This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Wollert, K. C. Articles by Drexler, H. Search for Related Content PubMed PubMed Citation Articles by Wollert, K. C. Articles by Drexler, H. Related Collections Apoptosis Growth factors/cytokines

(Circulation. 2000;101:1172.)
© 2000 American Heart Association, Inc.

Basic Science Reports

The Cardiac Fas (APO-1/CD95) Receptor/Fas Ligand System

Relation to Diastolic Wall Stress in Volume-Overload Hypertrophy In Vivo and Activation of the Transcription Factor AP-1 in Cardiac Myocytes

Kai C. Wollert, MD; Jörg Heineke, BS; Jürgen Westermann, MD; Mark Lüdde, BS; Beate Fiedler, PhD; Wolfgang Zierhut, MD; Didier Laurent, PhD; Manuel K. A. Bauer, PhD; Klaus Schulze-Osthoff, PhD; Helmut Drexler, MD

From the Department of Cardiology and Angiology (K.C.W., J.H., M.L., B.F., H.D.) and the Department of Anatomy (J.W.), Medizinische Hochschule Hannover, Hannover, the Department of Internal Medicine I, University of Tübingen (M.K.A.B., K.S.-O.), Tübingen, Germany, and the Department of Cardiovascular Research, Ciba-Geigy, Basel, Switzerland (W.Z., D.L.).

Correspondence to Helmut Drexler, MD, Department of Cardiology and Angiology, Medizinische Hochschule Hannover, Carl-Neuberg-Str 1, 30625 Hannover, Germany. E-mail drexler.helmut{at}mh-hannover.de

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background—Fas (APO-1/CD95) is a transmembrane receptor belonging to the tumor necrosis factor receptor superfamily. Cross-linking of Fas by Fas ligand (FasL), a tumor necrosis factor-–related cytokine, promotes apoptosis and/or transcription factor activation in a highly cell-type–specific manner. The biological consequences of Fas activation in cardiomyocytes and the regulation of Fas and FasL abundance in the myocardium in vivo remain largely unknown.

Methods and Results—As shown by immunohistochemistry, Fas was expressed on the sarcolemma of cardiomyocytes in left ventricular tissue sections. Moreover, FasL was constitutively expressed in the myocardium and in isolated cardiomyocytes, as revealed by reverse transcription polymerase chain reaction and Western blotting. Left ventricular abundance of Fas but not FasL was upregulated in a rat model of compensated volume-overload hypertrophy and was closely related to diastolic but not systolic wall stress as determined by MRI. Cardiomyocyte apoptosis was not enhanced in volume-overload hypertrophy despite the increased expression of Fas and the presence of FasL in the myocardium. Moreover, injection of mice with an agonistic anti-Fas antibody promoted hepatocyte but not cardiomyocyte apoptosis in vivo. Stimulation of isolated cardiomyocytes with recombinant FasL promoted an activation of the transcription factor AP-1 as shown by electrophoretic mobility shift assays but did not induce cell death.

Conclusions—Fas and FasL are constitutively expressed in the myocardium and in cardiomyocytes. Myocardial expression of Fas is closely related to diastolic loading conditions in vivo. Signaling pathways emanating from Fas are coupled to an activation of the transcription factor AP-1 in cardiomyocytes.

Key Words: receptors • myocytes • apoptosis • hypertrophy

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Cytokines including tumor necrosis factor- (TNF-) are emerging as potent regulators of cardiomyocyte hypertrophy and apoptosis and have been proposed to play an important role in the remodeling of the myocardium in response to chronic increases in hemodynamic load.^{1 2} Fas (APO-1/CD95) is a transmembrane receptor belonging to the TNF receptor superfamily.^{3 4 5 6} Activation of Fas requires cross-linking by Fas ligand (FasL), a TNF-–related cytokine.⁷ Fas is expressed rather abundantly in the myocardium and in cardiomyocytes, which indicates that cardiomyocytes are potential targets for FasL-mediated effects.^{4 8 9} Indeed, a recent study has demonstrated that short-term exposure of cardiomyocytes to FasL can alter [Ca²⁺]_i homeostasis.⁹ In many cell types, engagement of Fas by FasL is followed by caspase activation and apoptotic cell death.⁶ However, the response to Fas activation has been shown to be highly cell-type specific, with certain cells responding to Fas ligation with the induction of transcription factors and gene transcription rather than cell death.^{10 11 12} In this regard, the biological consequences of Fas activation in cardiomyocytes remain largely unknown. Overstretching of papillary muscles ex vivo has been shown to enhance cardiomyocyte Fas expression.¹³ Because Fas expression levels can determine cell susceptibility to FasL-mediated effects,⁶ it is crucial to understand whether more physiological levels of mechanical load that can be observed in cardiac hypertrophy in vivo are involved in the regulation of myocardial Fas expression.

In the present study, we investigated the regulation of Fas and FasL in the myocardium in relation to hemodynamic load in the in vivo setting, and we explored the functional significance of Fas activation in cardiomyocytes. With the use of a combined in vivo and in vitro approach, we demonstrated that the abundance of Fas in the myocardium is closely related to diastolic loading conditions and that signaling pathways emanating from Fas are coupled to an activation of the transcription factor AP-1 in cardiomyocytes.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Immunohistochemistry
The expression pattern of Fas in left ventricular (LV) myocardium was determined by immunohistochemistry.¹⁴ Rabbit polyclonal IgG(1:400) directed against the intracellular domain of mouse/rat Fas was used as the primary antibody (FAS-M20, Santa Cruz). Secondary (mouse anti-rabbit) and tertiary (goat anti-mouse alkaline phosphatase) antibodies were obtained from Dako. Fas expression was visualized with Fast blue. The slides were counterstained with hematoxylin. Fas peptide (FAS-M20P, Santa Cruz) was used in control experiments.

