(Received for publication, August 24, 1995; and in revised form, December 1, 1995)
From the
We have recently shown that mechanical stress induces
cardiomyocyte hypertrophy partly through the enhanced secretion of
angiotensin II (ATII). Endothelin-1 (ET-1) has been reported to be a
potent growth factor for a variety of cells, including cardiomyocytes.
In this study, we examined the role of ET-1 in mechanical
stress-induced cardiac hypertrophy by using cultured cardiomyocytes of
neonatal rats. ET-1 (1010
M) maximally induced the activation of both Raf-1 kinase
and mitogen-activated protein (MAP) kinases at 4 and 8 min,
respectively, followed by an increase in protein synthesis at 24 h. All
of these hypertrophic responses were completely blocked by pretreatment
with BQ123, an antagonist selective for the ET-1 type A receptor
subtype, but not by BQ788, an ET-1 type B receptor-specific antagonist.
BQ123 also suppressed stretch-induced activation of MAP kinases and an
increase in phenylalanine uptake by approximately 60 and 50%,
respectively, but BQ788 did not. ET-1 was constitutively secreted from
cultured cardiomyocytes, and a significant increase in ET-1
concentration was observed in the culture medium of cardiomyocytes
after stretching for 10 min. After 24 h, an
3-fold increase in
ET-1 concentration was observed in the conditioned medium of stretched
cardiomyocytes compared with that of unstretched cardiomyocytes. ET-1
mRNA levels were also increased at 30 min after stretching. Moreover,
ET-1 and ATII synergistically activated Raf-1 kinase and MAP kinases in
cultured cardiomyocytes. In conclusion, mechanical stretching
stimulates secretion and production of ET-1 in cultured cardiomyocytes,
and vasoconstrictive peptides such as ATII and ET-1 may play an
important role in mechanical stress-induced cardiac hypertrophy.
Cardiac hypertrophy, a major underlying cause of heart diseases
such as myocardial infarction and cardiac arrhythmias(1) , is
formed when increased external stimuli such as hemodynamic overload and
neurohumoral factors are continuously imposed on cardiac
myocytes(2, 3) . These external stimuli are generally
transduced into the nucleus through protein kinase cascades of
phosphorylation(4) , and Raf-1 kinase (Raf-1) ()(5) and mitogen-activated protein (MAP) kinases (6, 7) are important components in these cascades. We
have recently reported that stretching of cardiomyocytes sequentially
activates Raf-1 and MAP kinases, followed by an increase in protein
synthesis(8) . Interestingly, all of these events were
partially suppressed by a specific antagonist of the angiotensin II
(ATII) type 1 receptor, CV11974(9) . These results suggest that
mechanical stress exemplified by stretching might stimulate the
secretion of ATII from cardiomyocytes and that ATII participates in the
activation of the protein kinase cascades and the production of
cardiomyocyte hypertrophy through the ATII type 1 receptor. However,
because the inhibition of these hypertrophic events by CV11974 is
incomplete, factors other than ATII should also be involved in
cardiomyocyte hypertrophy induced by mechanical stress.
Endothelin-1
(ET-1) is a vasoactive peptide that contains 21 amino acids with two
intramolecular disulfide bond and was initially identified and purified
from porcine aortic endothelial cell cultures (10) . This
peptide is produced by endothelial and epithelial cells, macrophages,
fibroblasts, and many other types of cells, including cardiac myocytes
(for a review, see (11) ), and is not only a potent
vasoconstrictor, but also a potent growth factor for a variety of
cells, including cardiac
myocytes(12, 13, 14, 15) . It has
been reported that ET-1 increases the protein synthesis and the surface
area of cardiomyocytes without cell proliferation. Like many other
vasoactive peptides, ET-1 increases phosphoinositide turnover and
elevates diacylglycerol levels in rat cardiomyocytes(15) . Two
distinct ET-1 receptor subtypes (ET and ET
),
which have seven transmembrane-spanning regions and belong to the
superfamily of G protein-coupled receptors, have been cloned from cDNA
libraries of various cell
types(16, 17, 18, 19, 20, 21) ,
and both receptor subtypes have been shown to be expressed in the
heart(16, 17, 18, 21) .
