Hypertrophic effect of selective
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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ABSTRACT |
We
investigated whether selective
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
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
1-adrenoceptor blockade with atenolol (10 µM) or
metoprolol (10 µM), indicating that it was caused by selective
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
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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INTRODUCTION |
HYPERACTIVATION
of the sympathetic nerve system normally accompanies myocardial
hypertrophy and subsequent heart failure (18). In vivo,
-adrenoceptor stimulation by isoproterenol, a nonselective
-adrenoceptor agonist, causes myocardial hypertrophy, and, at least
in some cases, application of
-adrenoceptor antagonists reduces
myocardial hypertrophy independently of hemodynamic effects (1,2). In vitro, isolated
cardiomyocytes increase protein and RNA synthesis in response to the
-adrenoceptor agonist phenylephrine (14). They do not,
however, respond to isoproterenol in respect to hypertrophic growth
(21). The influence of
2-adrenoceptor inhibition on hypertrophy caused by isoproterenol, resulting in
1-adrenoceptor subtype-specific stimulation, has not
been investigated.
In the present study we investigated whether selective stimulation by
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
2-adrenoceptor
antagonist ICI-118551, an inhibitor of
2-adrenoceptors,
or with norepinephrine and an
-adrenoceptor blocker that also
produces a preferential
1-adrenoceptor stimulation.
These treatments were aimed to mimic a pathophysiological situation in
which autoantibodies directed against
1-adrenoceptors may selectively stimulate
1-adrenoceptors
(25). In vivo, the hypertrophic response to
-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
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
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
-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
1-adrenoceptor mediates its hypertrophic effect in a cAMP-independent manner. Because
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
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 G
i and
G
o, influences the hypertrophic response to selective
1-adrenoceptor stimulation, because pertussis toxin was
found to increase the sensitivity to
1-adrenoceptors
(13).
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MATERIALS |
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-
-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 ×
× length, assuming a cylindrical cell
shape. Cross-sectional area was determined by the following formula:
cross-sectional area = (radius)2 ×
. 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:
-actin, sense 5'-GAAGTGTGACGTTGACATCCG-3' and
antisense 5'-TGCTGATCCACATCTGCTGGA-3', for amplification between 2,731 and 3,081 bp of rat
-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
-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-
-D-arabinofuranoside, L-carnitine,
creatine, and taurine were obtained from Sigma (Deisenhofen, Germany).
All other chemicals were of analytic grade.
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RESULTS |
Hypertrophic effect of selective stimulation of
1-adrenoceptors.
Isolated ventricular cardiomyocytes from adult rat were used under
serum-free conditions to study the influence of
-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
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
-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.
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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
-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
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
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
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
1-adrenoceptor
antagonists atenolol or metoprolol (Fig.
4). It was not antagonized by the
-adrenoceptor antagonist prazosin (10 µM). The presence of
prazosin abolished, however, the hypertrophic effect of phenylephrine
(10 µM), an
-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 -adrenoceptor stimulation with Iso (10 µM), selective
1-adrenoceptor stimulation with Iso in the presence of
the 2-adrenoceptor antagonist ICI (10 µM) (Iso+ICI),
and selective 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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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
1-adrenoceptor stimulation with Iso (10 µM) plus ICI
(10 µM) and after addition of the 1-adrenoceptor
antagonists atenolol or metoprolol (0.1-10 µM, as indicated),
the -adrenoceptor antagonist prazosin (10 µM), or the
-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.
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In a further set of experiments yet another approach was used to
demonstrate the ability of
1-adrenoceptor stimulation to promote protein synthesis (Fig. 5).
Cardiomyocytes were exposed to norepinephrine (1 µM), which is known
to stimulate
1- and
-adrenoceptors. In the presence
of the
-blocker propranolol (10 µM), norepinephrine produced a
41% rise in [14C]phenylalanine incorporation, linked to
-adrenoceptor stimulation. In the presence of the
-blocker
prazosin (10 µM), norepinephrine produced a 21% rise, linked to
1-adrenoceptor stimulation. The combination of prazosin
(10 µM) and the
1-selective antagonist atenolol (10 µM) abolished the growth effect of norepinephrine. In contrast, the
combination of prazosin with the
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
2-adrenoceptor agonist procaterol, the growth effect of
norepinephrine was again abolished. These experiments showed that the
growth effect of norepinephrine remaining after
-blockade is caused by
1-adrenoceptor stimulation and can be inhibited by
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 -adrenoceptor antagonist propranolol (Prop) or
the -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.
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Induction of ornithine decarboxylase by
1-adrenoceptor stimulation.
