Hypertrophic effect of selective beta 1-adrenoceptor stimulation on ventricular cardiomyocytes from adult rat

Matthias Schäfer, Karen Frischkopf, Gerhild Taimor, Hans Michael Piper, and Klaus-Dieter Schlüter

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


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We investigated whether selective beta 1-adrenoceptor stimulation causes hypertrophic growth on isolated ventricular cardiomyocytes from adult rat. As parameters for the induction of hypertrophic growth, the increases of [14C]phenylalanine incorporation, protein and RNA mass, and cell size were determined. Isoproterenol (Iso, 10 µM) alone had no growth effect. In the presence of the beta 2-adrenoceptor antagonist ICI-118551 (ICI, 10 µM), Iso caused an increase in [14C]phenylalanine incorporation, protein and RNA mass, cell volume, and cross-sectional area. We showed for phenylalanine incorporation that the growth effect of Iso+ICI could be antagonized by beta 1-adrenoceptor blockade with atenolol (10 µM) or metoprolol (10 µM), indicating that it was caused by selective beta 1-adrenoceptor stimulation. The growth response to Iso+ICI was accompanied by an increase in ornithine decarboxylase (ODC) activity and expression. Inhibition of ODC by the ODC antagonist difluoromethylornithine (1 mM) attenuated this hypertrophic response, indicating that ODC induction is causally involved. The growth response to Iso+ICI was found to be cAMP independent but was sensitive to genistein (100 µM) or rapamycin (0.1 µM). The reaction was enhanced in the presence of pertussis toxin (10 µM). We conclude that selective beta 1-adrenoceptor stimulation causes hypertrophic growth of ventricular cardiomyocytes by a mechanism that is independent of cAMP but dependent on a tyrosine kinase and ODC.

ornithine decarboxylase; tyrosine kinase; p70s6k


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HYPERACTIVATION of the sympathetic nerve system normally accompanies myocardial hypertrophy and subsequent heart failure (18). In vivo, beta -adrenoceptor stimulation by isoproterenol, a nonselective beta -adrenoceptor agonist, causes myocardial hypertrophy, and, at least in some cases, application of beta -adrenoceptor antagonists reduces myocardial hypertrophy independently of hemodynamic effects (1,2). In vitro, isolated cardiomyocytes increase protein and RNA synthesis in response to the alpha -adrenoceptor agonist phenylephrine (14). They do not, however, respond to isoproterenol in respect to hypertrophic growth (21). The influence of beta 2-adrenoceptor inhibition on hypertrophy caused by isoproterenol, resulting in beta 1-adrenoceptor subtype-specific stimulation, has not been investigated.

In the present study we investigated whether selective stimulation by beta 1-adrenoceptors induces hypertrophic growth on cardiomyocytes isolated from ventricles of adult rats. The cells were stimulated under various pharmacological conditions, i.e., with isoproterenol in the presence of the beta 2-adrenoceptor antagonist ICI-118551, an inhibitor of beta 2-adrenoceptors, or with norepinephrine and an alpha -adrenoceptor blocker that also produces a preferential beta 1-adrenoceptor stimulation. These treatments were aimed to mimic a pathophysiological situation in which autoantibodies directed against beta 1-adrenoceptors may selectively stimulate beta 1-adrenoceptors (25). In vivo, the hypertrophic response to beta -adrenoceptor stimulation is accompanied by an induction of ornithine decarboxylase (ODC), and inhibition of ODC attenuates the hypertrophic response (2). Therefore, we further investigated, in isolated cardiomyocytes, whether selective beta 1-adrenoceptor stimulation induces ODC and whether ODC induction is causally involved in its hypertrophic effect.

