Cardiovascular Research

Cardiovascular Research 2000 45(2):410-417; doi:10.1016/S0008-6363(99)00351-X
© 2000 by European Society of Cardiology

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Copyright © 2000, European Society of Cardiology

Central role for ornithine decarboxylase in β-adrenoceptor mediated hypertrophy

Klaus-Dieter Schlüter^a,*, Karen Frischkopf^a, Markus Flesch^b, Stephan Rosenkranz^b, Gerhild Taimor^a and Hans Michael Piper^a

^aPhysiologisches Institut, Justus-Liebig-Universität, Aulweg 129, D-35392 Giessen, Germany
^bKlinik III für Innere Medizin der Universität zu Köln, Cologne, Germany

^* Corresponding author. Tel.: +49-641-994-7234; fax: +49-641-994-7239 Klaus-Dieter.Schlueter{at}physiologie.med.uni-giessen.de

Received 5 February 1999; accepted 24 September 1999

	Abstract

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Objective: TGF-β stimulation of cardiac myocytes induces a hypertrophic responsiveness to β-adrenoceptor stimulation. This study investigates whether this β-adrenoceptor mediated effect depends on induction of ornithine decarboxylase (ODC). Methods: Isolated adult ventricular cardiomyocytes from rats were used as an experimental model. Cells were either cultured in 20% (v/v) FCS to activate autocrine released TGF-β or used without pre-treatment. The hypertrophic response was characterized by an increased ¹⁴C-phenylalanine incorporation, RNA and protein mass or by an increased expression of atrionatriurectic factor and ODC. The results on cell cultures were compared to those achieved by isoprenaline perfused mice hearts from transgenic mice overexpressing TGF-β₁. Results: ODC activity and expression increased within 2 h in TGF-β₁ pre-treated cells under isoprenaline. In the presence of ODC inhibitors (-methylornithine or difluoromethylornithine) this increase remained absent and the increases in ¹⁴C-phenylalanine incorporation, protein and RNA mass under isoprenaline were abolished. In cells not exposed to TGF-β no induction of ODC was observed. Isoprenaline also induced ODC in isolated perfused ventricles from transgenic mice overexpressing TGF-β₁, but not in ventricles from their nontransgenic counterparts. Conclusions: This study shows first, a pivotal role for ODC induction in the hypertrophic response of cardiomyocytes to β-adrenoceptor stimulation and second, that ODC induction in vivo and in vitro requires pre-treatment of cardiomyocytes with TGF-β. It is concluded that TGF-β induces a hypertrophic responsiveness to β-adrenoceptor stimulation that is characterized by ODC induction.

KEYWORDS Adrenergic (ant)agonist; Cytokines; Hypertrophy; Myocytes; Signal transduction

	1 Introduction

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Hyperactivation of the sympathetic nerve system plays an important role in the pathogenesis of myocardial hypertrophy and heart failure [1]. It is generally accepted that excess of catecholamines causes myocardial hypertrophy in vivo and in vitro [2–4]. When adult ventricular cardiomyocytes are investigated in vitro, the hypertrophic effect of catecholamines depends on -adrenoceptor stimulation. Selective -adrenoceptor stimulation causes an increase in protein synthesis, but selective β-adrenoceptor stimulation does not [2,4]. This is contrasted by the in vivo situation, in which selective β-adrenoceptor stimulation can also cause myocardial hypertrophy [5]. On adult cardiomyocytes in vitro, specific culture procedures restore the in-vivo-like behavior in regard to the hypertrophic responsiveness to β-adrenoceptor stimulation, i.e., pre-cultivation of cardiomyocytes in the presence of low concentrations of isoprenaline [6] or pre-cultivation in the presence of fetal calf serum [7] induces hypertrophic responsiveness of adult ventricular cardiomyocytes to β-adrenoceptor stimulation. These culture systems, therefore, permit to analyze the cellular mechanisms by which β-adrenoceptor stimulation induces myocardial hypertrophy.

In cells with hypertrophic responsiveness to β-adrenoceptor stimulation, the hypertrophic effects of - and β-adrenoceptor stimulation are both characterized by an elevation of total RNA. Approximately 85% of total RNA mass represents ribosomal RNA and thereby the protein synthesis capacity of the cell [8]. Therefore, elevation of total RNA is a key factor in myocardial hypertrophy, that enables the cell to maintain a high rate of protein synthesis. In case of -adrenoceptor stimulation, RNA elevation is accompanied by an increased ¹⁴C-uridine incorporation. This indicates de novo synthesis of RNA. In case of β-adrenoceptor stimulation, however, RNA elevation is not accompanied by an increased ¹⁴C-uridine incorporation [7]. That let us conclude that the mechanism by which β-adrenoceptor stimulation elevates RNA mass seems to be due to a decrease of RNA degradation. Since polyamines are known to stabilize RNA [9] this may explain the observed elevation of RNA in the absence of elevated RNA synthesis.