Northern Blotting
Fas mRNA expression was quantified by Northern blotting with the use of a rat Fas cDNA probe obtained by polymerase chain reaction (PCR) cloning.^{8 15} Separate filters were hybridized to a rat atrial natriuretic factor probe.¹⁶ cDNA probes were [³²P]-labeled by random priming. To control for loading and transfer efficiency, filters were hybridized to a [³²P]–end-labeled oligonucleotide complementary to 18S rRNA.

Quantification of FasL mRNA Expression
FasL mRNA expression was quantified by standard calibrated reverse transcription (RT)-PCR.¹⁷ A 495-bp FasL cDNA fragment was amplified from LV total RNA and cloned into pCR2.1 (Invitrogen): forward primer nt371 to 392, reverse primer nt844 to 865.⁷ The resulting pCR2.1-FasL₍₄₉₅₎ plasmid was then amplified by PCR with the use of the reverse primer and a mutagenic forward primer composed of nt371 to 392 fused to nt493 to 512, thereby producing a FasL cDNA fragment with a 100-bp deletion. The 395-bp fragment was cloned into pCR2.1, generating pCR2.1-FasL₍₃₉₅₎. Competitor rat FasL cRNA molecules were synthesized by in vitro transcription from pCR2.1-FasL₍₃₉₅₎ (T7-MEGAshortscript, Ambion).

LV total RNA (625 ng) was reverse-transcribed into cDNA along with increasing quantities of FasL competitor cRNA molecules (1x10⁴ to 5x10⁵) and subsequently amplified by PCR by use of the FasL forward and reverse primers (36 cycles: 1 minute at 94°C, 1 minute at 53°C, 1 minute at 72°C each). In no case was a PCR product obtained when reverse transcriptase was omitted from the reaction. The FasL target and FasL competitor RT-PCR products were separated by ethidium bromide agarose gel electrophoresis and analyzed by laser densitometry.¹⁷

Western Blotting
FasL protein expression was analyzed by Western blotting, with a monoclonal mouse antibody generated against the extracellular domain of human FasL (Transduction Laboratories).¹⁸

Volume-Overload Cardiac Hypertrophy Model
Aortic regurgitation was induced by cusp perforation in male Wistar-Kyoto rats (weight 250 to 300 g).¹⁹ Control animals were instrumented but did not undergo cusp perforation. Eight weeks after the procedure, LV end-diastolic and end-systolic dimensions were determined by MRI.²⁰ After MRI data acquisition, an ultraminiature catheter pressure transducer (Millar) was inserted through the left carotid artery and advanced into the ascending aorta and LV cavity. Systolic blood pressure and LV end-diastolic pressure were recorded. On the basis of the hemodynamic data and the MRI-derived values for LV wall thickness and radius, in vivo end-diastolic and end-systolic wall stresses were calculated according to Laplace’s law.²⁰ After completion of the hemodynamic analyses, the heart was quickly removed and rinsed in ice-cold saline. The LV and the right ventricular free wall were separated and weighed. The LV free wall was divided into halves and snap-frozen for later isolation of total RNA and genomic DNA or embedded in OCT medium for later preparation of cryosections.

DNA Agarose Gel Electrophoresis
To detect internucleosomal cleavage of genomic DNA, a hallmark of apoptotic cell death, DNA was isolated from LV tissue and subjected to ethidium bromide agarose gel electrophoresis (10 µg/sample).²¹

In Situ Nick End-Labeling
Apoptotic nuclei were detected in LV myocardium by in situ terminal deoxynucleotidyl transferase (TdT)-mediated digoxigenin-conjugated dUTP nick end-labeling with the use of a commercially available kit (ApopTag Plus, Peroxidase, Oncor).²² LV cryostat sections (5 µm) were mounted onto glass slides, fixed in 10% buffered formalin, and postfixed in ethanol:acetic acid (2:1) at -20°C. Sections were then treated with 8 µg/mL proteinase K for 5 minutes at room temperature. End-labeling was carried out according to the manufacturer’s instructions, and tissue sections were finally counterstained with hematoxylin and eosin to allow a discrimination between cardiac myocyte and nonmyocyte nuclei. Five tissue sections per animal were examined at x400 magnification. Positive controls were prepared by treating selected slides with 0.5 µg/mL DNase I for 10 minutes at room temperature. dUTP labeling was never observed when TdT was omitted from the reaction.

Treatment of Mice With an Agonistic Anti-Fas Antibody
Five-week-old Balb/c mice were injected intraperitoneally with 10 µg of a monoclonal hamster anti-mouse Fas IgG (Jo2, Pharmingen).²³ Control mice were treated with anti-trinitrophenol IgG (Pharmingen). After 24 hours, cryosections were prepared from the LV and liver and analyzed by in situ nick end-labeling.

Cardiomyocyte Culture
Neonatal rat ventricular cardiomyocytes and nonmyocytes were isolated from 1- to 3-day-old Sprague-Dawley rats.^{24 25} Myocytes were plated at a density of 5x10⁴ cells/cm² in gelatin-coated tissue culture plates. After overnight incubation in serum-containing medium, cardiomyocytes were switched to serum-free medium and stimulated with various agents. Cell survival was assessed by trypan blue exclusion under a phase contrast microscope. Nonmyocytes were enriched by differential plating for 1 hour in 10-cm tissue culture plates. Cells adhering to the culture dish were grown to confluence over a period of 2 to 3 days.²⁵ Adult rat ventricular cardiomyocytes were isolated from male Sprague-Dawley rats (weight 380 to 420 g).²⁶ Adult cardiomyocytes were plated in laminin-coated tissue culture plates (1x10⁴ cells/cm²). More than 98% of the cells displayed a rod-shaped morphology. Cells were stimulated in serum-free medium with various agents. Cell viability was determined by counting rod-shaped cells per field.²⁷ Recombinant human soluble FasL expressed as an epitope tag fusion protein and a cross-linking mouse monoclonal IgG directed against the epitope tag were purchased from Upstate Biotechnology.