In another series of experiments we characterized the hypertrophic response of cardiomyocytes to beta 1-adrenoceptor stimulation in respect to several key elements of intracellular signal transduction. In previous studies from our group on the same experimental model (21), we found that neither a nonselective beta -adrenoceptor stimulation nor direct stimulation of cAMP-dependent protein kinases by dibutyryl-cAMP increases protein or RNA synthesis. This led us to hypothesize that the beta 1-adrenoceptor mediates its hypertrophic effect in a cAMP-independent manner. Because beta 1-adrenoceptor-mediated signaling pathways have also been shown to depend on the activation of genistein-sensitive tyrosine kinases (26), we further investigated whether tyrosine kinases participate in the intracellular signaling pathway. In a more general way, activation of phosphatidylinositol 3-kinase (PI 3-kinase) and p70s6k need to be activated by different intracellular signaling pathways that lead to hypertrophic growth of adult cardiomyocytes (20, 24). Therefore, we hypothesized that these two kinases are part of the intracellular signaling event caused by selective beta 1-adrenoceptor stimulation and leading to hypertrophic growth. The different intracellular signaling steps were inhibited with the use of specific inhibitors. Finally, we investigated whether pertussis toxin, an inhibitor of Galpha i and Galpha o, influences the hypertrophic response to selective beta 1-adrenoceptor stimulation, because pertussis toxin was found to increase the sensitivity to beta 1-adrenoceptors (13).


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Cell culture. Ventricular heart muscle cells were isolated from 200- to 250-g male Wistar rats as previously described (21). Isolated cells were suspended in fetal calf serum (FCS)-free culture medium and plated at a density of 1.4 × 105 elongated cells/35-mm culture dish (Falcon type 3001). The culture dishes had been preincubated overnight with 4% FCS in medium 199. The basic culture medium consisted of medium 199 with Earle's salts, 5 mM creatine, 2 mM L-carnitine, 5 mM taurine, 100 IU/ml penicillin, and 100 µg/ml streptomycin. To prevent growth of nonmyocytes, media were also supplemented with 10 µM cytosine-beta -D-arabinofuranoside.

Four hours after plating, cultures were washed twice with culture medium to remove round and nonattached cells and were supplied with FCS-free experimental medium, in which cells were incubated for a 24-h period at 37°C. The experiments were carried out in basic culture medium (control), with additions of agonists at the concentrations indicated. Ascorbic acid (100 µM) was added to all cultures as an antioxidant.

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 measuring the incorporation of radioactivity into an acid-insoluble cell mass as described previously (21). Nonradioactive phenylalanine (0.3 mM) 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 removing the supernatant medium from the cultures and washing three times with ice-cold phosphate-buffered saline (PBS; composition in mM: 1.5 KH2PO4, 137 NaCl, 2.7 KCl, and 1.0 Na2HPO4, pH 7.4). Subsequently, ice-cold 10% (wt/vol) 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 represent 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 N NaOH-0.01% (wt/vol) sodium dodecyl sulfate by an incubation for 2 h at 37°C. In these samples, protein (6) and DNA contents (10) were determined, and the radioactivity was counted. RNA was determined from an aliquot of these samples after precipitation with an equal volume of 10% (wt/vol) perchloric acid in the remaining supernatant (15). The RNA content was expressed relative to the DNA content of the samples.

Cell morphology. Cell morphology was determined as described earlier (22). Myocyte growth was determined on phase-contrast micrographs recorded on tape using a charge-coupled device video camera. Cell volumes were calculated by the following formula: volume = (radius)2 × pi × length, assuming a cylindrical cell shape. Cross-sectional area was determined by the following formula: cross-sectional area = (radius)2 × pi . Cell viability was determined using trypan blue extrusion assay.

ODC activity. The activity of ODC was determined as described in Ref. 16. Cardiomyocyte cultures were washed twice with ice-cold PBS, scraped off, and centrifuged for 2 min at 3,000 g. The pellet was resuspended in lysis buffer (composition in mM: 10 Tris · HCl, 250 sucrose, 5 dithiothreitol, 1 EDTA, and 0.5 pyridoxalphosphate, pH 7.3) and homogenized by sonification. The homogenate was centrifuged again for 2 min at 3,000 g. This supernatant was used to determine ODC activity. ODC activity in the supernatants was determined by the release of [14C]CO2 from L-[14C]ornithine. The supernatant (50 µl) and lysis buffer (250 µl) including 0.1 µCi/ml L-[14C]-ornithine and 3 mM ornithine were incubated for 30 min in a closed tube equipped with filter paper wetted in 1 N KOH to trap released CO2. The reaction was terminated by incubation overnight at 4°C to adsorb released CO2. To estimate nonspecific CO2 release during the incubation, blank tubes were set up and the nonspecific release was subtracted from the sample release. The filter papers were removed, and trapped [14C]CO2 was counted by liquid scintillation.