Ornithine decarboxylase (ODC) represents the rate limiting enzyme of the polyamine metabolism. An induction of ODC is causally involved in the mechanism by which β-adrenoceptor stimulation elevates cellular RNA mass [10]. β-Adrenoceptor mediated hypertrophy in vivo is, indeed, accompanied by induction of ODC [5]. The present study investigates on the cellular level, whether ODC is induced under β-adrenoceptor stimulation in cardiomyocytes with hypertrophic responsiveness to β-adrenoceptor stimulation and if this induction is responsible for the hypertrophic effect of β-adrenoceptor stimulation in this cell system.

This study was performed in a well-described in vitro system in which ventricular cardiomyocytes isolated from adult rats were cultured for 6 days in serum supplemented culture media. These cells exhibit hypertrophic responsiveness to β₂-adrenoceptor stimulation [11]. It was previously shown that induction of hypertrophic responsiveness depends on the autocrine stimulation with TGF-β of the cultured cells [12]. In accordance with the aforementioned aims, we have analyzed whether pre-stimulation with TGF-β also promotes the β-adrenergic inducibility of ODC in cardiomyocytes. The cell cultures system used in this study exhibits resemblance to the situation of the myocardium in vivo at the transition from compensated to non-compensated cardiac hypertrophy where an increased expression of TGF-β₁ indicates an activation of the local TGF-β system [13]. We were interested to compare the cell culture data to the in vivo situation. We chose to compare hearts from transgenic mice over-expressing TGF-β₁ with hearts from normal mice. The hearts were perfused and responsiveness to β₂-adrenoceptor stimulation was studied in respect to ODC activity and expression. As a molecular marker for myocardial hypertrophy the expression of ANF was also analyzed in these hearts.

	2 Methods

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
All animal studies were performed in accordance with guidelines described in the NIH Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH, publication no. 85-23, revised 1996).

2.1 Cell culture
Ventricular heart muscle cells were isolated from 200- to 250-g male Wistar rats as previously described [4]. Isolated cells were suspended in FCS-free culture medium and plated at a density of 1.4x10⁵ elongated cells/35-mm culture dish (Falcon type 3001). The culture dishes had been pre-incubated overnight with 4% fetal calf serum (FCS) in medium 199. The basic culture medium consisted of medium 199 with Earle's salts, 5 mmol/l creatine, 2 mmol/l L-carnitine, 5 mmol/l taurine, 100 I.U./ml penicillin, and 100 µg/ml streptomycin. To prevent growth of nonmyocytes, media were also supplemented with 10 µmol/l cytosine-β-D-arabinofuranoside.

Four hours after plating, cultures were washed twice with culture medium to remove round and nonattached cells and either supplied with FCS-free experimental media, in which cells were incubated for up to 24 h at 37°C or with basic culture medium supplemented with 20% fetal calf serum, in which cells were incubated for 6 days at 37°C. In both cases, the experiments were carried out in basic culture medium under serum free conditions (control), with addition of phenylephrine, isoprenaline or dibutyryl-cyclo-AMP, at concentrations indicated. Ascorbic acid (100 µmol/l) was added to all cultures as an antioxidant.

2.2 Treatment protocol for cardiomyocytes (TGF-β exposure)
Adult ventricular cardiomyocytes from rat were isolated, plated on culture dishes, and cultured either for 24 h under serum-free conditions or for 6 days in presence of 20% (v/v) fetal calf serum (FCS) and thereafter for 24 h under serum-free conditions. TGF-β activity was determined in both culture systems. After 24 h, the media of cardiomyocytes cultured under serum-free conditions had low TGF-β activities (0.3±0.3 ng active TGF-β/ml) and those cultured in presence of high FCS had high TGF-β activities (1.5±0.2 ng active TGF-β/ml; n=4, P<0.05 vs. serum-free cultures).

2.3 Incorporation of ¹⁴C-phenylalanine and changes in cellular protein and RNA mass
Incorporation of phenylalanine into cells was determined by exposing cultures to L-¹⁴C-phenylalanine (0.1 µCi/ml) for 24 h and determination of the incorporation of radioactivity into acid-insoluble cell mass as described before [7]. As shown previously ¹⁴C-phenylalanine incorporation increases constantly for 36 h under these conditions [7]. Nonradioactive phenylalanine (0.3 mmol/l) was added to the medium to minimize variations in the specific activity of the precursor pool responsible for protein synthesis. In incorporation studies, experiments were terminated by removal of the supernatant medium from the cultures and washed three times with ice-cold phosphate-buffered saline (PBS; composition in mmol/l: 1.5 KH₂PO₄, 137 NaCl, 2.7 KCl, and 1.0 Na₂HPO₄, pH 7.4). Subsequently, ice-cold 10% (w/v) trichloroacetic acid was added. After storage overnight at 4°C, the acid was removed from the dishes. Radioactivity contained in this acid fraction was taken to present the intracellular precursor pool. The dishes were then washed twice with ice-cold PBS. The remaining precipitate on the culture dishes was dissolved in 1 M NaOH–0.01% (w/v) sodium dodecylsulphate (S.D.S) by an incubation for 2 h at 37°C. In these samples protein contents [14] and DNA contents [15] were determined, and the radioactivity was counted. RNA was determined from an aliquot of these samples after precipitation with an equal volume of 10% (w/v) perchloric acid in the remaining supernatant [8]. The RNA content was also expressed relative to the DNA content of the samples.