Electrophoretic Mobility Shift Assay
Neonatal rat ventricular cardiomyocytes were plated into 6-cm dishes and serum-starved for 24 hours before stimulation with recombinant FasL and cross-linking IgG. AP-1 and nuclear factor-B DNA binding was detected by electrophoretic mobility shift assay.^{28 29 30} Supershift experiments were performed with rabbit polyclonal IgG directed against c-Jun. Rabbit polyclonal IgG directed against retinoblastoma protein was used as a control (both antibodies from Santa Cruz).

Statistical Analysis
Data are presented as mean±SEM. The unpaired Student’s t test was used for intergroup comparisons. Linear regression analysis was performed to test for a correlation between 2 variables. A 2-tailed P value of <0.05 was considered to indicate statistical significance.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Expression of Fas and FasL in Left Ventricular Myocardium
As shown by immunohistochemistry, Fas was localized primarily on the sarcolemma of cardiomyocytes (Figure 1B). No staining could be detected when the primary antibody was omitted from the reaction (Figure 1A). Furthermore, Fas staining was abolished by coincubation with the Fas peptide used for immunization (Figure 1C). FasL mRNA (reverse transcription–polymerase chain reaction) and protein (Western blotting) were readily detectable in the myocardium as well as in isolated cardiomyocytes and nonmyocytes (Figure 2, A and B).

View larger version (93K): [in this window] [in a new window]   Figure 1. Detection of LV Fas expression by immunohistochemistry. No staining was detectable when the primary antibody was omitted from the reaction (A). In a tissue section stained with anti-Fas, Fas can be detected on the sarcolemma of cardiomyocytes (B). Coincubation of anti-Fas with Fas peptide abolished Fas immunoreactivity (C).

View larger version (59K): [in this window] [in a new window]   Figure 2. Detection of FasL expression by RT-PCR and Western blotting. A, 495-bp FasL cDNA fragment was amplified from LV myocardium (lane 1), purified adult rat cardiomyocytes (lane 2), and neonatal rat ventricular nonmyocytes (lane 3) by a 36-cycle, non–standard-calibrated RT-PCR (625 ng of total RNA per reaction). On left, DNA ladder was loaded. By Western blotting, FasL could be detected as 32-kDa protein in LV myocardium (B, lane 1), purified adult rat cardiomyocytes (lane 2), and neonatal rat ventricular nonmyocytes (lane 3). Forty micrograms of protein was loaded per lane. On left, positions of molecular weight standards are shown.

Volume-Overload Hypertrophy Model
To analyze the abundance of Fas and FasL in the myocardium in relation to hemodynamic load in vivo, we used a rat model of volume-overload hypertrophy (Table 1). Left ventricular (LV) end-diastolic and end-systolic wall stresses were increased in volume-overloaded rats. As determined in post mortem examination, volume overload resulted in an increase in LV–to–body weight ratio and an upregulation of LV atrial natriuretic factor mRNA expression.

View this table: [in this window] [in a new window]   Table 1. Volume-Overload Hypertrophy Model

Regulation of Fas and FasL in Volume-Overload Hypertrophy
Chronic volume overload resulted in an upregulation of LV Fas mRNA expression (Figure 3A). LV abundance of Fas was closely related to end-diastolic (Figure 3B) but not end-systolic wall stress (not shown) in volume-overload hypertrophy. As shown by standard-calibrated RT-PCR, LV expression of FasL mRNA did not differ significantly between control and volume-overloaded animals (7.3±1.2 vs 6.6±1.0x10⁴ mRNA transcripts/625 ng RNA, respectively) and did not correlate with ventricular wall stress (not shown).

View larger version (14K): [in this window] [in a new window]   Figure 3. Fas mRNA expression normalized to 18S in LV myocardium from control rats and rats with volume-overload hypertrophy (A). Correlation between LV end-diastolic wall stress and LV Fas mRNA expression in rats with volume-overload hypertrophy (B) is shown.

Cardiomyocyte Apoptosis in Volume-Overload Hypertrophy
We next investigated whether enhanced Fas expression levels in volume-overload hypertrophy were associated with an increase in apoptotic cell death in the myocardium. However, no evidence of DNA fragmentation was found in control hearts and in hearts with volume-overload hypertrophy (not shown). Because the DNA-laddering technique is rather insensitive, we used in situ nick end-labeling to detect apoptotic cardiomyocyte nuclei in LV myocardium: There was no significant difference in the prevalence of dUTP-labeled cardiomyocyte nuclei between control and volume-overloaded rats (Figure 4).

View larger version (46K): [in this window] [in a new window]   Figure 4. In situ detection of DNA strand breaks in LV myocardium. The majority of cardiomyocyte nuclei are labeled with dUTP in DNase-treated positive control slide (A). dUTP-positive cardiomyocyte nucleus in tissue section from volume-overloaded heart is depicted in B (arrow). Prevalence of dUTP-labeled cardiomyocyte nuclei in control rats and in rats with volume-overload hypertrophy is summarized in C.

Effect of Fas Activation on Cardiomyocyte Viability
To assess whether Fas activation can trigger cardiomyocyte death in vivo, mice were injected with an agonistic anti-Fas antibody. In mice treated with control antibody, dUTP-labeled cells were very rare in the LV and the liver (Figure 5, A and C). Injection of anti-Fas induced severe liver damage resulting from extensive hepatocyte apoptosis (Figure 5D) but did not promote cardiomyocyte apoptosis in LV myocardium (Figure 5B). Conceivably, the lack of apoptosis in the heart after injection of anti-Fas may reflect a different efficiency of penetration of the antibody into cardiac versus hepatic tissues. To address this possibility, cardiomyocytes were isolated from neonatal and adult rats and exposed to recombinant soluble FasL in vitro for up to 48 hours. Even in the presence of cross-linking antibodies, however, FasL did not promote cell death in cultured cardiomyocytes (Table 2).