Reverse transcriptase-polymerase chain reaction. Total RNA from cardiomyocytes was extracted with RNA-Clean (AGS, Heidelberg, Germany) as described by the manufacturer. Reverse transcription 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 (Boehringer Mannheim, Germany), 1 mM dNTPs (GIBCO BRL), 8 units of RNase Block (Promega, Mannheim, Germany), and 60 units of Moloney murine leukemia virus 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 containing 1.5 µM primer pairs, 0.4 mM dNTPs, 1.5 mM MgCl2, and 1 unit of Taq polymerase (GIBCO BRL). Amplification was performed under the following cycle conditions: 1 min at 93°C, 1 min at 57°C, and 3 min at 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: beta -actin, sense 5'-GAAGTGTGACGTTGACATCCG-3' and antisense 5'-TGCTGATCCACATCTGCTGGA-3', for amplification between 2,731 and 3,081 bp of rat beta -actin gene (19); and ODC, sense 5'-GAAGATGAGTCAAACGAGCA-3' and antisense 5'-AGTAGATGTTTGGCCTCTGG-3', for amplification between 5,777 and 6,352 bp of rat ODC gene (27).

After amplification reaction products were separated on 5% polyacrylamide gels, they were stained with ethidium bromide and photographed under ultraviolet illumination. For quantification, the densities of the DNA fragments were determined by ImageQuant (Molecular Dynamics, Krefeld, Germany). The results for ODC expression were normalized for beta -actin expression.

Statistics. Data are given as means ± SE from n different culture preparations. Statistical comparisons were performed by one-way analysis of variance and with the use of the Student-Newman-Keuls test for post hoc analysis (11). In exceptional cases only two groups were compared. In these cases, Student's t-test was performed. Differences with P < 0.05 were regarded as statistically significant. All data were computed using SAS software (version 6.11; SAS Institute, Cary, NC).

Cell contraction. Cell contractions were analyzed as described previously (17, 22). Briefly, cardiomyocytes were paced at a constant frequency (0.5 Hz), and cell contractions were monitored by using a line camera. The contractile responses were expressed as cell shortening in percentage of diastolic cell length.

Materials. Falcon tissue culture dishes were obtained from Becton Dickinson (Heidelberg, Germany). Boehringer Mannheim (Mannheim, Germany) was the source for glutamine-free medium 199 and FCS. Cytosine-beta -D-arabinofuranoside, L-carnitine, creatine, and taurine were obtained from Sigma (Deisenhofen, Germany). All other chemicals were of analytic grade.


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Hypertrophic effect of selective stimulation of beta 1-adrenoceptors. Isolated ventricular cardiomyocytes from adult rat were used under serum-free conditions to study the influence of beta -adrenoceptor stimulation on [14C]phenylalanine incorporation. The cells remained mechanically quiescent throughout the experiments. Isoproterenol, applied at a saturating concentration of 10 µM (17), had no effect on phenylalanine incorporation. The additional presence of the beta 2-adrenoceptor antagonist ICI-118551 gradually increased [14C]phenylalanine incorporation when applied at increasing concentrations. A maximal increase was obtained at 1 µM ICI-118551, and no further change was observed at 10 µM ICI-118551 (Fig. 1A). The maximal effect achieved by isoproterenol in the presence of ICI- 118551 reached 47% of the maximal effect evoked by alpha -adrenoceptor stimulation with phenylephrine (10 µM), which was used as a control (100%).


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Fig. 1.   [14C]phenylalanine (14Phe) incorporation of cardiomyocytes 24 h after incubation with isoproterenol (Iso, 10 µM) in the presence of increasing concentrations of ICI-118551 (ICI) (A) or with ICI (10 µM) in the presence of increasing concentrations of Iso (B). Data are expressed relative to maximal responsiveness to phenylephrine (100% corresponded to 721 ± 31 dpm/µg DNA). Basal values (0%) corresponded to 468 ± 19 dpm/µg DNA. Data are means ± SE of n = 16 cultures. *P < 0.05 vs. nontreated control cultures.

In a second series of experiments the concentration of ICI-118551 was fixed at 10 µM, and the concentration of isoproterenol was increased. In the presence of ICI-118551, isoproterenol caused a concentration-dependent increase in protein synthesis, reaching significance at 3 µM (Fig. 1B). The isoproterenol effect was not saturated up to 30 µM. Because it is known that isoproterenol at concentrations >10 µM can stimulate alpha -adrenoceptors, all further experiments were performed with 10 µM isoproterenol. In some of the experiments this was combined with an equimolar dose of ICI-118551.