2.4 TGF-β activity
TGF-β activity in the media was determined as described [16] by the growth inhibitory effect of TGF-β on the proliferation of microvascular endothelial cells. Isolation and cultivation of microvascular endothelial cells was described earlier [17]. Supernatants of the cell culture media were diluted and added to subconfluent monolayers of microvascular endothelial cells plated on 96-well dishes. After 48-h protein contents of the wells were determined. The cultures were fixed by addition of 3% (v/v) paraformaldehyde, stained by replacement of the paraformaldehyde by 1 (% v/v) methylene blue dissolved in TBE buffer (composition: in mM 89 tris(hydroxymethyl)aminomethane (Tris), 89 borate, 2 EDTA). Finally, the dishes were washed twice and incubated with ethanol–HCl (1:1, v/v). The density of the cultures was determined at 630 nm. The growth inhibitory effect was compared to a standard curve using activated TGF-β isolated from porcine platelets (British Biotechnology Products, Oxon, UK). The results are expressed as ng active TGF-β/ml. Specificity of the inhibitory effect of media supernatants to TGF-β was further demonstrated by the use of a neutralizing antibody to TGF-β₁ (British Biotechnology Products), which abolished the growth inhibitory effect.

2.5 ODC activity
The activity of ODC was determined as described by Meilhoc et al. [18]. Cardiomyocyte cultures were washed twice with ice-cold phosphate buffered saline, scrapped off, and centrifuged for 2-min at 3000 g. The pellet was resuspended in lysis buffer (composition in mM: 10 tris(hydroxymethyl)aminomethane (Tris)·HCl, 250 sucrose, 5 dithiothreitol, 1 EDTA, 0.5 pyridoxalphosphate, pH 7.3) and homogenized by sonification. The homogenate was centrifuged again for 2 min at 3000 g. This supernatant was used to determine ODC activity. ODC activity from ventricles of mice hearts was determined by homogenization of ventricle samples in lysis buffer (composition as above) using a ultravox homogenizer, and the suspension was used as described for cardiomyocyte cultures. ODC activity in the supernatants was determined by the release of ¹⁴CO₂ from 1-¹⁴C-ornithine. A 50-µl volume of the supernatant and 250 µl of lysis buffer including 0.1 µCi/ml 1-¹⁴C-ornithine and 3 mM ornithine was incubated for 30 min in a closed tube equipped with filter-paper wetted in 1 M KOH to trap released CO₂. The incubation was terminated by incubation over night at 4°C to adsorb released ¹⁴CO₂. To estimate non-specific CO₂ release during the incubation, blank tubes were set up and the nonspecific release was subtracted from sample release. The filter papers were removed, and trapped ¹⁴CO₂ was counted by liquid scintillation.

2.6 RT-PCR
Total RNA from cardiomyocytes and ventricular tissue was extracted with RNA-Clean (AGS, Heidelberg, Germany) as described by the manufacturers. Reverse transcription reactions were performed for 1 h at 37°C in a final volume of 10 µl RNA, 100 ng oligo(dT)₁₅ (Boehringer Mannheim, Germany), 1 mM dNTPs (Gibco-BRL,) 8 U RNAse Block (Promega, Mannheim, Germany) and 60 U M-MLV reverse transcriptase (Gibco-BRL). Aliquots (1.5 µl) of the synthesized cDNA were used for polymerase chain reaction (PCR) in a final volume of 10 µl 1.5 µM of primer pairs, 0.4 mM dNTPs, 1.5 mM MgCl₂, and 1 U Taq-polymerase (Gibco-BRL). Amplification was performed under the following cycle conditions: 1 min 93°C, 1 min 57°C, 3 min 72°C. For each assayed gene the number of cycles resulting in a linear amplification range was tested. Oligonucleotide primers were synthesized by Gibco-BRL and had the following sequences: β-actin sense 5'-GAAGTGTGACGTTGACATCCG-3' and antisense 5'-TGCTGATCCACATCTGCTGGA-3', for amplification between bp 2731 and 3081 of rat β-actin gene [19]; ODC sense 5'-GAAGATGAGTCAAACGAGCA-3' and antisense 5'-AGTAGATGTTTGGCCTCTGG-3', for amplification between bp 5777 and 6352 [20]; ANF sense 5'-ATGGGCTCCTTCTCCATCAC-3' and antisense TCTTCGGTACCGGAAGCT-3', for amplification between bp 64 and 520 [21]; c-fos sense 5'-TGCCAGATGTGGACCTGTCTG-3' and antisense 5'-CCACAGCTTGGTGTGTTTCAC-3', for amplification between bp 999 and 1390 [22].

