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Am J Physiol Heart Circ Physiol 286: H2369-H2375, 2004. First published February 5, 2004; doi:10.1152/ajpheart.00741.2003
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Transcription activator protein 1 mediates {alpha}- but not {beta}-adrenergic hypertrophic growth responses in adult cardiomyocytes

G. Taimor, K.-D. Schlüter, P. Best, S. Helmig, and H. M. Piper

Physiologisches Institut, Justus-Liebig-Universität, 35392 Giessen, Germany

Submitted 4 August 2003 ; accepted in final form 30 January 2004


   ABSTRACT
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 ABSTRACT
METHODS
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 DISCUSSION
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In some models of cardiac hypertrophy, activation of activator protein 1 (AP-1) correlates with growth. However, AP-1 is also activated by stimuli not involved in cardiac growth. This raises the following questions: does AP-1 plays a causal role for cardiomyocyte growth, and is this role model or stimulus dependent? We used a single model to address these questions, i.e., ventricular cardiomyocytes of adult rats, and two growth stimuli, i.e., {alpha}- and {beta}-adrenoceptor agonists [10 µM phenylephrine (PE) and 1 µM isoprenaline (Iso), respectively]. After 1 h of stimulation with PE, mRNA expression of c-Fos and c-Jun was upregulated to 185 ± 32 and 132 ± 13% of control. Fos and Jun proteins formed the AP-1 complex. PE stimulated DNA binding activity of AP-1 to 165 ± 22% of control within 2 h and increased protein synthesis to 161 ± 27% of control and cross-sectional area to 126 ± 4% of control. Inhibition of AP-1 binding activity by cAMP response element (CRE) decoy oligonucleotides abolished both of these growth responses. Iso stimulated AP-1 binding activity to 203 ± 19% of control within 2 h and stimulated protein synthesis to 145 ± 17% of control. However, the growth effect of Iso was not abolished by CRE decoys: Iso increased protein synthesis to 158 ± 17% of control in the presence of CRE. In conclusion, AP-1 is a causal mediator of the {alpha}-adrenergic, but not the {beta}-adrenergic, growth response of cardiomyocytes.

hypertrophy; adrenoceptors; immediate early genes; transcription factors

HYPERTROPHIC GROWTH of the heart is accompanied by an increase in the expression of Fos and Jun family members. This has been documented in several models of cardiac hypertrophy: in hearts after pressure overload in vivo (2, 18) and in isolated hearts or cardiomyocytes after adrenergic stimulation (1, 5). Jun and Fos family members can dimerize to form the transcription factor activator protein 1 (AP-1) (7). Correlations between formation of AP-1 and hypertrophic growth have also been shown in several experimental models, e.g., in hearts of stroke-prone spontaneously hypertensive rats and in hearts of rats infused with angiotensin II or isoprenaline (Iso) (6, 27, 24), as well as in phenylephrine (PE)- or endothelin-stimulated neonatal cardiomyocytes (12). These studies have suggested a causal role of AP-1 in cardiac hypertrophy. However, this seems not to be generally true. In neonatal cardiomyocytes, expression of Fos and Jun family members can be upregulated by ATP without promoting myocyte hypertrophy (28). In adult cardiomyocytes, we previously identified AP-1 as a mediator of nitric oxide-induced apoptotic cell death (25). Additionally, the transcription of atrial natriuretic factor, a marker gene for hypertrophic growth that contains an AP-1 binding site in its promoter, can be stimulated by overexpression of Jun but is decreased by overexpression of Fos (10). These examples show that there are exceptions from the correlation of AP-1 formation with cardiac hypertrophy.

It is unclear whether these differences in the role of AP-1 in cardiac hypertrophy are due to differences in the stimuli producing cardiac hypertrophy or differences in the investigated models. In the present study, our aim was to investigate, on the cardiomyocyte level, whether two different hypertrophic growth stimuli, i.e., {alpha}- and {beta}-adrenoceptor agonists, exhibit differences in AP-1 dependence. To exclude model differences between the stimuli, the same cell type, i.e., cardiomyocytes isolated from the ventricles of adult rats, was used.

