in vivo results, we found that neonatalcardiomyocytes from lpr mice not only are unable toreact to Fas ligand but also exhibit an impaired hyper-trophy induction in response to angiotensin II. It isconceivable that the Fas receptor is involved in thetransactivation of G protein–coupled receptors, a phe-nomenon recently described for the bradykinin B2receptor and VEGF receptor KDR/Flk-1 (24).

We now identified GSK3β as an important down-stream cytoplasmic phosphorylation target of Fasreceptor signaling. Phosphorylation and inactivationof GSK3β, a negative regulator of cardiomyocyte hyper-trophy, was recently identified as necessary and suffi-cient for hypertrophy induction by angiotensin II andendothelin-1 in vitro (18). Our in vitro studies demon-strate that increased ANF transcription induced by Fasactivation as a hallmark of the hypertrophic responsewas dependent on GSK3β phosphorylation. Impor-tantly, when compared with wild-type mice, lpr micedisplayed a differential GSK3β regulation with anincrease rather than an inhibition of GSK3β activityfollowing the induction of pressure overload. There-fore, inhibition of GSK3β activity appears to be also relevant in our in vivo model and may provide a molecular explanation for Fas activation–inducedhypertrophy, which is defective in lpr mice.

GSK3β is an established target of the PI3K/Akt sig-naling pathway with Akt phosphorylating and therebyinactivating GSK3β (19). Fas receptor activationinduced a profound and transient increase of Akt phos-phorylation preceding the PI3K-dependent GSK3βphosphorylation. Downstream of GSK3β, we observeda prominent nuclear translocation of NF-AT followingFas receptor activation. These data support previousstudies in cardiomyocytes showing that GSK3β isdownstream of Akt and that GSK3β phosphorylationoccurs in a PI3K-dependent manner (19). However, thesignaling pathway connecting the Fas receptor with thePI 3- and Akt-kinases remains to be elucidated.

Because cardiac hypertrophy is mediated by multiplesignaling cascades, other signaling may also be alteredin lpr mice in response to pressure overload. Indeed, wefound that the potent JNK1 activation observed inwild-type mice after aortic banding was significantlyattenuated in lpr mice. Given that JNKs are known reg-ulators of cardiac hypertrophy in vivo (16, 21), JNK1dysregulation may also contribute to the lpr mouseheart phenotype observed. In this regard, it is notablethat GSK3β is a negative regulator of the JNK target c-jun (25). Therefore, our JNK results are well compat-ible with an essential role for GSK3β in Fas-inducedcardiomyocyte hypertrophy.

In summary, we identified Fas receptor signaling as anovel signal necessary for cardiac hypertrophy inresponse to the biomechanical stress of pressure over-

380 The Journal of Clinical Investigation | February 2002 | Volume 109 | Number 3

load. In vitro, Fas activation–induced hypertrophydepends on increased GSK3β phosphorylation, andthis mechanism may be causative in vivo as well.

AcknowledgmentsWe thank Isabel Lassotta-Klasen for expert technicalassistance. This work was supported by the DeutscheForschungsgemeinschaft (Ba1668/3-1, Ju241/2-1).

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