After amplification reaction products were separated on 5% polyacrylaminde gels, stained with ethidium bromide and photographed under UV illumination. For quantification density of the DNA fragments were determined by IMAGE QUANT (Molecular Dynamics, Krefeld, Germany). The results for c-fos, ODC, and ANF expression were normalized for equal loading by β-actin amplification.

2.7 Transgenic animals
The trangenic animals used in this study have been described in detail by Sanderson et al. [23]. They over-express TGF-β₁ in which Cys²²³ and Cys²²⁵ codons were replaced with serine codons, resulting in preferential secretion of the mature form of TGF-β₁ [24]. Their TGF-β₁ plasma concentrations have been documented from the second week on [23]. All animals used in this study were used at an age of 8 weeks and compared to non-transgenic control animals from the same strain.

2.8 Heart perfusion
Hearts from mice were isolated as described above for rat hearts to prepare cardiomyocytes preparations and quickly connected to a Langendorff perfusion system. Hearts were perfused at 37°C and at a flow-rate of 0.5 ml/min. The composition of the perfusion buffer was as follows (mM): 145 NaCl, 25 NaHCO₃, 5.9 KCl, 1.2 mM KH₂PO₄, 1.2 MgSO₄, 2.5 CaCl₂, 0.5 EDTA, 0.1 ascorbic acid, 5.5 glucose, 0.001 isoprenaline, 0.01 atenolol, gassed with 95% O₂–5% CO₂. Hearts were allowed to beat spontaneously. After an initial equilibration period (5 min) the first heart was removed and taken as time 0. The perfusion was prolonged for up to 2 h. Release of lactate dehydrogenase was monitored continuously as described before [25] to control sarcolemmal integrity throughout the whole perfusion time.

2.9 Statistics
Data are given as means±S.E.M. from n different culture preparations. Statistical comparisons were performed by one-way analysis of variance and use of the Student–Newman–Keuls test for post hoc analysis [26]. Differences with P<0.05 were regarded as statistical significant. All data analyses were computed using SAS software, version 6.11 (SAS Institute, Cary, NC, USA).

2.10 Materials
Falcon tissue culture dishes were obtained from Becton-Dickinson (Heidelberg, Germany). Boehringer Mannheim was the source for glutamine-free medium 199 and fetal calf serum. Cytosine-β-D-arabinofuranoside, L-carnitine, creatine, taurine, L-phenylephrine hydrochloride, ICI 118,551, and DL-isoproterenol hydrochloride were obtained from Sigma (Deisenhofen, Germany). All other chemicals were of analytical grade.

	3 Results

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
3.1 Effect of active TGF-β on hypertrophic responsiveness and ODC induction under isoprenaline
On cardiomyocytes cultured with low TGF-β activity, isoprenaline did not increase ¹⁴C-phenylalanine incorporation, indicating hypertrophic growth of cardiomyocytes, and did not induce ANF expression, a molecular marker of hypertrophy (Fig. 1). In contrast, isoprenaline increased ¹⁴C-phenylalanine incorporation and ANF expression in cardiomyocytes pre-exposed to high TGF-β activity (Fig. 1).

View larger version (15K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Incorporation of 14C-phenylalanine and expression of atrionatriuretic factor (ANF) in cardiomyocytes cultured either for 24 h under serum free conditions with low TGF-β activity in absence (C) and presence of isoprenaline (ISO, 1 µM) or in cardiomyocytes cultured for 6 days in high TGF-β activity and subsequently for 24 h under serum free conditions in the absence or presence of ISO. In case of 14C-phenylalanine incorporation, 14C-phenylalanine was included in the medium and its incorporation into the protein fraction was calculated as 14C-phenylalanine per µg DNA and expressed as percentage of the controls. S.E.M. bars of the controls represent variations between four different culture preparations. In case of ANF expression, cells were harvested at the end of the incubation time and the mRNA content of ANF was determined by RT-PCR (23 cycles). In each case, data are given as percent values relative to the controls and the S.E.M. bars of the controls represent the variations between four different culture preparations. *, P<0.05 vs. C; n=4 cultures.