In the absence of ICI-118551, isoproterenol (10 µM) did not increase [14C]phenylalanine incorporation, protein mass, or RNA mass (Fig. 2). In the presence of this beta 2-adrenoceptor antagonist (10 µM), however, isoproterenol increased all three parameters. Similar results were also observed when the concentrations of isoproterenol and ICI-118551 were reduced from 10 µM to 100 nM. Again, isoproterenol in the presence of equimolar amounts of ICI-118551 caused a significant increase in [14C]phenylalanine incorporation of 18 ± 7% (P < 0.05 vs. untreated control cultures, n = 16). A selective beta 2-adrenoceptor stimulation with procaterol (10 µM) did not cause hypertrophic growth (Fig. 2). All the hypertrophic parameters investigated were normalized to the DNA content of the culture dishes to diminish variations in cell number between different culture preparations. DNA contents did not change among groups. The mean DNA content in control cultures was 29.0 ± 1.1 µg; in the presence of isoproterenol, isoproterenol plus ICI-118551, and procaterol it amounted to 31.5 ± 3.2, 30.8 ± 3.7, and 30.4 ± 6.1 µg, respectively. Induction of hypertrophy under equimolar concentrations of isoproterenol and ICI-118551 was confirmed by the analysis of cell morphology. Stimulation by isoproterenol plus ICI-118551 increased cell length by 9.7%, cell width by 16.8%, cell volume by 46.2%, and cross-sectional area by 31.3% within 24 h (Table 1). Treatment with isoproterenol plus ICI- 118551 did not change the gross morphology of the cells except for the aforementioned changes in cell size (Fig. 3). Cell viability was not changed. Under control conditions, 0.4 ± 0.1% of all cells stained positively with trypan blue. In the presence of isoproterenol plus ICI-118551 the number of trypan blue-positive cells was 0.6 ± 0.2% [not significant (NS) vs. control, n = 5 preparations]. In conclusion, these studies indicated that selective beta 1-adrenoceptor stimulation is required to stimulate a cellular growth response. This was also confirmed by the following experiments. The effect of isoproterenol plus ICI-118551 on [14C]phenylalanine incorporation could be antagonized concentration dependently by the beta 1-adrenoceptor antagonists atenolol or metoprolol (Fig. 4). It was not antagonized by the alpha -adrenoceptor antagonist prazosin (10 µM). The presence of prazosin abolished, however, the hypertrophic effect of phenylephrine (10 µM), an alpha -adrenoceptor agonist used as a positive control.


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Fig. 2.   [14C]phenylephrine incorporation (A), protein mass (B), and RNA mass (C) of cardiomyocytes 24 h after incubation under nonselective beta -adrenoceptor stimulation with Iso (10 µM), selective beta 1-adrenoceptor stimulation with Iso in the presence of the beta 2-adrenoceptor antagonist ICI (10 µM) (Iso+ICI), and selective beta 2-adrenoceptors stimulation with procaterol (Proc, 10 µM). Data are expressed as percent increase relative to nontreated controls. Basal values corresponded to 322 ± 13 dpm/µg DNA (A), 34.2 ± 1.4 µg protein/µg DNA (B), and 2.63 ± 0.08 µg RNA/µg DNA (C). Data are means ± SE of n = 16 cultures. *P < 0.05; **P < 0.01 vs. nontreated control cultures.


                              
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Table 1.   Influence of beta 1-adrenoceptor stimulation on cell shape



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Fig. 3.   Phase-contrast micrographs from cardiomyocyte cultures after 24 h without further additions (control) (A) or after 24 h in the presence of Iso (10 µM) and ICI (10 µM) (B).



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Fig. 4.   [14C]phenylephrine incorporation of cardiomyocytes 24 h after incubation under selective beta 1-adrenoceptor stimulation with Iso (10 µM) plus ICI (10 µM) and after addition of the beta 1-adrenoceptor antagonists atenolol or metoprolol (0.1-10 µM, as indicated), the alpha -adrenoceptor antagonist prazosin (10 µM), or the alpha -adrenoceptor agonist phenylephrine (PE, 10 µM), and in combinations as indicated. Data are expressed as percent increase relative to nontreated controls. Basal values corresponded to 418 ± 23 dpm/µg DNA. Data are means ± SE of n = 16 cultures. *P < 0.05 vs. Iso+ICI. #P < 0.01 vs. PE.