View larger version (115K): [in this window] [in a new window]   Figure 5. Detection of apoptotic cell death in LV and liver after injection of agonistic anti-Fas antibody. As revealed by in situ nick end-labeling, no dUTP-positive nuclei are detectable in LV (A) and liver (C) of mice injected with control antibody. Injection of anti-Fas did not promote cardiomyocyte apoptosis in myocardium (B). By contrast, anti-Fas induced extensive hepatocyte apoptosis, as revealed by dUTP-labeling of hepatocyte nuclei (arrows in D).

View this table: [in this window] [in a new window]   Table 2. Effect of Recombinant FasL on Cardiomyocyte Viability

Effect of FasL on Activation of AP-1 and Nuclear Factor-B
To investigate whether FasL can trigger transcription factor activation in cardiomyocytes, DNA-binding activities of AP-1 and nuclear factor (NF)-B were analyzed by electrophoretic mobility shift assay. Stimulation of neonatal rat ventricular myocytes with recombinant FasL and cross-linking antibodies induced AP-1 DNA-binding activity (Figure 6) but did not enhance NF-B DNA binding (not shown). The specificity of AP-1 DNA binding was supported by competition experiments with 100-fold molar excess of unlabeled AP-1. As expected, unlabeled NF-B did not compete for AP-1 DNA binding. The AP-1/DNA complex was supershifted in part by an antibody directed against c-Jun but not by a control antibody directed against the unrelated retinoblastoma protein (Figure 6).

View larger version (101K): [in this window] [in a new window]   Figure 6. Effect of recombinant FasL on AP-1 DNA-binding activity. Cellular extracts (10 µg of protein) from untreated control cardiomyocytes (lane 1), cardiomyocytes stimulated for 1 hour with cross-linking antibody (1.5 µg/mL) alone (lane 2), or cross-linking antibody and recombinant FasL (50 ng/mL) (lanes 3 to 7) were analyzed by EMSA with a 32P-labeled double-stranded oligonucleotide encompassing the AP-1 binding site. A 100-fold molar excess of unlabeled AP-1 (lane 4) but not unlabeled NF-B (lane 5) competed for AP-1 DNA binding. The AP-1/DNA complex was supershifted in part by antibody directed against c-Jun (lane 6) but not by control antibody directed against retinoblastoma protein (lane 7).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Cytokines, including TNF- and members of the interleukin-6 cytokine family, have been recognized as potent regulators of cardiomyocyte growth and apoptosis.^{24 27 31 32} Fas is a member of the TNF receptor superfamily of structurally related cytokine receptors.^{3 4 5} In patients with heart failure, circulating levels of soluble Fas and FasL are increased, which suggests a potential role of Fas in this setting.^{33 34} Although Fas promotes caspase activation and apoptosis in susceptible cell types, Fas can mediate biological functions unrelated to cell death, such as transcription factor activation and the induction of cell growth and differentiation.^{10 11 12} In the present study, we investigated the regulation of Fas and FasL in the myocardium in relation to hemodynamic load in vivo and explored the functional significance of Fas activation in cardiomyocytes.

Fas and FasL Are Expressed in Myocardium and in Cardiomyocytes
As shown by immunohistochemistry in our study, Fas was localized on the sarcolemma of cardiomyocytes, supporting the idea that cardiomyocytes are targets for Fas-mediated effects.^{4 8 9} By using RT-PCR and Western blotting, we could also detect constitutive FasL mRNA and protein expression in the myocardium. Both cardiomyocytes and nonmyocytes contribute to FasL expression in the heart. It has been shown in patients with heart failure that the concentration of soluble FasL in coronary sinus exceeds the concentration in aortic blood.³³ Collectively, these data indicate that FasL synthesized locally within the myocardium represents a potential mechanism for cardiomyocyte Fas activation.

Regulation of Fas and FasL in Volume-Overload Hypertrophy
It has previously been shown that overstretching induces Fas expression in isolated papillary muscles.¹³ To study the regulation of myocardial Fas and FasL abundance in relation to more physiological levels of mechanical load in vivo, we used a rat model of volume-overload hypertrophy. As shown by MRI, volume-overload hypertrophy was characterized by LV chamber dilation but no change in wall thickness. The changes in LV geometry combined with the increases in LV end-diastolic pressure and systolic blood pressure translated into a significant elevation of LV diastolic and systolic wall stress. The increase in LV mass was associated with enhanced atrial natriuretic factor expression levels, confirming hypertrophy of the cardiomyocyte compartment. Right ventricular–to–body weight ratio was unchanged and mean arterial pressure was preserved, which indicates a compensated stage of LV hypertrophy (data not shown). LV expression of Fas was upregulated in relation to diastolic but not systolic wall stress in volume-overload hypertrophy. FasL, by contrast, was not induced, which indicates distinct regulatory mechanisms. Although a causal relation cannot be inferred from these data, the close relation between Fas expression and diastolic wall stress suggests that Fas expression levels in the myocardium are modulated by loading conditions in vivo.