 
ODC induction under isoprenaline was investigated next. On cells cultured under low TGF-β activity, isoprenaline did not increase ODC activity or expression (Fig. 2). On cells pre-cultivated with high TGF-β₁-activity, however, enzyme activity and expression increased within 2 h after addition of isoprenaline (Fig. 2). In these cells, actinomycin D, a transcriptional inhibitor, attenuated ODC induction under isoprenaline (Table 1), indicating that the increase in ODC mRNA under isoprenaline requires transcriptional activation. The effect of isoprenaline on ODC induction could be mimicked by dibutyryl-cAMP and antagonized by the β₂-adrenoceptor antagonist ICI 118,551 (Table 1). Two different ODC inhibitors, namely -methylornithine and difluoromethylornithine (DFMO), abolished ODC induction in these cells (Table 1). In contrast, stimulation of -adrenoceptors by phenylephrine did not induce ODC activity (Table 1).

View larger version (16K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Expression and activity of ornithine decarboxylase (ODC) in cardiomyoytes following treatment with isoprenaline (1 µM). Cells were cultured as described for Fig. 1. ODC enzyme activity was determined in cell extracts from the respective cultures as the release of 14CO2 from 1-14C-ornithine and expressed as mU/min. ODC mRNA expression was determined by RT-PCR (25 cycles). Data are means±S.E.M. from n=4 cultures. *, P<0.05 vs. 0 h; n=4 cultures.

 

View this table: [in this window] [in a new window]   Table 1 ODC activity in cardiomyocytes pre-treated with TGF-β for 6 daysa

 
3.2 Causal relationship between ODC induction and hypertrophy
The above reported results show that β-adrenoceptor stimulation is accompanied by ODC induction. Whether ODC induction and induction of hypertrophy are causally linked was investigated by pharmacological inhibition of ODC. In this set of experiments, cardiomyocytes with hypertrophic responsiveness to β-adrenoceptor stimulation were used, namely 6-day-old cultures with high TGF-β activity. -Methylornithine dose-dependently attenuated the increase in protein and RNA mass in response to isoprenaline (Fig. 3). The inhibitory effect of -methylornithine on enlargement of protein and RNA mass was accompanied by an inhibition of ¹⁴C-phenylalanine incorporation (Table 2). A similar result was obtained by use of DFMO that attenuated ¹⁴C-phenylalanine incorporation in response to isoprenaline. In contrast, under -adrenoceptor stimulation by phenylephrine, the presence of -methylornithine did not attenuate this hypertrophic effect (Table 2).

View larger version (14K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Influence of -methylornithine on isoprenaline induced increases of protein and RNA mass. Cardiomyocytes were pre-cultured for 6 days in the presence of high TGF-β activity and subsequently for 24 h under serum free conditions with isoprenaline (1 µM) and addition of -methylornithine at concentrations as indicated. Protein and RNA mass of the cultures were calculated relative to the DNA content of the dishes and expressed as percentage relative to untreated controls. Data are means±S.E.M. of n=4 cultures. *, P<0.05 vs. without -methylornithine.

 

View this table: [in this window] [in a new window]   Table 2 Influence of ODC inhibitors on 14C-phenylalanine incorporation in cardiomyocytes pre-treated with TGF-β for 6 daysa

 
3.3 Effect of β-adrenoceptor stimulation on ODC induction in isolated perfused mice hearts
To investigate, whether the observed induction of hypertrophic responsiveness to β-adrenoceptor stimulation by TGF-β on cultured cardiomyocytes bears relevance to the whole heart, we determined the expression of ANF, a molecular marker for the onset of myocardial hypertrophy in hearts from transgenic mice over-expressing TGF-β₁ and non-transgenic control mice from the same strain. Hearts from transgenic animals exhibited already elevated ANF mRNA levels compared to non-transgenic animals (Fig. 4). Hearts were connected to a Langendorff perfusion system and perfused with a constant flow (0.5 ml/min) for 2 h. Isoprenaline (1 µM) and, to abolish β₁-adrenoceptor stimulation, the β₁-adrenoceptor antagonist atenolol (10 µM) were added to the perfusion system. Atenolol was added because on the isolated cell system atenolol did not abolished the hypertrophic response to isoprenaline in responsive cells and the perfusion conditions were aimed to mimic closely those previously used on the cell culture system [11]. In response to isoprenaline and atenolol ANF mRNA contents increased in hearts from transgenic mice but not in control hearts, indicating hypertrophic responsiveness of hearts from transgenic mice. In hearts from transgenic animals and from non transgenic control mice, isoprenaline induced c-fos mRNA, indicating that even the control hearts were able to respond to isoprenaline with expressional changes. Finally, the induction of ODC in whole hearts isolated from transgenic mice over-expressing TGF-β₁ and control mice was investigated. ODC activity and expression was found significantly increased within 2 h in hearts from transgenic animals. In hearts from control mice, however, ODC activity and expression did not increase (Fig. 5).