In a further set of experiments yet another approach was used to demonstrate the ability of beta 1-adrenoceptor stimulation to promote protein synthesis (Fig. 5). Cardiomyocytes were exposed to norepinephrine (1 µM), which is known to stimulate beta 1- and alpha -adrenoceptors. In the presence of the beta -blocker propranolol (10 µM), norepinephrine produced a 41% rise in [14C]phenylalanine incorporation, linked to alpha -adrenoceptor stimulation. In the presence of the alpha -blocker prazosin (10 µM), norepinephrine produced a 21% rise, linked to beta 1-adrenoceptor stimulation. The combination of prazosin (10 µM) and the beta 1-selective antagonist atenolol (10 µM) abolished the growth effect of norepinephrine. In contrast, the combination of prazosin with the beta 2-selective antagonist ICI-118551 (1 µM) left the same growth effect of norepinephrine as that of prazosin alone. When prazosin was combined with the beta 2-adrenoceptor agonist procaterol, the growth effect of norepinephrine was again abolished. These experiments showed that the growth effect of norepinephrine remaining after alpha -blockade is caused by beta 1-adrenoceptor stimulation and can be inhibited by beta 2-adrenoceptor stimulation.


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Fig. 5.   [14C]phenylephrine incorporation of cardiomyocytes 24 h after incubation with norepinephrine (1 µM) and addition of the beta -adrenoceptor antagonist propranolol (Prop) or the alpha -adrenoceptor antagonist prazosin alone (Praz, 10 µM) or with the further addition of atenolol (Praz+Ate, 10 µM), ICI-118551 (Praz+ICI, 1 µM), or procaterol (Praz+procaterol, 10 µM). Data are expressed as percent increase relative to nontreated controls. Basal values corresponded to 358 ± 33 dpm/µg DNA. Data are means ± SE of n = 16 cultures. *P < 0.05 vs. control cultures. #P < 0.05 vs. all other groups.

Induction of ornithine decarboxylase by beta 1-adrenoceptor stimulation. We investigated whether selective beta 1-adrenoceptor stimulation induces ODC in isolated cardiomyocytes and whether this induction is causally involved in the mechanism by which beta 1-adrenoceptor stimulation increases protein and RNA mass. In the presence of isoproterenol plus ICI-118551, ODC activity doubled within 2 h after agonist addition and remained elevated for the following 2 h (Fig. 6). An increase in ODC mRNA accompanied the increase in ODC activity as illustrated in Fig. 7A. Nonselective stimulation of beta -adrenoceptors with isoproterenol alone did not induce ODC activity or increase its expression (Fig. 7B). Addition of the ODC inhibitor difluoromethylornithine (DFMO, 1 mM) abolished the induction of ODC activity (93 ± 12% compared with the 100% value of nontreated control cultures, n = 4 cultures, NS), and the increments in [14C]phenylalanine incorporation, protein mass, and RNA mass were also attenuated (Fig. 8). The DNA content of the culture dishes was not modified by DFMO: the mean DNA content under control conditions, isoproterenol plus ICI-118551, DFMO, and isoproterenol plus ICI-118551 plus DFMO was 31.2 ± 5.1, 34.2 ± 2.4, 32.8 ± 4.8, and 34.5 ± 3.6 µg, respectively.


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Fig. 6.   Ornithine decarboxylase (ODC) activity in cardiomyocytes under selective beta 1-adrenoceptor stimulation. Cardiomyocytes were cultured for the indicated times with Iso (10 µM) in the presence of ICI (10 µM) to stimulate beta 1-adrenoceptors. ODC enzyme activity was determined in cell extracts from respective cultures as the release of [14C]CO2 from [1-14C]ornithine and was expressed as percentage of basal activity (0.6 ± 0.8 mU/mg protein). Data are means ± SE from n = 16 cultures. *P < 0.05; **P < 0.01 vs. time 0.



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Fig. 7.   A: Representative gel of RT-PCR samples to quantify ODC mRNA expression in cardiomyocytes 2 h after beta 1-adrenoceptor stimulation. Cardiomyocytes were cultured for 2 h with Iso (10 µM) plus ICI (10 µM) to stimulate beta 1-adrenoceptors. RT-PCR for ODC mRNA was run for 25 cycles, and RT-PCR for beta -actin was run for 18 cycles. C, control. B: ODC activity and expression in cardiomyocytes cultured for 2 h in the presence of Iso alone (10 µM) and Iso plus ICI (10 µM). ODC activity was determined as described in Fig. 6, and ODC expression was determined as described in A. Data are normalized to nontreated controls and represent means ± SE from n = 4 cultures. *P < 0.05; **P < 0.01 vs. nontreated controls.