Both {alpha}- and {beta}-adrenoceptor agonists have been shown to increase cardiac AP-1 formation and stimulate hypertrophic growth in cardiomyocytes. The respective signaling pathways have been partially analyzed previously. The hypertrophic growth response of adult rat ventricular cardiomyocytes to 10 µM PE is due to {alpha}1-adrenoceptor stimulation, because the growth effect can be antagonized by the {alpha}-adrenoceptor blocker prazosine, but not by inhibition of {beta}-adrenoceptors with propranolol. This growth response is mediated by an increase in the rate of RNA synthesis (16). The hypertrophic growth response to stimulation with 1 µM Iso is mediated through {beta}2-adrenoceptors (29) and requires preexposure of the cells to transforming growth factor-{beta}. This can be achieved by culturing the cells for 6 days in serum-containing medium, because under these conditions an autocrine stimulation of cardiomyocytes with transforming growth factor-{beta} takes place (22). {beta}2-Adrenoceptor stimulation increases RNA stability without changing the rate of RNA synthesis (16, 21). For either case of cardiomyocyte growth stimulation, the role of AP-1-mediated signaling is unclear. To identify the causal role of AP-1, we transformed cardiomyocytes with decoy oligonucleotides that interfere with AP-1 binding at its specific promoter elements (13).


   METHODS
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This investigation conforms with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Pub. No. 85-23, Revised 1996).

Cell isolation and cardiomyocyte cultures. Ventricular cardiomyocytes were isolated from 200- to 250-g male Wistar rats, suspended in basal culture medium, and plated on culture dishes, which were preincubated overnight with 4% FCS in medium 199 as previously described (17). The basal culture (CCT) medium was modified medium 199 including Earle's salts, 2 mM L-carnitine, 5 mM taurine, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 10 µM cytosine-{beta}-D-arabinofuranoside (pH 7.4). At 3 h after the cardiomyocytes were plated, the dishes were washed twice with CCT medium. This results in cultures of an average of ~90% quiescent rod-shaped cells. The cells were directly stimulated, or cardiomyocytes were supplied with CCT medium containing 20% FCS and incubated for 6 days without change of medium.

For adrenergic stimulation, media were replaced by FCS-free CCT medium with the addition of 10 µM PE to 1-day-old cardiomyocytes, 1 µM Iso to 6-day-old cardiomyocytes, or vehicle and incubated at 37°C. Ascorbic acid (100 µM) was added to all cultures as an antioxidant.

Semiquantitative RT-PCR analysis. Total RNA from cardiomyocytes or left ventricles was extracted with RNA-Clean (AGS) as described by the manufacturer. RT reactions were performed for 1 h at 37°C in a final volume of 10 µl using 1 µg of RNA, 100 ng of oligo(dT)15, 1 mM dNTPs, 8 units of RNase block, and 60 units of Moloney murine leukemia virus reverse transcriptase. Aliquots (1.5 µl) of the synthesized cDNA were used for PCR in a final volume of 10 µl containing primer pairs at 1.5 µM, 0.4 mM dNTPs, 1.5 mM MgCl2, and 1 unit of Taq polymerase. Primers were 5'-ACG CCA ACC TCA GCA ACT TCA-3' and 5'-GCT GCG TTA GCA TGA GTT GGC-3' for c-Jun detection, 5'-TGC CAG ATG TGG ACC TGT CTG-3' and 5'-CCA CAG CTT GGT GTG TTT CAC-3' for c-Fos detection, and 5'-GAAGTGTGACGTTGACATCG-3' and 5'-TGCTGATCCACATCTGCTGGA-3' for {beta}-actin. For each assayed gene, annealing temperature and the number of cycles resulting in a linear amplification range were tested. After amplification reaction, products were separated on 5% polyacrylamide gels, stained with ethidium bromide, and photographed under UV illumination. For quantification, density of the DNA fragments was determined by Image Quant (Molecular Dynamics, Krefeld, Germany). A normalization quotient between the respective cDNA and {beta}-actin amplification products was determined.