In this
study, we first elucidated that ET-1 induces the activation of
hypertrophic signals such as Raf-1 and MAP kinases through the ET receptor, followed by an increase in protein synthesis in
neonatal rat cardiomyocytes. These responses were quite similar to
those observed when mechanical stress was imposed on cardiac myocytes (8) . Then, we examined the involvement of ET-1 in mechanical
stretch-induced hypertrophic responses by using ET-1 receptor-specific
antagonists. The ET
receptor-specific antagonist, BQ123,
significantly inhibited stretch-induced activation of Raf-1 and MAP
kinases and uptake of phenylalanine into cells. We have further
demonstrated that ET-1 is constitutively secreted from the cultured
cardiomyocytes of neonatal rats and that mechanical stretching enhances
the ET-1 release from the cells. Moreover, mRNA levels of ET-1 were
also increased by stretching of cardiomyocytes. These results suggest
that not only ATII, but also ET-1 plays an important role in mechanical
stress-induced cardiac hypertrophy.
Figure 1:
Dose dependence of ET-1-induced MAP
kinase activation. A, neonatal rat cardiomyocytes were
stimulated with various doses of ET-1 for 8 min. Cardiomyocyte lysates
were immunoprecipitated with an antibody against MAP kinases, Y91,
and the immunoprecipitates were subjected to Western blot analysis (upper panel) or were electrophoresed on an SDS-polyacrylamide
gel containing MBP. After denaturation and renaturation of MAP kinases,
phosphorylation of MBP was assayed by incubating the gel with
[
-
P]ATP. After incubation, the gel was
dried and subjected to autoradiography (lower panel). B, the data presented are an average from two independent
experiments. The activity of 44-kDa MAP kinase in control lysates is
designated as 1.0.
Figure 2:
Time course of ET-1-induced MAP kinase
activation. A and B, cardiomyocytes were exposed to
10M ET-1 for the indicated periods of
time. MAP kinase activities were measured as described in the Fig. 1legend. C, the data are presented as -fold
increases as compared with the activity of 44-kDa MAP kinase without
stimulation (1.0, 0 min) from four independent experiments (mean
± S.E.). Statistical analysis was performed using the paired
sample t test with p values corrected by the
Bonferroni method (see (46) ). *, p < 0.05 versus control.
Figure 3:
Activation of Raf-1 by ET-1 stimulation.
Cardiac myocytes were stimulated with 10M ET-1 for the indicated periods of time. Aliquots of the
cardiomyocyte lysates were incubated with substrate (syntide-2) and
[
-
P]ATP. After terminating the reaction,
the reaction mixture was spotted on P-81 paper. The incorporation of
P into syntide-2 was assessed by Cerenkov counting. The
data are presented as means ± S.E. from four independent
experiments compared with controls (100%). *, p < 0.05 versus control.
Figure 4: Effect of ET-1-neutralizing antibody on ET-1-induced MAP kinase activation. ET-1 was added to cardiac myocytes after pretreatment with a neutralizing antibody against ET-1 (HPE37B11) at the indicated concentrations or with control mouse IgG (100 µg/ml) for 30 min. MAP kinase assays were performed as described in the Fig. 1legend. A representative autoradiogram from three independent experiments is shown.
Figure 5:
Effect of ET or ET
receptor antagonist on ET-1- or stretch-induced MAP kinase
activation. A, after pretreatment with BQ123 (10
M) or BQ788 (10
M) for 30
min, cardiomyocytes were stimulated with 10
M ET-1 for 8 min. MAP kinase assays were performed as described in
the Fig. 1legend. B, the intensities of the 42- and
44-kDa bands were measured by densitometric scanning of the
autoradiogram. Values represent the average from two independent
experiments. The intensity of 44-kDa MAP kinase in unstimulated
myocytes is designated as 1.0. C, after pretreatment with
BQ123 (10
M) and CV11974 (10
M) for 30 min, cardiomyocytes were stretched by 20% for
8 min, and MAP kinase activities were measured as described in the Fig. 1legend. A representative autoradiogram from three
independent experiments is shown. D, the intensities of the
42- and 44-kDa bands in the autoradiogram (C) were measured by
densitometric scanning. The data represent the average percentage of
the controls (100%) from three independent experiments (mean ±
S.E.). *, p < 0.05 versus control.