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Fig. 8.   [14C]phenylalanine incorporation (A), protein mass (B), and RNA mass (C) of cardiomyocytes 24 h after incubation with the beta -adrenoceptor agonist Iso (10 µM) in the presence of the beta 2-adrenoceptor antagonist ICI (10 µM) or in the presence of difluoromethylornithine (DFMO, 1 mM). Data are expressed as percent increase relative to nontreated controls. Basal values corresponded to 320 ± 19 dpm/µg DNA (A), 36.2 ± 2.2 µg protein/µg DNA (B), and 2.38 ± 0.01 mg RNA/µg DNA (C). Data are means ± SE of n = 16 cultures. *P < 0.05; **P < 0.01 vs. control cultures. #P < 0.05 vs. each other.

Intracellular signaling pathways involved in the hypertrophic response to beta 1-adrenoceptor stimulation. Key elements of intracellular signaling that might be involved in the hypertrophic effect of beta 1-adrenoceptor stimulation were investigated. First, cAMP-dependent protein kinase activation was inhibited by the Rp diastereomer of adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS, 10 µM), but this did not attenuate the increases of [14C]phenylalanine incorporation, protein mass, or RNA mass (Fig. 9 and Table 2). As a positive control for the inhibitory effect of Rp-cAMPS (10 µM), its effect on the isoproterenol-stimulated, cAMP-dependent increase in twitch amplitude of electrically paced cardiomyocytes was analyzed. The control value for the twitch amplitude was 4.12 ± 1.44% of diastolic cell length, the twitch amplitude in the presence of isoproterenol was 8.39 ± 2.12% of diastolic cell length (P < 0.05 vs. control), and the twitch amplitude in the presence of isoproterenol plus Rp-cAMPS was only 5.12 ± 2.11% of diastolic cell length (NS vs. control, each n = 12 cells).


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Fig. 9.   [14C]phenylalanine incorporation of cardiomyocytes 24 h after incubation with the beta -adrenoceptor agonist Iso (10 µM) in the presence of the beta 2-adrenoceptor antagonist ICI (10 µM) or in the presence of the Rp diastereomer of adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS, 10 µM), genistein (Gen, 100 µM), or rapamycin (Rapa, 100 nM) where indicated. Data are expressed as percent increase relative to nontreated controls. Basal values corresponded to 320 ± 21 dpm/µg DNA. Data are means ± SE of n = 16 cultures. *P < 0.05; **P < 0.01 vs. control cultures. #P < 0.05 vs. Iso+ICI.


                              
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Table 2.   Influence of Rp-cAMPS, genistein, rapamycin, and wortmannin on protein and RNA mass of cardiomyocytes under selective beta 1-adrenoceptor stimulation

Second, tyrosine kinase activation was inhibited by genistein (100 µM). Genistein attenuated the beta 1-adrenoceptor-mediated hypertrophic effect (Fig. 9 and Table 2). Third, rapamycin (0.1 µM) was used to inhibit activation of p70s6k. Rapamycin completely abolished the hypertrophic effect of beta 1-adrenoceptor stimulation (Fig. 9 and Table 2). Fourth, wortmannin (0.1 µM) was used to inhibit PI 3-kinase activation. Wortmannin did not modify the increase in [14C]phenylalanine incorporation in response to beta 1-adrenoceptor stimulation (Table 2). In contrast, wortmannin inhibited the hypertrophic response evoked by phenylephrine in the same culture model (20). When applied alone, none of these inhibitors changed the basal levels of [14C]phenylalanine incorporation, protein mass, or RNA mass (Fig. 9 and Table 2).

Finally, we investigated whether an inhibition of pertussis toxin-sensitive G proteins influences the growth response to beta 1-adrenoceptor stimulation (Fig. 10). In pertussis toxin-treated cardiomyocytes, [14C]phenylalanine incorporation under beta 1-adrenoceptor stimulation was increased compared with that in nontreated cardiomyocytes. Pertussis toxin alone had no effect on basal [14C]phenylalanine incorporation.