Retardation assay. For generation of nuclear extracts, cardiomyocytes were homogenized in swelling buffer (10 mM Tris·HCl, pH 7.9, 10 mM KCl, 1 mM MgCl2, and 1 mM DTT). After incubation for 1 h on ice, nuclei were pelleted by centrifugation at 900 rpm for 10 min. Pellets were homogenized in 10 mM Tris·HCl, pH 7.9, 300 mM saccharose, 1.5 mM MgCl2, 1 mM DTT, and 0.3% Triton X-100 and again centrifuged as described above. Pellets were suspended in storage buffer (10 mM HEPES, pH 7.5, 50 mM KCl, 300 mM NaCl, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, and 20% glycerol) on ice for 30 min and centrifuged at 13,000 rpm at 4°C for 5 min. The resulting supernatants were used for retardation assays: TPA response element (TRE) oligonucleotides, containing complementary sequences of the AP-1 binding domain (5'-CATCCGCTTGATGAGTCAGCCGGAA-3') were hybridized, radioactively labeled, and incubated with nuclear extracts in the presence of 1 µg of poly(dIdC) at 30°C for 30 min. The samples were run on 4% native polyacrylamide gels. For supershift assays, subsequent to the incubation of nuclear extracts with the oligonucleotide, 0.5 µg of antibodies was added to the reaction mixture and incubated for another 30 min at 30°C. Dried gels were exposed on PhosphorImager (Molecular Dynamics).

Decoy oligonucleotides. For intracellular scavenging of transcription-promoting, DNA-binding proteins, cardiomyocytes were preincubated for 5 h with 500 nM decoy oligonucleotides containing the specific binding sequences. Phosphorothioate-modified nucleotides at each end of the oligonucleotides increased their stability in cells (exonuclease resistance). For blockage of AP-1-directed transcription, we used a palindromic CRE decoy oligonucleotide as described by Park et al. (13) (5'-TGACGTCATGACGTCATGACGTCA-3'). This oligonucleotide has been shown previously to inhibit AP-1 activity in adult cardiomyocytes (25). As a control, corresponding scrambled (SCR) oligonucleotides (5'-TGACGATCTGCAGTACAGTCGTCA-3') were used.

Incorporation of [14C]phenylalanine. To determine the rate of protein synthesis, incorporation of phenylalanine was measured by exposure of the cultures to [L-14C]phenylalanine (0.1 µCi/ml) for 24 h. Incorporation of radioactivity into acid-insoluble cell mass was determined as described previously (16).

Cell morphology. Myocyte size was determined on micrographs digitalized by a charge-coupled device camera as described elsewhere (20). Five micrographs were taken randomly per sample, and all rod-shaped myocytes in these fields were measured. This results in a total of 60–100 cells that were measured for one condition of one preparation. Width/diameter of myocytes was determined at the widest point of each myocyte using the software program Analysis from SIS. Cross-sectional area of cardiomyocytes was calculated by the following formula: (radius)2 x {pi}.

Statistics. Values are means ± SE from n different culture preparations. Statistical comparisons were performed by one-way analysis of variance, and the Student-Newman-Keuls test was used for post hoc analysis (3). P < 0.05 was considered statistically significant. SPSS software (version 11.5.1, SAS Institute, Cary, NC) was used to analyze data.

Materials. Medium 199 was obtained from Boehringer (Mannheim, Germany), FCS from PAA (Linz, Austria), crude collagenase from Biochrom (Berlin, Germany), antibodies from Santa Cruz Biotechnology, and PCR reagents from Invitrogen (Karlsruhe, Germany).