Figure 6:
Release of ET-1 in culture medium and ET-1
gene expression in stretched myocytes. A, after changing the
medium, cardiomyocytes were stimulated with stretching (20%) for the
indicated periods of time. The amount of ET-1 was determined by enzyme
immunoassay as previously reported(29) . The results are
indicated as means ± S.E. for six independent experiments. Asterisks indicate undetectable levels (<1 pM). B, cardiomyocytes were stretched by 20% for the indicated
periods of time. Northern blot hybridization was performed using P-labeled ET-1 riboprobe. Similar results were obtained
from three independent experiments, and a representative autoradiogram
is shown.
Figure 7:
ATII and ET-1 independently induce MAP
kinase activation, and both agents synergistically activate MAP kinases
and Raf-1. A and B, after pretreatment with
10M BQ123 (A) or 10
M CV11974 (B) for 30 min, cardiac myocytes were
stimulated with 10
M ATII (AngII; A) or 10
M ET-1 (B) for 8
min. Analysis of MAP kinase activities was performed as described in
the Fig. 1legend. A representative autoradiogram from two
independent experiments is shown. C, cardiomyocytes were
stimulated with ET-1 (10
M) and/or with
ATII (10
M) for 8 min, and MAP kinase
activities were determined. A representative autoradiogram from two
independent experiments is shown in the upper panel, and the
densitometric scanning data of the 42- and 44-kDa bands are shown in
the lower panel. Values represent the means from two
independent experiments. The intensity of 44-kDa MAP kinase in
unstimulated cardiomyocytes is designated as 1.0. D, after
stimulation with ET-1 (10
M) and/or with
ATII (10
M) for 4 min, Raf-1 activities
were determined in cardiomyocytes. The data represent the average
percentage of the controls (100%) from four independent experiments
(mean ± S.E.) *, p < 0.05 versus control.
We next investigated the mutual effects of ET-1
and ATII. Although 10M ET-1 did not
significantly activate MAP kinases by itself, the presence of
10
M ET-1 dramatically augmented ATII
(10
M)-induced MAP kinase activation (Fig. 7C). With regard to Raf-1, similar synergistic
effects were observed. The addition of either 10
M ET-1 or 10
M ATII slightly
activated Raf-1; however, the simultaneous addition of ET-1 and ATII
markedly activated Raf-1 (Fig. 7D).
We (9, 38) and others (37) have
reported that endogenous ATII plays an important role in
stretch-induced cardiomyocyte hypertrophy. However, incomplete
inhibitions of stretch-induced hypertrophic responses by ATII receptor
antagonists suggest the involvement of other factors in producing
hypertrophy(9, 38) . ET-1 has been reported to be not
only a vasoconstrictor, but also a potent hypertrophy-promoting factor.
ET-1 produces cardiomyocyte hypertrophy in vitro as well as in
vivo(13, 14, 15, 40, 41) .
Therefore, to elucidate the involvement of ET-1 in stretch-induced
cardiac hypertrophy, we first investigated whether ET-1 activates the
protein kinase cascade of phosphorylation and induces cardiac
hypertrophy. ET-1 activated MAP kinases and Raf-1 in a dose-dependent
manner through the ET receptor, followed by an increase in
protein synthesis. We next examined the involvement of ET-1 in
stretch-induced cardiac hypertrophy by using ET-1 receptor antagonists.
The ET
receptor-specific antagonist, BQ123, significantly
inhibited stretch-induced Raf-1 and MAP kinase activation, suggesting
that ET-1 mediates stretch-induced cardiomyocyte hypertrophy through
the ET
receptor. ET-1 was constitutively secreted from
cardiomyocytes, and the secretion was enhanced by mechanical stress. In
addition, Northern blot analysis revealed that mRNA levels of ET-1 were
also increased by stretching. Finally, we have demonstrated that ET-1
and ATII synergistically activate Raf-1 and MAP kinases.