Functional Significance of Fas Activation in Cardiomyocytes
In the present study, recombinant soluble FasL did not promote cell death in isolated cardiomyocytes. Like TNF-, FasL is synthesized as a membrane-bound protein that can be converted by proteolytic cleavage into a soluble form that is then released into the circulation.³⁵ Although soluble FasL has been shown to induce apoptosis in susceptible cell types,^{36 37} it has been reported that membrane-bound FasL is more active as compared with soluble FasL and that the proapoptotic activity of soluble FasL can be restored by cross-linking antibodies.³⁸ As shown in the present study, however, cross-linking of soluble FasL did not enhance its cytotoxic activity in cultured cardiomyocytes. Moreover, supernatants from a murine neuroblastoma cell line stably transfected with a murine FasL expression vector did not promote cardiomyocyte death in vitro (data not shown), although FasL is released from these cells in vesicles, that is, in a membrane-bound, unprocessed form.^{38 39} In agreement with our in vitro results, an agonistic anti-Fas antibody induced hepatocyte apoptosis but did not promote cardiomyocyte apoptosis in vivo. Taken together, it appears that the signaling cascade coupling Fas to the induction of cell death is inhibited at some crucial point(s) in isolated cardiomyocytes and in normal myocardium. However, a general resistance of cardiomyocytes to Fas-mediated apoptosis cannot be inferred from these data. In the present study, increased Fas expression levels were not associated with enhanced cardiomyocyte apoptosis in rats with compensated volume-overload hypertrophy. By contrast, increased levels of Fas are accompanied by pronounced increases in cardiomyocyte apoptosis in overstretched and in ischemic myocardium.^{13 40} It is conceivable, therefore, that cardiomyocytes may become susceptible to Fas-mediated cell death under certain pathophysiological conditions.

As shown by EMSA in the present study, Fas-dependent signaling pathways are coupled to the activation of the transcription factor AP-1 in isolated cardiomyocytes. By contrast, Fas ligation did not result in NF-B activation. AP-1 is a transcriptional activator composed of Jun and Fos gene family members.⁴¹ Indeed, supershift experiments indicated that c-Jun is an integral part of the AP-1/DNA complex induced by FasL in cardiomyocytes. FasL has been shown to activate AP-1 in other cell types as well, and in some cases, stimulation of AP-1 appears to be independent from the induction of apoptosis.^{10 42} In cardiomyocytes, AP-1 has been implicated in the transcriptional regulation of several genes associated with a hypertrophic response.^{43 44} Our data therefore raise the intriguing possibility that the Fas receptor can modulate gene expression through AP-1 in cardiomyocytes. In this regard, a preliminary study has recently demonstrated that cardiac-specific overexpression of FasL promotes cardiomyocyte hypertrophy in transgenic mice.⁴⁵

	Acknowledgments

 
This study was supported by the Deutsche Forschungsgemeinschaft (Dr Wollert and Dr Drexler, SFB244 and Dr 148/6-4).

Received July 23, 1999; revision received September 24, 1999; accepted September 29, 1999.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Packer M. Is tumor necrosis factor an important neurohormonal mechanism in chronic heart failure? Circulation. 1995;92:1379–1382.[Free Full Text]
   
2. Meldrum DR. Tumor necrosis factor in the heart. Am J Physiol. 1998;274:R577–R595.
   
3. Itoh N, Yonehara S, Ishii A, Yonehara M, Mizushima SI, Sameshima M, Hase A, Seto Y, Nagata S. The polypeptide encoded by the cDNA for human cell surface antigen Fas can mediate apoptosis. Cell. 1991;66:233–243.[Medline] [Order article via Infotrieve]
   
4. Watanabe-Fukunaga R, Brannan CI, Itoh N, Yonehara S, Copeland NG, Jenkins NA, Nagata S. The cDNA structure, expression, and chromosomal assignment of the mouse Fas antigen. J Immunol. 1992;148:1274–1279.[Abstract]
   
5. Oehm A, Behrmann I, Falk W, Pawlita M, Maier G, Klas C, Li-Weber M, Richards S, Dhein J, Trauth BC, Ponstingl H, Krammer PH. Purification and molecular cloning of the APO-1 cell surface antigen, a member of the tumor necrosis factor/nerve growth factor receptor superfamily: sequence identity with the Fas antigen. J Biol Chem. 1992;267:10709–10715.[Abstract/Free Full Text]
   
6. Schulze-Osthoff K, Ferrari D, Los M, Wesselborg S, Peter ME. Apoptosis signaling by death receptors. Eur J Biochem. 1998;254:439–459.[Medline] [Order article via Infotrieve]
   
7. Suda T, Takahashi T, Golstein P, Nagata S. Molecular cloning and expression of the Fas ligand, a novel member of the tumor necrosis factor family. Cell. 1993;75:1169–1178.[Medline] [Order article via Infotrieve]
   
8. Tanaka M, Ito H, Adachi S, Akimoto H, Nishikawa T, Kasajima T, Marumo F, Hiroe M. Hypoxia induces apoptosis with enhanced expression of Fas antigen messenger RNA in cultured neonatal rat cardiomyocytes. Circ Res. 1994;75:426–433.[Abstract/Free Full Text]
   
9. Felzen B, Shilkrut M, Less H, Sarapov I, Maor G, Coleman R, Robinson RB, Berke G, Binah O. Fas (CD95/APO-1)-mediated damage to ventricular myocytes induced by cytotoxic T lymphocytes from perforin-deficient mice: a major role for inositol 1,4,5-triphosphate. Circ Res. 1998;82:438–450.[Abstract/Free Full Text]
   
10. Okamoto K, Fujisawa K, Hasunuma T, Kobata T, Sumida T, Nishioka K. Selective activation of the JNK/AP-1 pathway in Fas-mediated apoptosis of rheumatoid arthritis synoviocytes. Arthritis Rheum. 1997;40:919–926.[Medline] [Order article via Infotrieve]
    
11. Ponton A, Clement MV, Stamenkovic I. The CD95(APO-1/Fas) receptor activates NF-B independently of its cytotoxic function. J Biol Chem. 1996;271:8991–8995.[Abstract/Free Full Text]
    
12. Malinin NL, Boldin MP, Kovalenko AV, Wallach D. MAP3K-related kinase involved in NF-B induction by TNF, CD95 and IL-1. Nature. 1997;385:540–544.[Medline] [Order article via Infotrieve]
    
13. Cheng W, Li B, Kajstura J, Li P, Wolin MS, Sonnenblick EH, Hintze TH, Olivetti G, Anversa P. Stretch-induced programmed myocyte cell death. J Clin Invest. 1995;96:2247–2259.
    