We investigated whether selective
1-adrenoceptor
stimulation induces ODC in isolated cardiomyocytes and whether this
induction is causally involved in the mechanism by which
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
-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 1-adrenoceptor stimulation.
Cardiomyocytes were cultured for the indicated times with Iso (10 µM)
in the presence of ICI (10 µM) to stimulate
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
1-adrenoceptor stimulation. Cardiomyocytes were cultured
for 2 h with Iso (10 µM) plus ICI (10 µM) to stimulate
1-adrenoceptors. RT-PCR for ODC mRNA was run for 25 cycles, and RT-PCR for -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
-adrenoceptor agonist Iso (10 µM) in the presence of the
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.
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Intracellular signaling pathways involved in the hypertrophic
response to
1-adrenoceptor stimulation.
Key elements of intracellular signaling that might be involved in the
hypertrophic effect of
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 -adrenoceptor agonist Iso (10 µM) in the presence of the 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
1-adrenoceptor stimulation
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Second, tyrosine kinase activation was inhibited by genistein (100 µM). Genistein attenuated the
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
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
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
1-adrenoceptor stimulation (Fig.
10). In pertussis toxin-treated
cardiomyocytes, [14C]phenylalanine incorporation under
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 -adrenoceptor
agonist Iso (10 µM) in the presence of the
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.
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DISCUSSION |
It is the main finding of the present study that
-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
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
-adrenoceptor stimulation. A direct and contractile independent
hypertrophic effect on adult ventricular cardiomyocytes by
-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
-adrenoceptor stimulation was found. In contrast to growth effects
mediated by
-receptors, the findings on growth effects of
-adrenoceptor stimulation are consistent. In all investigated models
-adrenoceptor stimulation promotes protein synthesis and cell
growth. In adult cardiomyocytes from rats this effect is mediated
through
1-adrenoceptors (4). In the present
study this hypertrophic effect to
1-adrenoceptor stimulation was used as a reference.
In contrast to previous experiments on the growth-promoting effects of
-adrenoceptor stimulation in cardiomyocytes, the experiments presented here were performed with isoproterenol in the presence of an
equimolar dose of the selective
2-adrenoceptor
antagonist ICI-118551. The resulting hypertrophic response was
antagonized by the addition of either atenolol or metoprolol, two
chemically distinct
1-adrenoceptor antagonists.
Nonspecific effects on
-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
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
1-adrenoceptor stimulation. This interpretation of our
data, however, is limited by the uncertainty regarding side effects of
these agents on putative
3- and
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
1-adrenoceptor stimulation with the
inefficiency of nonselective
-adrenoceptor stimulation leads to the
conclusion that nonselective
-adrenoceptor stimulation confers an
inhibitory action on the growth promotion by selective
1-adrenoceptor stimulation. The fact that the
2-antagonist ICI- 118551 is sufficient to unravel this
growth promotion indicates that
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
2-mediated action was not investigated in this study.
The sole presence of the
2-adrenoceptor agonist
procaterol did not induce a growth effect on its own. We showed
previously that on exposure to transforming growth factor-
(TGF-
), adult cardiomyocytes in culture can develop a specific
hypertrophic responsiveness to
2-adrenoceptor
stimulation (23, 28). In these
TGF-
-treated cultures, cardiomyocytes no longer exhibit a growth
response to
1-adrenoceptor stimulation, and
2-adrenoceptor stimulation no longer inhibits this
response. Under the influence of TGF-
, the hypertrophic
responsiveness of cardiomyocytes to
-adrenoceptor stimulation
therefore changes markedly.
In vivo, effects of
-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
-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
1-adrenoceptor stimulation causes induction of
ODC in vitro. Inhibition of ODC by DFMO inhibited the hypertrophic
response to
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
1-adrenoceptor stimulation.
The
1-adrenoceptor-mediated hypertrophic effect
identified in the present study is genistein sensitive, indicating that
this
1-adrenoceptor-mediated signaling pathway may
include an activation of tyrosine kinases. A genistein-sensitive
signaling pathway under
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
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.,
-adrenoceptor stimulation
(12, 20).
We also found that the hypertrophic response to selective
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
1-adrenoceptors in adult
cardiomyocytes from the guinea pig (13). This could be
explained by a competition between tonically active G
i
(pertussis toxin sensitive) and G
s (pertussis toxin
insensitive) for adenylate cyclase. Because the hypertrophic response
to selective
1-adrenoceptor stimulation described here
is cAMP independent, another explanation must be sought. It is, at
present, unclear how
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
-adrenoceptor
stimulation can induce hypertrophy of ventricular cardiomyocytes from
adult rats if
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
1-adrenoceptors
(25).
In summary, our study describes a cAMP-independent hypertrophic effect
of selective
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.
 |
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