   RESULTS
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Formation of AP-1 in cardiomyocytes under {alpha}-adrenergic growth stimulation. Stimulation of 1-day-old cardiomyocytes with the {alpha}-adrenoceptor agonist PE (10 µM) upregulated mRNAs coding for Fos and Jun. After 1 h, Fos mRNA was maximally increased by 85 ± 23% and Jun mRNA by 32 ± 13% of control (n = 4, P < 0.05 vs. control). In retardation assays, we analyzed whether this increased mRNA expression results in enhanced AP-1 activity. For this purpose, nuclear extracts of cardiomyocytes were prepared and tested for activity of AP-1 binding to its consensus binding site TRE. AP-1 binding activity increased to 132 ± 10% of control within 1 h after the addition of PE (n = 5, P < 0.05 vs. unstimulated, time-matched controls; Fig. 1). Longer incubation times increased AP-1 binding activity to 177 ± 29% of control after 4 h (n = 5, P < 0.05 vs. unstimulated, time-matched controls). Composition of PE-induced AP-1 was analyzed in supershift assays (Fig. 2A). Panselective Jun and Fos antibodies blocked the AP-1 shift when added to the nuclear extracts (Fig. 2A, lanes 4 and 5). Among the specific antibodies for Jun and Fos family members, c-Jun, JunB, c-Fos, and FosB antibodies reduced the AP-1 shift (Fig. 2A, lanes 8, 9, 13, and 14), whereas JunD and Fra1 antibodies had no influence on AP-1 binding activity induced by PE.



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  		Fig. 1. Activation of activator protein 1 (AP-1) binding by phenylephrine (PE). Cardiomyocytes were incubated in the presence of 10 µM PE for up to 4 h. Nuclear extracts of the cells were prepared at 0.5, 1, 2, and 4 h. These extracts were tested in retardation assays using radioactively labeled TPA response element (TRE) oligonucleotides with the specific binding sequence for AP-1. A: retardation gel. Time-dependent induction of AP-1 binding results in increased band shifts. B: quantification of AP-1 binding activity. AP-1 shifts in retardation gels were analyzed densitometrically. Data are expressed as percent increase relative to untreated controls. Values are means ± SE of 5 independent culture preparations. *Significantly different from unstimulated controls (P < 0.05).

 


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  		Fig. 2. AP-1 complex is composed of Jun and Fos. Cardiomyocytes were stimulated for 2 h with 10 µM PE. Nuclear extracts of the cells were prepared and tested for their AP-1 binding activity to the TRE oligonucleotide. A: before loading, samples on a retardation gel for antibodies panselective (lanes 4 and 5) or specific for Jun or Fos family members (lanes 6–15) were added to the binding reaction. B: AP-1 binding activity induced by PE was analyzed in cardiomyocytes transformed with cAMP response element (CRE) decoy oligonucleotides. C, control; F, free probe; TRE, oligonucleotide without nuclear extract.

 
Function of AP-1 in cardiomyocytes under {alpha}-adrenergic growth stimulation. To investigate the functional role of AP-1 activation in the {alpha}-adrenergic growth response, cardiomyocytes were transformed with CRE decoy oligonucleotides, which contain AP-1 binding sites. In CRE-transformed cardiomyocytes, activation of AP-1 binding by PE was inhibited (Fig. 2B, lane 18). Under these conditions, the rate of protein synthesis was determined. Stimulation of cardiomyocytes with PE for 24 h increased the rate of protein synthesis to 161 ± 27% of control (n = 4, P < 0.05 vs. control; Fig. 3). Preincubation of cardiomyocytes with CRE decoy oligonucleotides abolished the PE-induced increase in the rate of protein synthesis (123 ± 13% of control in CRE + PE-treated cells vs. 126 ± 19% in CRE-treated cells, n = 4; not significant). Transformation of cardiomyocytes with SCR oligonucleotides did not inhibit the growth response after stimulation with PE (143 ± 12%, n = 4, P < 0.05 vs. control).