14. Westermann J, Geismar U, Bode U, Sparshott S, Bell E. In vivo CD4⁺ T cells of both the naive and the memory phenotype enter rat lymph nodes and Peyer’s patches via high endothelial venules: within the tissue their migratory behaviour differs. Eur J Immunol. 1997;27:3174–3181.[Medline] [Order article via Infotrieve]
    
15. Kimura K, Wakatsuki T, Yamamoto M. A variant mRNA species encoding a truncated form of Fas antigen in the rat liver. Biochem Biophys Res Commun. 1994;198:666–674.[Medline] [Order article via Infotrieve]
    
16. Seidman CE, Duby AD, Choi E, Graham RM, Haber E, Homcy C, Smith JA, Seidman JG. The structure of rat preproatrial natriuretic factor as defined by a complementary DNA clone. Science. 1984;225:324–326.[Abstract/Free Full Text]
    
17. Wollert KC, Studer R, Doerfer K, Schieffer E, Holubarsch C, Just H, Drexler H. Differential effects of kinins on cardiomyocyte hypertrophy and interstitial collagen matrix in the surviving myocardium after myocardial infarction in the rat. Circulation. 1997;95:1910–1917.[Abstract/Free Full Text]
    
18. Vogt M, Bauer MKA, Ferrari D, Schulze-Osthoff K. Oxidative stress and hypoxia/reoxygenation trigger CD95 (APO-1/Fas) ligand expression in microglial cells. FEBS Lett. 1998;429:67–72.[Medline] [Order article via Infotrieve]
    
19. Uematsu T, Yamazaki T, Matsuno H, Hayashi Y, Nakashima M. A simple method for producing graded aortic insufficiencies in rats and subsequent development of cardiac hypertrophy. J Pharmacol Methods. 1989;22:249–257.[Medline] [Order article via Infotrieve]
    
20. Zierhut W, Studer R, Laurent D, Kästner S, Allegrini P, Whitebread S, Cumin F, Baum HP, de Gasparo M, Drexler H. Left ventricular wall stress and sarcoplasmic reticulum Ca²⁺-ATPase gene expression in renal hypertensive rats: dose-dependent effects of ACE inhibition and AT₁-receptor blockade. Cardiovasc Res. 1996;31:758–768.[Medline] [Order article via Infotrieve]
    
21. Piot CA, Padmanaban D, Ursell PC, Sievers RE, Wolfe CL. Ischemic preconditioning decreases apoptosis in rat hearts in vivo. Circulation. 1997;96:1598–1604.[Abstract/Free Full Text]
    
22. Itoh G, Tamura J, Suzuki M, Suzuki Y, Ikeda H, Koike M, Nomura M, Jie T, Ito K. DNA fragmentation of human infarcted myocardial cells demonstrated by the nick end labeling method and DNA agarose gel electrophoresis. Am J Pathol. 1995;146:1325–1331.[Abstract]
    
23. Ogasawara J, Watanabe-Fukunaga R, Adachi M, Matsuzawa A, Kasugai T, Kitamura Y, Itoh N, Suda T, Nagata S. Lethal effect of the anti-Fas antibody in mice. Nature. 1993;364:806–809.[Medline] [Order article via Infotrieve]
    
24. Wollert KC, Taga T, Saito M, Narazaki M, Kishimoto T, Glembotski CC, Vernallis AB, Heath JK, Pennica D, Wood WI, Chien KR. Cardiotrophin-1 activates a distinct form of cardiac muscle cell hypertrophy: assembly of sarcomeric units in series via gp130/leukemia inhibitory factor receptor-dependent pathways. J Biol Chem. 1996;271:9535–9545.[Abstract/Free Full Text]
    
25. Kim NN, Villarreal FJ, Printz MP, Lee AA, Dillmann WH. Trophic effects of angiotensin II on neonatal rat cardiac myocytes are mediated by cardiac fibroblasts. Am J Physiol. 1995;269:E426–437.[Abstract/Free Full Text]
    
26. Reinecke H, Vetter R, Drexler H. Effects of -adrenergic stimulation on the sarcolemmal Na⁺/Ca²⁺-exchanger in adult rat ventricular cardiocytes. Cardiovasc Res. 1997;36:216–222.[Abstract/Free Full Text]
    
27. Yokoyama T, Nakano M, Bednarczyk JL, McIntyre BW, Entmann M, Mann DL. Tumor necrosis factor- provokes a hypertrophic growth response in adult cardiac myocytes. Circulation. 1997;95:1247–1252.[Abstract/Free Full Text]
    
28. Schulze-Osthoff K, Beyaert R, Vandevoorde V, Haegeman G, Fiers W. Depletion of the mitochondrial electron transport abrogates the cytotoxic and gene-inductive effects of TNF. EMBO J. 1993;12:3095–3104.[Medline] [Order article via Infotrieve]
    
29. Angel P, Imagawa M, Chiu R, Stein B, Imbra RJ, Rahmsdorf HJ, Jonat C, Herrlich P, Karin M. Phorbol ester-inducible genes contain a common cis element recognized by a TPA-modulated trans-acting factor. Cell. 1987;49:729–739.[Medline] [Order article via Infotrieve]
    
30. Zabel U, Schreck R, Baeuerle PA. DNA binding of purified transcription factor NF-B: affinity, specificity, Zn²⁺ dependence, and differential half-site recognition. J Biol Chem. 1991;266:252–260.[Abstract/Free Full Text]
    