Recently,
Ito et al.(41) have reported that continuous
administration of BQ123 in rats with aortic banding blocks both cardiac
hypertrophy and the increase in skeletal -actin and atrial
natriuretic peptide gene expression. These results suggest that ET-1
may play an important role in producing cardiac hypertrophy during
pressure overload in vivo. It remains uncertain, however, how
ET-1 is involved in mechanical stress-induced cardiac hypertrophy. We
have demonstrated for the first time by using deformable silicone
dishes that ET-1 is constitutively released from cardiomyocytes and
that the release is enhanced by mechanical stress. Recently, Ito et
al.(39) have shown that ATII not only up-regulates
prepro-ET-1 mRNA levels at 30 min, but also stimulates ET-1 release
from neonatal rat cardiomyocytes at 60 min. They hypothesized that ATII
activates the transcription of the ET-1 gene, resulting in the
increased secretion of the ET-1 protein. In the present study, we have
shown that ET-1 is constitutively secreted from cardiomyocytes and that
mechanical stretching enhances the secretion from as early as 10 min
after stretching (Fig. 6A). We have also observed the
increase in ET-1 mRNA levels at 30 min after stretching (Fig. 6B). Collectively, these results suggest that
ET-1 may be stored in cardiomyocytes like ATII (37) and that
mechanical stretching directly induces the secretion of ET-1 as well as
increases the production of ET-1. It has been thought that ET-1 is
synthesized and released from the cell surface by exocytosis without
prior concentration and storage in secretory granules(42) .
Namely, ET-1 production is thought to be regulated at the level of
transcription rather than at the level of protein secretion. To
elucidate the intracellular localization of ET-1 in cultured
cardiomyocytes, we attempted an immunohistochemical analysis using
ET-1-specific antibodies, but failed to detect ET-1 in secretory
granules (data not shown). Some recent reports, however, have shown
that ET-1 is stored in secretory granules after segregation from the
Golgi cisterns of bone cells(43) , endothelial cells of
umbilical veins(44) , and aortic endothelial
cells(45) . Further studies are necessary to elucidate the
existence of ET-1 in secretory granules of cardiac myocytes and the
mechanism of the increase in ET-1 secretion during stretch-induced
cardiomyocyte hypertrophy.
In this study, we have shown that
mechanical stretching increases the secretion of ET-1 from
cardiomyocytes and that stretch-induced activation of Raf-1 and MAP
kinases is in part dependent on ET-1 through the ET receptor. It has been reported that unlike ATII, the secretion of
ET-1 is not enhanced by stretching(37) . Although we do not
know the reason for the discrepancy, our very sensitive assay for ET-1,
as well as the experiment using the specific antagonist, strongly
suggests that ET-1 is increased in the medium after stretching. We have
not determined exactly the local concentrations of ET-1 around the
cultured cardiac myocytes; however, the levels of ET-1 after stretching
were not as high as ATII levels reported previously(37) . As
shown in Fig. 7(C and D), however, even a low
level of ET-1 was able to induce hypertrophic responses in the presence
of ATII. We have shown that ET-1 activates Raf-1 and MAP kinases in a
synergistic manner with ATII in cultured cardiac myocytes. Interaction
between ET-1 and several growth factors has been shown to have
synergistic stimulatory effects on proliferation of vascular smooth
muscle cells or fibroblasts in culture (11) . Although it has
been well established that both ET-1 and ATII activate protein kinase C
in cardiac myocytes, other signal transduction pathways such as
Ca
-calmodulin and tyrosine kinases have been
demonstrated in other cell types. The mechanisms underlying the
synergistic action of ET-1 with different growth factors have yet to be
established; however, ET-1 may induce the hypertrophic responses
through different pathways compared with ATII.
Although the precise mechanisms by which the secretion of ET-1 and ATII is enhanced in stretched cardiomyocytes and by which ET-1 and ATII synergistically activate hypertrophic responses remain unknown, this study shows that not only ATII, but also ET-1 is involved in stretch-induced cardiac hypertrophy. In conclusion, these results suggest that vasoactive peptides play an important role in mechanical stress-induced cardiac hypertrophy, and this new finding may pave the way to develop new therapeutic strategies for cardiac hypertrophy.