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  		Fig. 3. AP-1-decoy oligonucleotides inhibit stimulation of protein synthesis stimulated by PE. Incorporation of [14C]phenylalanine (14C-Phe) during 24 h of stimulation with 10 µM PE was determined under control conditions and after 5 h of preincubation with 500 nM CRE decoy oligonucleotides containing specific binding sites for AP-1 or with a control scrambled (SCR) oligonucleotide. Data are expressed as percent increase relative to untreated controls. Values are means ± SE of 4 independent culture preparations. *Different from unstimulated controls, P < 0.05.

 
As an additional marker of hypertrophic growth, cell size of cardiomyocytes was determined. Enlargement of cardiomyocytes after stimulation with PE is shown in Fig. 4A. This response is abrogated in cardiomyocytes transformed with CRE decoy oligonucleotides, but not in SCR-transformed cells. Calculation of cross-sectional area of myocytes revealed an increase to 126 ± 4% under stimulation with PE (n = 6, P < 0.05 vs. control; Fig. 4B). This growth response to PE was abolished in cardiomyocytes transformed with CRE oligonucleotides (104 ± 3%). Transformation with SCR oligonucleotides did not inhibit the enlargement of myocytes due to PE (129 ± 4%, P < 0.05 vs. control).



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  		Fig. 4. AP-1 decoy oligonucleotides inhibit PE-induced enlargement of cell size. After cardiomyocytes were incubated with PE for 24 h, cells were photographed and their cross-sectional area was calculated. A: representative photographs of cardiomyocytes. B: quantification of cross-sectional area of cardiomyocytes. Data are expressed as percent increase relative to untreated controls. Values are means ± SE of 6 independent culture preparations. Size of ~480 cells per condition was determined. *Different from unstimulated controls, P < 0.05.

 
This demonstrates that intracellular scavenging of AP-1 with CRE decoy oligonucleotides inhibits the {alpha}-adrenergic growth stimulation in cardiomyocytes. It identifies AP-1 as an essential mediator in this signaling pathway.

Formation and function of AP-1 in cardiomyocytes under {beta}-adrenergic growth stimulation. In 6-day-old cardiomyocyte cultures, {beta}-adrenergic stimulation promotes hypertrophic growth. We investigated whether AP-1 activation is also part of this signaling cascade resulting in hypertrophic growth. Incubation of these cultures with 1 µM Iso increased AP-1 binding activity to 203 ± 19% of control within 2 h (n = 5, P < 0.05 vs. control; Fig. 5). This AP-1 complex was composed of Jun and Fos family members, because preincubation of nuclear extracts with panselective antibodies for these protein families blocked the AP-1 binding in retardation assays (Fig. 6A, lanes 4 and 5). Antibodies specific for c-Jun, JunD, c-Fos, and Fra1 reduced the AP-1 shift (Fig. 6A, lanes 8, 10, 12, and 15), whereas antibodies specific for JunB and FosB had no influence on AP-1 binding activity induced by Iso (Fig. 6A, lanes 9 and 14). AP-1 binding activity induced by Iso could be intracellularly scavenged by transformation of cardiomyocytes with CRE decoy oligonucleotides. This is demonstrated in retardation assays, because nuclear extracts from these cells do not form AP-1 shifts (Fig. 6B, lane 18).



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  		Fig. 5. Activation of AP-1 binding by isoprenaline (Iso). Cardiomyocytes were incubated in the presence of 1 µM Iso for up to 2 h. At 0.5, 1, and 2 h, nuclear extracts of the cells were prepared. These extracts were tested in retardation assays using radioactively labeled TRE oligonucleotides with the specific binding sequence for AP-1. A: retardation gel. Time-dependent induction of AP-1 binding results in increased band shifts. C, control. B: quantification of AP-1 binding activity. AP-1 shifts in retardation gels were analyzed densitometrically. Data are expressed as percent increase relative to untreated controls. Values are means ± SE of 5 independent culture preparations. *Different from unstimulated controls, P < 0.05.