31. Krown KA, Page MT, Nguyen C, Zechner D, Gutierrez V, Comstock KL, Glembotski CC, Quintana PJE, Sabbadini RA. Tumor necrosis factor alpha-induced apoptosis in cardiac myocytes: involvement of the sphingolipid signaling cascade in cardiac cell death. J Clin Invest. 1996;98:2854–2865.[Medline] [Order article via Infotrieve]
    
32. Sheng Z, Knowlton K, Chen J, Hoshijima M, Brown JH, Chien KR. Cardiotrophin-1 (CT-1) inhibition of cardiac myocyte apoptosis via a mitogen-activated protein kinase-dependent pathway. J Biol Chem. 1997;272:5783–5791.[Abstract/Free Full Text]
    
33. Yamaguchi S, Yamaoka M, Okuyama M, Nitoube J, Fukui A, Shirakabe M, Shirakawa K, Nakamura N, Tomoike H. Elevated circulating levels and cardiac secretion of soluble Fas ligand in patients with congestive heart failure. Am J Cardiol. 1999;83:1500–1503.[Medline] [Order article via Infotrieve]
    
34. Nishigaki K, Minatoguchi S, Seishima M, Asano K, Noda T, Yasuda N, Sano H, Kumada H, Takemura M, Noma A, Tanaka T, Watanabe S, Fujiwara H. Plasma Fas ligand, an inducer of apoptosis, and plasma soluble Fas, an inhibitor of apoptosis, in patients with chronic congestive heart failure. J Am Coll Cardiol. 1997;29:1214–1220.[Abstract]
    
35. Kayagaki N, Kawasaki A, Ebata T, Ohmoto H, Ikeda S, Inoue S, Yoshino K, Okumura K, Yagita H. Metalloproteinase-mediated release of human Fas ligand. J Exp Med. 1995;182:1777–1783.[Abstract/Free Full Text]
    
36. Tanaka M, Suda T, Takahashi T, Nagata S. Expression of the functional soluble form of human Fas ligand in activated lymphocytes. EMBO J. 1995;14:1129–1135.[Medline] [Order article via Infotrieve]
    
37. Tanaka M, Suda T, Haze K, Nakamura N, Sato K, Kimura F, Motoyoshi K, Mizuki S, Tagawa S, Ohga S, Hatake K, Drummond AH, Nagata S. Fas ligand in human serum. Nature Med. 1996;2:317–322.[Medline] [Order article via Infotrieve]
    
38. Schneider P, Holler N, Bodmer JL, Hahne M, Frei K, Fontana A, Tschopp J. Conversion of membrane-bound Fas(CD95) ligand to its soluble form is associated with downregulation of its proapoptotic activity and loss of liver toxicity. J Exp Med. 1998;187:1205–1213.[Abstract/Free Full Text]
    
39. Rensing-Ehl A, Frei K, Flury R, Matiba B, Mariani SM, Weller M, Aebischer P, Krammer PH, Fontana A. Local Fas/APO-1 (CD95) ligand-mediated tumor cell killing in vivo. Eur J Immunol. 1995;25:2253–2258.[Medline] [Order article via Infotrieve]
    
40. Kajstura J, Cheng W, Reiss K, Clark WA, Sonnenblick EH, Krajewski S, Reed JC, Olivetti G, Anversa P. Apoptotic and necrotic myocyte cell deaths are independent contributing variables of infarct size in rats. Lab Invest. 1996;74:86–107.[Medline] [Order article via Infotrieve]
    
41. Karin M. The regulation of AP-1 activity by mitogen-activated protein kinases. J Biol Chem. 1995;270:16483–16486.[Free Full Text]
    
42. Lenczowski JM, Dominguez L, Eder AM, King LB, Zacharchuk CM, Ashwell JD. Lack of a role for jun kinase and AP-1 in Fas-induced apoptosis. Mol Cell Biol. 1997;17:170–181.[Abstract]
    
43. Paradis P, MacLellan WR, Belaguli NS, Schwartz RJ, Schneider MD. Serum response factor mediates AP-1-dependent induction of the skeletal -actin promotor in ventricular myocytes. J Biol Chem. 1996;271:10827–10833.[Abstract/Free Full Text]
    
44. Kovacic-Milivojevic B, Wong VSH, Gardner DG. Selective regulation of the atrial natriuretic peptide gene by individual components of the activator protein-1 complex. Endocrinology. 1996;137:1108–1117.[Abstract]
    
45. Nelson DP, Hall DG, Setser E, Akanbi A, Klevitsky R, Osinska H, Sussman MA, Schwartz SM, Robbins J. Transgenic cardiac-specific overexpression of Fas ligand: a model of cytokine-mediated hypertrophy. Circulation. 1998;98(suppl I):I-345. Abstract.
    

This article has been cited by other articles:

				D. Sanchis, M. Llovera, M. Ballester, and J. X. Comella An alternative view of apoptosis in heart development and disease Cardiovasc Res, February 1, 2008; 77(3): 448 - 451. [Full Text] [PDF]

				F. Roubille, S. Combes, J. Leal-Sanchez, C. Barrere;, F. Cransac, C. Sportouch-Dukhan, G. Gahide, I. Serre, E. Kupfer, S. Richard, et al. Myocardial Expression of a Dominant-Negative Form of Daxx Decreases Infarct Size and Attenuates Apoptosis in an In Vivo Mouse Model of Ischemia/Reperfusion Injury Circulation, December 4, 2007; 116(23): 2709 - 2717. [Abstract] [Full Text] [PDF]

				Y. Iwanaga, I. Nishi, S. Furuichi, T. Noguchi, K. Sase, Y. Kihara, Y. Goto, and H. Nonogi B-Type Natriuretic Peptide Strongly Reflects Diastolic Wall Stress in Patients With Chronic Heart Failure: Comparison Between Systolic and Diastolic Heart Failure J. Am. Coll. Cardiol., February 21, 2006; 47(4): 742 - 748. [Abstract] [Full Text] [PDF]