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Fig. 10.   [14C]phenylalanine incorporation of cardiomyocytes 24 h after incubation with the beta -adrenoceptor agonist Iso (10 µM) in the presence of the beta 2-adrenoceptor antagonist ICI (10 µM) and pertussis toxin (Ptx, 10 µM) where indicated. Data are expressed as percent increase relative to nontreated controls. Basal values corresponded to 362 ± 31 dpm/µg DNA. Data are means ± SE of n = 16 cultures. *P < 0.05; **P < 0.01 vs. control cultures. #P < 0.05 vs. Iso+ICI alone.


    DISCUSSION
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ABSTRACT
INTRODUCTION
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DISCUSSION
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It is the main finding of the present study that beta -adrenoceptor stimulation by isoproterenol exerts a hypertrophic effect of variable magnitude on mechanically quiescent ventricular cardiomyocytes isolated from adult rat in copresence with equimolar concentrations of the beta 2-adrenoceptor antagonist ICI-118551. Induction of ornithine decarboxylase is causally involved in this hypertrophic response. In addition, this hypertrophic response is genistein and rapamycin sensitive but cAMP independent. It is enhanced when pertussis toxin-sensitive G proteins are inhibited.

This study provides the first evidence for hypertrophic responsiveness of ventricular cardiomyocytes from adult rats to beta -adrenoceptor stimulation. A direct and contractile independent hypertrophic effect on adult ventricular cardiomyocytes by beta -adrenoceptor stimulation has not been shown before. Such effects on cardiac growth have been shown previously in vivo (2, 5), in neonatal cardiomyocytes, where they were linked to cell density and contraction (3, 14), or in adult cardiomyocytes after precultivation and stimulation of mechanical performance (7) or pretreatment with isoproterenol (9). In newly isolated ventricular cardiomyocytes from adult animals, however, no direct effect of nonspecific beta -adrenoceptor stimulation was found. In contrast to growth effects mediated by beta -receptors, the findings on growth effects of alpha -adrenoceptor stimulation are consistent. In all investigated models alpha -adrenoceptor stimulation promotes protein synthesis and cell growth. In adult cardiomyocytes from rats this effect is mediated through alpha 1-adrenoceptors (4). In the present study this hypertrophic effect to alpha 1-adrenoceptor stimulation was used as a reference.

In contrast to previous experiments on the growth-promoting effects of beta -adrenoceptor stimulation in cardiomyocytes, the experiments presented here were performed with isoproterenol in the presence of an equimolar dose of the selective beta 2-adrenoceptor antagonist ICI-118551. The resulting hypertrophic response was antagonized by the addition of either atenolol or metoprolol, two chemically distinct beta 1-adrenoceptor antagonists. Nonspecific effects on alpha -receptors were excluded by showing the inability of prazosin to influence the hypertrophic effect of isoproterenol plus ICI-118551. These data, together with the finding that the beta 2-adrenoceptor agonist procaterol has no effect on its own, indicate that the observed hypertrophic effect of isoproterenol plus ICI-118551 is caused by a selective beta 1-adrenoceptor stimulation. This interpretation of our data, however, is limited by the uncertainty regarding side effects of these agents on putative beta 3- and beta 4-adrenoceptors on cardiomyocytes. Because the cardiomyocytes are quiescent in the model system, the effects are independent of contractile performance. The hypertrophic effect includes an acceleration of protein synthesis, an increase in cellular protein and RNA mass, and enlargements of length, width, volume, and cross-sectional area of the cells. Comparison of the growth-stimulating action of beta 1-adrenoceptor stimulation with the inefficiency of nonselective beta -adrenoceptor stimulation leads to the conclusion that nonselective beta -adrenoceptor stimulation confers an inhibitory action on the growth promotion by selective beta 1-adrenoceptor stimulation. The fact that the beta 2-antagonist ICI- 118551 is sufficient to unravel this growth promotion indicates that beta 2-adrenoceptor stimulation exerts this inhibitory action. This is confirmed by the observation that procaterol antagonizes the growth effect of norepinephrine plus prazosin. The mechanism of the inhibitory beta 2-mediated action was not investigated in this study.