 


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  		Fig. 6. AP-1 complex is composed of Jun and Fos. Cardiomyocytes were stimulated for 2 h with 1 µM Iso. Nuclear extracts of the cells were prepared and tested for their AP-1 binding activity to the TRE oligonucleotide. Before samples were loaded on a retardation gel, antibodies panselective (lanes 4 and 5) or specific for Jun or Fos family members (lanes 6–15) were added to the binding reaction. B: AP-1 binding activity induced by PE was analyzed in cardiomyocytes transformed with CRE decoy oligonucleotides.

 
ISO increased the rate of protein synthesis to 145 ± 17% of control during 24 h of incubation (n = 8, P < 0.05 vs. control; Fig. 7). In contrast to the {alpha}-adrenergic effect, {beta}-adrenergic stimulation of hypertrophic growth was not inhibited by preincubation of cardiomyocytes with CRE decoy oligonucleotides (158 ± 17% of control, n = 8, P < 0.05 vs. CRE-treated control). This demonstrates that formation of AP-1 under {beta}-adrenergic stimulation is not involved in the growth processes induced by these receptors. This conclusion is supported by our findings that Iso stimulated AP-1 also in 1-day-old cardiomyocytes, which do not exhibit a growth response to {beta}-adrenergic stimulation.



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  		Fig. 7. AP-1 decoy oligonucleotides do not inhibit stimulation of protein synthesis by ISO. Incorporation of [14C]phenylalanine during 24 h of stimulation of cardiomyocytes with 1 µM Iso was determined under control conditions and after 5 h of preincubation with 500 nM CRE decoy oligonucleotides containing specific binding sites for AP-1. Data are expressed as percent increase relative to untreated controls. Values are means ± SE of 8 independent culture preparations. *Different from unstimulated controls, P < 0.05; #different from CRE-treated controls, P < 0.05.

 

   DISCUSSION
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Although AP-1 activation often correlates with hypertrophic growth conditions, its induction does not always result in the development of cardiac hypertrophy. The central question of this study was, therefore, whether AP-1 plays a causal role in either form of growth stimulation in one and the same cell type, i.e., in ventricular cardiomyocytes of the rat, under {alpha}- or {beta}-adrenergic stimulation. The main finding is that AP-1 is a causal mediator of the {alpha}-adrenergic, but not the {beta}-adrenergic, growth response of cardiomyocytes.

Stimulation with {alpha}-adrenoceptor agonists is known to induce the immediate early genes Fos and Jun, AP-1, and hypertrophic growth in cardiac myocytes (9, 23). Recently, overexpression of dominant-negative c-Jun mutants in neonatal cardiomyocytes was shown to inhibit the {alpha}-adrenergic growth response (12). This suggests causal involvement of AP-1 in the growth response, because c-Jun can dimerize with itself or other factors such as Fos to build AP-1. However, dimerization with different Jun and Fos family members can change the binding and transcription activation potential of AP-1 (7, 26). Jun can also interact with diverse transcriptional repressors such as TG-interacting factor or Ski or with other transcription factors, e.g., signal transducer and activator of transcription 3 (14, 15, 23). Depending on these binding partners, c-Jun changes its promoter specificity and the genes it regulates. The cited study (12) left open, therefore, whether the hypertrophy-related function of c-Jun is indeed due to formation of AP-1 under {alpha}-adrenergic stimulation. We now demonstrate in adult cardiomyocytes that AP-1 binding is activated on {alpha}-adrenergic stimulation within 2 h. The AP-1 complex is composed of Jun/Fos heterodimers. This was analyzed by use of specific antibodies that blocked AP-1 binding in retardation assays. Transformation of cardiomyocytes with decoy oligonucleotides, which contain the AP-1 consensus binding site and inhibit AP-1 binding activity in cardiomyocytes, blocked the {alpha}-adrenergic-stimulated growth response. For the first time, these experiments provide direct evidence that AP-1 is an essential mediator for {alpha}-adrenergic growth stimulation in cardiomyocytes.