View larger version (14K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 ANF and c-fos expression in mice ventricles. Hearts from transgenic mice over-expressing TGF-β1 and their non-transgenic counterparts were perfused for 2 h without additions (C, control), or with isoprenaline (ISO, 1 µM) and atenolol (10 µM). The expression of c-fos and ANF was determined by RT-PCR (23 cycles). Data are means±S.E.M. from n=4 hearts. *, P<0.05 vs. control hearts from non-transgenic hearts; #, P<0.05 vs. control hearts from transgenic hearts.

 

View larger version (13K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Expression and activity of ornithine decarboxylase (ODC) in mice ventricles. Hearts were perfused as indicated in Fig. 4. ODC enzyme activity was determined in extracts from the ventricles as the release of 14CO2 from 1-14C-ornithine and expressed as mU/min. ODC mRNA expression was determined by RT-PCR (25 cycles). Data are means±S.E.M. from n=4 hearts. *, P=0.05 vs. control hearts from non-transgenic and transgenic mice.

 

	4 Discussion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
This study investigated whether induction of ODC, the rate limiting enzyme of the polyamine metabolism, is causally involved in the hypertrophic response to β-adrenoceptor stimulation in cardiomyocytes with hypertrophic responsiveness to β-adrenoceptor stimulation. Induction of such a hypertrophic responsiveness depends on TGF-β activity. The main findings are first, that β-adrenoceptor mediated hypertrophy is indeed dependent on the induction of ODC in responsive cardiomyocytes, and, second, that such an induction of ODC is observed only when cells have been pre-exposed to TGF-β. Experiments in whole hearts from normal or TGF-β₁ over-expressing mice confirmed these conclusions.

This study has used a previously well characterized cell system in which hypertrophic responsiveness to β-adrenoceptor stimulation is inducible by addition of serum [7]. The cultured cardiomyocytes release TGF-β into the medium which, in presence of a serum supplement, is activated. Active TGF-β₁ induces the hypertrophic responsiveness to β-adrenoceptor stimulation as characterized by elevation of protein and RNA mass and ¹⁴C-phenylalanine incorporation and induction of ODC and ANF expression. We now demonstrate further on transgenic animals over-expressing TGF-β₁, that TGF-β₁ also induces hypertrophic responsiveness to β-adrenoceptor stimulation in vivo. Induction of ODC and ANF expression in the ventricles of transgenic and non-transgenic animals were used as molecular markers for myocardial hypertrophy

In TGF-β exposed cells and transgenic animals, β-adrenoceptor stimulation evoked the induction of ODC. It was found in the cell system that the induction of ODC could be abolished by the presence of two chemically distinct ODC inhibitors (-methylornithine and difluoromethylornithine). With either one of these inhibitors present, the stimulatory action of β-adrenoceptor stimulation on ¹⁴C-phenylalanine incorporation was suppressed. For -methylornithine it was also shown that the increase in cellular protein and RNA mass was dose-dependently reduced. These results suggest, that an activation of the polyamine metabolism, as indicated by ODC induction, is involved in the elevation of RNA that is observed under β-adrenoceptor stimulation. As a control for the specificity of ODC inhibition on the β-adrenoceptor hypertrophic response, the same inhibitors were applied under -adrenoceptor stimulation. In this case, they had no effect on the increase in RNA mass and protein synthesis. In summary, these results indicate that the hypertrophic effect of β-adrenoceptor stimulation in responsive cardiomyocytes depends specifically on the induction of ODC.

The role of ODC in cellular growth has been investigated in several cell culture systems. Inhibition of ODC by DFMO depletes cellular polyamines and thereby reduces the proliferation rate, e.g. in Ehrlich ascites tumor cells [27]. High levels of polyamines seem to be required for the initiation of the cell cycle. Adult ventricular cardiomyocytes are unable to divide and thus grow by hypertrophy. One common feature of hypertrophic and hyperplastic growth is, that under both conditions an increase in protein synthesis is necessary. This requires an elevation of the translational machinery as indicated by an increase in rRNA and, since this is >80% of all RNA, of total RNA. Our data indicate, that an induction of ODC is causally involved in this process in adult cardiomyocytes. A correlation between ODC induction and increase in tissue content of the polyamines spermidine, spermine, putrescine, and total RNA has been shown before on stimulated bovine lymphocytes as well [10].

The hypertrophic effect of β-adrenoceptor stimulation in this model of cultured cardiomyocytes is due to a stimulation of β₂-adrenoceptors [11]. Likewise the hypertrophic response characterized before, ODC induction by isoprenaline in responsive cells could be antagonized by ICI 118,551, a β₂-adrenoceptor antagonist. Together with the aforementioned results this suggests that ODC induction in these cells is due to β₂-adrenoceptor stimulation. In this context, we also were able to induce ODC in hearts from transgenic mice over-expressing TGF-β₁ by isoprenaline in the presence of a β₁-adrenoceptor antagonist, again suggesting that most likely β₂-adrenoceptors are involved in the observed induction of hypertrophic responsiveness by TGF-β. In agreement with this conclusion, it has been found that clenbuterol, a β₂-adrenoceptor agonist, can induce ODC in vivo [28]. The β₂-adrenoceptor mediated hypertrophic effect is known to be cAMP dependent [11]. Consistent with this finding and the conclusion that hypertrophic growth depends on ODC induction is the present observation, that ODC induction can also be achieved by addition of the cell permeable cAMP analogue db-cAMP.