				Y. J. Kang Cardiac Hypertrophy: A Risk Factor for QT-Prolongation and Cardiac Sudden Death Toxicol Pathol, January 1, 2006; 34(1): 58 - 66. [Abstract] [Full Text] [PDF]

				J. Wohlschlaeger, K. J. Schmitz, C. Schmid, K. W. Schmid, P. Keul, A. Takeda, S. Weis, B. Levkau, and H. A. Baba Reverse remodeling following insertion of left ventricular assist devices (LVAD): A review of the morphological and molecular changes Cardiovasc Res, December 1, 2005; 68(3): 376 - 386. [Abstract] [Full Text] [PDF]

				Y. D. Barac, N. Zeevi-Levin, G. Yaniv, I. Reiter, F. Milman, M. Shilkrut, R. Coleman, Z. Abassi, and O. Binah The 1,4,5-inositol trisphosphate pathway is a key component in Fas-mediated hypertrophy in neonatal rat ventricular myocytes Cardiovasc Res, October 1, 2005; 68(1): 75 - 86. [Abstract] [Full Text] [PDF]

				C. Hasel, S. Durr, A. Bauer, R. Heydrich, S. Bruderlein, T. Tambi, U. Bhanot, and P. Moller Pathologically elevated cyclic hydrostatic pressure induces CD95-mediated apoptotic cell death in vascular endothelial cells Am J Physiol Cell Physiol, August 1, 2005; 289(2): C312 - C322. [Abstract] [Full Text] [PDF]

				R. S. Misra, D. M. Jelley-Gibbs, J. Q. Russell, G. Huston, S. L. Swain, and R. C. Budd Effector CD4+ T Cells Generate Intermediate Caspase Activity and Cleavage of Caspase-8 Substrates J. Immunol., April 1, 2005; 174(7): 3999 - 4009. [Abstract] [Full Text] [PDF]

				R. von Harsdorf "Fas-ten" Your Seat Belt: Anti-apoptotic Treatment in Heart Failure Takes Off Circ. Res., September 17, 2004; 95(6): 554 - 556. [Full Text] [PDF]

				Y. Li, G. Takemura, K.-i. Kosai, T. Takahashi, H. Okada, S. Miyata, K. Yuge, S. Nagano, M. Esaki, N. C. Khai, et al. Critical Roles for the Fas/Fas Ligand System in Postinfarction Ventricular Remodeling and Heart Failure Circ. Res., September 17, 2004; 95(6): 627 - 636. [Abstract] [Full Text] [PDF]

				D. Hilfiker-Kleiner, A. Hilfiker, M. Fuchs, K. Kaminski, A. Schaefer, B. Schieffer, A. Hillmer, A. Schmiedl, Z. Ding, E. Podewski, et al. Signal Transducer and Activator of Transcription 3 Is Required for Myocardial Capillary Growth, Control of Interstitial Matrix Deposition, and Heart Protection From Ischemic Injury Circ. Res., July 23, 2004; 95(2): 187 - 195. [Abstract] [Full Text] [PDF]

				Y. Takemura, K. Fukuo, O. Yasuda, T. Inoue, N. Inomata, T. Yokoi, H. Kawamoto, T. Suhara, and T. Ogihara Fas Signaling Induces Akt Activation and Upregulation of Endothelial Nitric Oxide Synthase Expression Hypertension, April 1, 2004; 43(4): 880 - 884. [Abstract] [Full Text] [PDF]

				S. Gupta, R. Natarajan, S. G. Payne, E. J. Studer, S. Spiegel, P. Dent, and P. B. Hylemon Deoxycholic Acid Activates the c-Jun N-terminal Kinase Pathway via FAS Receptor Activation in Primary Hepatocytes: ROLE OF ACIDIC SPHINGOMYELINASE-MEDIATED CERAMIDE GENERATION IN FAS RECEPTOR ACTIVATION J. Biol. Chem., February 13, 2004; 279(7): 5821 - 5828. [Abstract] [Full Text] [PDF]

				Q Z Feng, T D Li, L X Wei, X Qiao, J Yi, L Wang, and T S Yang Tempero-spatial dissociation between the expression of Fas and apoptosis after coronary occlusion Mol. Pathol., December 1, 2003; 56(6): 362 - 367. [Abstract] [Full Text] [PDF]

				L. Jiang, Y. Huang, S. Hunyor, and C.G. dos Remedios Cardiomyocyte apoptosis is associated with increased wall stress in chronic failing left ventricle Eur. Heart J., April 2, 2003; 24(8): 742 - 751. [Abstract] [Full Text] [PDF]

				P. Lee, M. Sata, D. J. Lefer, S. M. Factor, K. Walsh, and R. N. Kitsis Fas pathway is a critical mediator of cardiac myocyte death and MI during ischemia-reperfusion in vivo Am J Physiol Heart Circ Physiol, February 1, 2003; 284(2): H456 - H463. [Abstract] [Full Text] [PDF]

				T. Aoyama, G. Takemura, R. Maruyama, K.-i. Kosai, T. Takahashi, M. Koda, K. Hayakawa, Y. Kawase, S. Minatoguchi, and H. Fujiwara Molecular mechanisms of non-apoptosis by Fas stimulation alone versus apoptosis with an additional actinomycin D in cultured cardiomyocytes Cardiovasc Res, September 1, 2002; 55(4): 787 - 798. [Abstract] [Full Text] [PDF]

W. Chao, Y. Shen, L. Li, and A. Rosenzweig Importance of FADD Signaling in Serum Deprivation- and Hypoxia-induced Cardiomyocyte Apoptosis J. Biol. Chem., August 23, 2002; 277(35): 31639 - 31645. [Abstract] [Full Text] [PDF]