As a second point of our analysis, we investigated whether AP-1 is also involved in {beta}-adrenergic growth stimulation. This was done because infusion of hearts with {beta}-adrenoceptor agonists is known to induce the immediate early genes Fos and Jun, AP-1, and hypertrophic growth in cardiomyocytes in a manner similar to that induced by {alpha}-adrenergic agonists (1, 24). We now demonstrate that {beta}-adrenergic stimulation of adult cardiomyocytes indeed activates AP-1 binding with kinetics similar to those seen under {alpha}-adrenergic stimulation and that the AP-1 complex also contains Fos and Jun subunits. However, intracellular inhibition of AP-1 binding by transformation of cardiomyocytes with AP-1 decoy oligonucleotides did not block the {beta}-adrenergic growth response. This demonstrates that, although AP-1 is activated, hypertrophic growth under {beta}-adrenergic stimulation is not dependent on AP-1 activation.

Although correlations of AP-1 induction and hypertrophic growth have been shown in several models of hypertrophy, including Iso-infused rats (13), and also in the present study under {alpha}- and {beta}-adrenergic stimulation, functional involvement of AP-1 in hypertrophic growth could be demonstrated here only for {alpha}-adrenergic stimulation. Our findings raise the following question: why does activation of AP-1 not always induce similar pathways, thereby stimulating hypertrophic growth in cardiomyocytes? In this context, it must be noted that activation of AP-1 in this and other studies has been determined by the binding of AP-1 to its consensus binding element TRE. In the intact cell, however, promoter specificity of AP-1 can be influenced by the composition of the AP-1 complex itself or by other transcription factors interacting with AP-1. In this study, we found that the AP-1 complexes were Jun/Fos heterodimers under {alpha}- and {beta}-adrenergic growth stimulation. Looking at the composition of AP-1 dimers in further detail, we observed c-Jun and c-Fos in both complexes. However, the complexes differ in other subunits. Under {alpha}-adrenergic stimulation, JunB and FosB are additionally found, whereas in {beta}-adrenergic-stimulated complexes, JunD and Fra1 were observed. These differences in the AP-1 complex may be a reason for the different roles of AP-1 in {alpha}- or {beta}-adrenergic growth stimulation. Activation of further transcription factors may also cause the different outcome of AP-1 activation. Potential candidates influencing the action of AP-1 are the transcription factors nuclear factor of activated T cells 3 (NFAT-3) and GATA-4. Both are able to associate with AP-1 (8, 19) and, in doing so, change the pattern of AP-1-driven gene expression. NFAT-3 and GATA-4 are found activated in {alpha}-adrenergic-stimulated cardiomyocytes (4, 11). The simultaneous activation of these factors together with AP-1 may result in AP-1-dependent hypertrophic growth under {alpha}-adrenergic stimulation. Another possibility is the necessity of other pathways acting in parallel to that activated by AP-1 to stimulate hypertrophic growth.

In summary, we identified the transcription factor AP-1 as an essential mediator of {alpha}-adrenergic-induced hypertrophic growth in adult cardiomyocytes. However, activation of AP-1 does not generally result in hypertrophic growth as shown by {beta}-adrenergic stimulation. Mechanisms underlying these different outcomes of AP-1 activation remain to be elucidated.


   GRANTS
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This study was supported by Philip Morris USA.


   ACKNOWLEDGMENTS
 
The authors thank Daniela Schreiber and Birgit Störr for excellent technical assistance.

This work is part of the thesis submitted by P. Best.


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. Taimor, Physiologisches Institut, Justus-Liebig-Universität, Aulweg 129, 35392 Giessen, Germany (E-mail: Gerhild.Taimor{at}physiologie.med.uni-giessen.de).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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