In the cell culture system investigated here induction of ODC and hypertrophy upon β-adrenoceptor stimulation requires a previous exposure of the cells to TGF-β. We used a whole animal model of cardiac pre-exposure to TGF-β₁ to test the hypothesis that in intact myocardium the induction of ODC by β-adrenoceptor stimulation also depends on a pre-exposure to TGF-β₁. For this purpose hearts from normal animals were compared to those from animals with elevated circulating TGF-β levels. The latter animals were transgenic animals with an increased expression and release of active TGF-β₁. The hearts from transgenic animals showed a similar induction of ANF expression, a molecular marker of myocardial hypertrophy, upon β₂-adrenoceptor stimulation than the isolated cardiomyocytes upon isoprenaline treatment. These hearts, however, had already elevated ANF expression prior to β-adrenoceptor stimulation. They had fibrosis, as expected from the known effect of TGF-β₁ on fibroblasts, and this seems to cause a hypertrophic response caused by increased stiffness of the ventricles. However, stimulation of β-adrenoceptors further induces ANF and ODC expression only in hearts from transgenic animals.

Hearts undergoing myocardial hypertrophy show a transitory increased expression of TGF-β₁ [13]. In these hearts, the expression of TGF-β₁ is increased at the transition from compensatory hypertrophy to heart failure. The results of the present study suggest that in this pathophysiological transition stage, TGF-β₁ stimulation renders cardiomyocytes responsive to a hypertrophic action of β-adrenoceptor stimulation. This adds a further receptor-mediated stimulus to the growth promotion of the myocardial cells, which under normal conditions would not be found. Recently we showed that neuropeptide Y, which under normal conditions does not elevate protein synthesis either, can also increase protein synthesis of cardiomyocytes after pre-exposure to TGF-β [29]. Therefore, a pivotal role for TGF-β₁ for cardiomyocyte hypertrophy seems more important than expected from the present study alone.

In summary, our study demonstrates a causal role for ODC in the hypertrophic response to β-adrenoceptor stimulation.

Time for primary review 28 days.

	Acknowledgements

 
This study was supported by the Deutsche Forschungsgemeinschaft, grant Schl 324/3-1. We thank Dr. Thorgeirsson for his kindly help and for the transgenic animals and their genetic counterparts. This study was part of the thesis of K. Frischkopf.