The sole presence of the beta 2-adrenoceptor agonist procaterol did not induce a growth effect on its own. We showed previously that on exposure to transforming growth factor-beta (TGF-beta ), adult cardiomyocytes in culture can develop a specific hypertrophic responsiveness to beta 2-adrenoceptor stimulation (23, 28). In these TGF-beta -treated cultures, cardiomyocytes no longer exhibit a growth response to beta 1-adrenoceptor stimulation, and beta 2-adrenoceptor stimulation no longer inhibits this response. Under the influence of TGF-beta , the hypertrophic responsiveness of cardiomyocytes to beta -adrenoceptor stimulation therefore changes markedly.

In vivo, effects of beta -adrenoceptor stimulation on myocardial hypertrophy have been shown to be accompanied by induction of ODC (2). ODC represents the rate-limiting enzyme of the polyamine metabolism. Elevated tissue contents of polyamines are found in hypertrophic hearts on beta -adrenoceptor stimulation (8). Polyamines are able to stabilize nucleic acids such as RNA, because of their cationic character, and prolong their half-life. This may explain the mechanism by which ODC induces the elevation of the cellular RNA mass. In the present study we found that selective beta 1-adrenoceptor stimulation causes induction of ODC in vitro. Inhibition of ODC by DFMO inhibited the hypertrophic response to beta 1-adrenoceptor stimulation, i.e., it attenuated the increase in total RNA mass, protein synthesis, and protein mass. These results demonstrate a supportive role for ODC in the hypertrophic response to beta 1-adrenoceptor stimulation.

The beta 1-adrenoceptor-mediated hypertrophic effect identified in the present study is genistein sensitive, indicating that this beta 1-adrenoceptor-mediated signaling pathway may include an activation of tyrosine kinases. A genistein-sensitive signaling pathway under beta 1-adrenoceptor stimulation was recently also found for the activation of KCl channels (26). The kind of genistein-sensitive kinases involved here have yet to be identified. The beta 1-adrenoceptor-mediated hypertrophic effect on newly isolated cardiomyocytes is cAMP independent, in agreement with our previous observations that dibutyryl-cAMP does not increase protein synthesis in newly isolated cardiomyocytes from adult rats (21) and the results that Bogoyevitch and colleagues (4) found with isolated perfused rat hearts. The hypertrophic effect is not sensitive to wortmannin, a PI 3-kinase inhibitor. PI 3-kinase activation is part of the intracellular signaling pathway leading to myocardial hypertrophic growth caused by other growth promoters, e.g., alpha -adrenoceptor stimulation (12, 20).

We also found that the hypertrophic response to selective beta 1-adrenoceptor stimulation is sensitive to pertussis toxin. The presence of pertussis toxin enlarged the stimulation of protein synthesis. Others observed before that pertussis toxin can increase the sensitivity of beta 1-adrenoceptors in adult cardiomyocytes from the guinea pig (13). This could be explained by a competition between tonically active Galpha i (pertussis toxin sensitive) and Galpha s (pertussis toxin insensitive) for adenylate cyclase. Because the hypertrophic response to selective beta 1-adrenoceptor stimulation described here is cAMP independent, another explanation must be sought. It is, at present, unclear how beta 1-adrenoceptors couple to tyrosine kinases (as discussed above). The data for pertussis toxin may suggest that pertussis toxin-sensitive proteins tonically inhibit this coupling.

The results of the present study demonstrate that beta -adrenoceptor stimulation can induce hypertrophy of ventricular cardiomyocytes from adult rats if beta 1-adrenoceptors are preferentially activated. One pathomechanism by which this can occur in vivo has been described. In patients with heart failure, autoantibodies are frequently found in plasma that confer an intrinsic activity directed selectively against the beta 1-adrenoceptors (25).

In summary, our study describes a cAMP-independent hypertrophic effect of selective beta 1-adrenoceptor stimulation on adult cardiomyocytes that requires an activation of the polyamine metabolism as indicated by its dependency on ODC induction.


    ACKNOWLEDGEMENTS

This study was supported by the Deutsche Forschungsgemeinschaft Grants Schl 324/3-1 and Pi 162/11-2.


    FOOTNOTES

Address for reprint requests and other correspondence: K.-D. Schlüter, Physiologisches Institut, Justus-Liebig-Universität, Aulweg 129, D-35392 Giessen, Germany (E-mail: Klaus-Dieter.Schlueter{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. §1734 solely to indicate this fact.

Received 28 May 1999; accepted in final form 8 March 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS
RESULTS
DISCUSSION
REFERENCES

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