	References

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 

1. Morgan H.E., Baker K.M. Cardiac hypertrophy: mechanical, neural and endocrine dependence. Circulation (1991) 83:13–25.[Free Full Text]
2. Fuller S.J., Gaitanaki C.J., Sugden P.H. Effects of catecholamines on protein synthesis in cardiac myocytes and perfused hearts isolated from adult rats. Stimulation of translation is stimulated by alpha-1-adrenoceptor. Biochem J (1990) 266:727–736.[Web of Science][Medline]
3. Decker R.S., Cook M.G., Behnke-Barclay M.M., Decker M.L., Lesch M., Samarel A.M. Catecholamines modulate protein turnover in cultured, quiescent rabbit cardiac myocytes. Am J Physiol (1993) 265:H239–H339.
4. Schlüter K.-D., Piper H.M. Trophic effects of catecholamines and parathyroid hormone on adult ventricular cardiomyocytes. Am J Physiol (1992) 263:H1739–H1746.[Web of Science][Medline]
5. Bartolome J., Guguenard J., Slotkin T.A. Role of ornithine decarboxylase in cardiac growth and hypertrophy. Science (1980) 210:793–794.[Abstract/Free Full Text]
6. Dubus I., Samuel J.-L., Marott F., Delcayre C., Rappaport L. Beta-adrenergic agonists stimulate the synthesis of noncontractile but not contractile proteins in cultured myocytes isolated from adult rat heart. Circ Res (1990) 66:867–874.[Abstract/Free Full Text]
7. Pinson A., Schlüter K.-D., Zhou X.J., Schwartz P., Kessler-Icekson G., Piper H.M. Alpha- and beta-adrenergic stimulation in cultured adult ventricular cardiomyocytes. J Mol Cell Cardiol (1993) 25:477–490.[CrossRef][Web of Science][Medline]
8. McDermott P.J., Rothblum L.I., Smith S.D., Morgan H.E. Accelerated rates of ribosomal RNA synthesis during growth of contracting heart cells in culture. J Biol Chem (1989) 264:18220–18227.[Abstract/Free Full Text]
9. Igarashi K., Kakegawa T., Hirose S. Stabilization of 30 S ribosomal subunits of Bacillus subtilis W168 by spermidine and magnesium ions. Biochim Biophys Acta (1982) 755:326–331.
10. Cohen S.S. A guide to the polyamines. Cohen S.S., ed. (1998) New York: Elsevier Science. 184–203.
11. Zhou X.J., Schlüter K.-D., Piper H.M. Hypertrophic responsiveness to β₂-adrenoceptor stimulation on adult ventricular cardiomyocytes. Mol Cell Biochem (1996) 163-164:211–216.
12. Schlüter K.-D., Zhou X.J., Piper H.M. Induction of hypertrophic responsiveness to isoproterenol by TGF-β in adult rat cardiomyocytes. Am J Physiol (1995) 269:1311–1316.
13. Boluyt M.O., O’Neill L., Meredith A.L., et al. Alterations in cardiac gene expression during the transition from stable hypertrophy to heart failure. Circ Res (1994) 75:23–32.[Abstract/Free Full Text]
14. Bradford M.M. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem (1976) 72:248–254.[CrossRef][Web of Science][Medline]
15. Giles K.W., Myers A. An improved diphenylamine method for the estimation of deoxyribonucleic acid. Nature (1965) 206:93.[Medline]
16. Flaumenhaft R., Abe M., Mignatti P., Rifkin D.B. Basic fibroblast growth factor-induced activation of latent transforming growth factor β in endothelial cells: regulation of plasminogen activator activity. J Cell Biol (1992) 118:901–909.[Abstract/Free Full Text]
17. Piper H.M., Spahr R., Mertens S., Krützfeld A., Watanabe H. Cell culture techniques in heart and vessel research. Piper H.M., ed. (1990) Heidelberg: Springer-Verlag. 158–177.
18. Meilhoc E., Moutin M.-J., Romani H.B., Osborne B.H. Relationship between ornithine decarboxylase activity and hexamethylene bisacetamine-induced differentiation of murine erythroleukemia cells. Exp Cell Res (1986) 162:142–150.[CrossRef][Web of Science][Medline]
19. Nudel U., Zakut R., Shani M., Neuman S., Levy Z., Yaffe D. The nucleotide sequence of the rat cytoplasmic beta-actin gene. Nucleic Acids Res (1983) 11:1759–1771.[Abstract/Free Full Text]
20. Blackshear P.J., Manzella J.M., Stumpo D.J., et al. High level, cell-specific expression of ornithine decarboxylase transcripts in rat genitourinary tissues. Mol Endocrinol (1989) 3:68–78.[Abstract/Free Full Text]
21. Flynn T.G. The elucidation of the structure of atrial natriuretic factor, a new peptide hormone. Can J Physiol Pharmacol (1987) 65:2013–2020.[Web of Science][Medline]
22. Curran T., Gordon M.B., Rubino K.L., Sambucetti L.C. Isolation and characterization of the c-fos(rat) cDNA and analysis of post-translational modification in vitro. Oncogene (1987) 2:79–84.[Web of Science][Medline]
23. Sanderson N., Factor V., Nagy P., et al. Hepatic expression of mature TGF-β1 in transgenic mice results in multiple tissue lesions. Proc Natl Acad Sci (1995) 92:2572–2576.[Abstract/Free Full Text]
24. Samuel S.K., Hurta R.A., Kondaiah P., et al. Autocrine induction of tumor protease production and invasion by a metallothionein-regulated TGF-beta 1 (Ser223, 225). EMBO J (1992) 11:1599–1605.[Web of Science][Medline]
25. Schlüter K.-D., Schwartz P., Siegmund B., Piper H.M. Prevention of the oxygen paradox in hypoxic–reoxygenated hearts. Am J Physiol (1991) 261:H416–H423.[Web of Science][Medline]
26. Godfrey K. Comparing the means of several groups. New Engl J Med (1985) 313:1450–1456.[Abstract]
27. Oredsson S.M., Anehus S., Heby O. Reversal of the growth inhibitory effect of alpha-difluoromethylornithine by putrescine but not by other divalent cations. Mol Cell Biochem (1984) 64:163–172.[Web of Science][Medline]
28. Cubria J.C., Reguera R., Balana-Fouce R., Ordonez C., Ordonez D. Polyamine-mediated heart hypertrophy induced by clenbuterol in the mouse. J Pharm Pharmacol (1998) 50:91–96.[Web of Science][Medline]
29. Goldberg Y., Taimor G., Piper H.M., Schlüter K-D. Intracellular signaling leads to the hypertrophic effect of neuropeptide Y. Am J Physiol (1988) 275:C1207–C1215.

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