Download PDF Article

Journal of the American College of Cardiology
© 1999 by the American College of Cardiology
Published by Elsevier Science Inc.
Vol. 33, No. 2, 1999
ISSN 0735-1097/99/$20.00
PII S0735-1097(98)00564-6
Upregulated Expression of Cardiac
Endothelin-1 Participates in
Myocardial Cell Growth in
Bio14.6 Syrian Cardiomyopathic Hamsters
Tsukasa Inada, MD,* Hisayoshi Fujiwara, MD,* Koji Hasegawa, MD,* Makoto Araki, MD,*
Rikako Yamauchi-Kohno, MS,† Hideo Yabana, PHD,† Takako Fujiwara, MD,‡ Masaru Tanaka, MD,*
Shigetake Sasayama, MD, FACC*
Kyoto, Japan
OBJECTIVES
The purpose of this study is to investigate the role of endogenous endothelin-1 (ET-1) in
myocardial growth in Bio 14.6 Syrian cardiomyopathic hamsters (Bio).
BACKGROUND While ET-1, as a growth-promoting peptide, has been implicated in the development of
secondary cardiac hypertrophy, the role of endogenous ET-1 in cardiac growth in primary
myocardial disease is unknown.
METHODS
We measured left ventricular ET-1 levels by a specific sandwich enzyme-linked immunosorbent assay. Furthermore, we examined the chronic effect of T0201, an ET type A
receptor-specific antagonist.
RESULTS
The ET-1 levels in the left ventricles were 1.8-fold higher (p , 0.0005) at 20 weeks and
6.4-fold higher (p , 0.0001) at 35 weeks in the Bio compared to age-matched control F1B
hamsters (F1B). The Bio ET-1 levels in the lungs exhibited an only 1.3-fold elevation at 35
weeks. Immunohistochemistry demonstrated the localization of ET-1 mainly in the cardiac
myocytes. The treatment with T0201 significantly reduced the heart weight/body weight
ratio in the Bio, but did not affect the heart weight/body weight ratio in the F1B.
Histologically, T0201 reduced the myocyte diameter of Bio to a level similar with that of
F1B. However, T0201 did not affect the extent of fibrosis in Bio or F1B.
CONCLUSIONS The ET-1 level in the heart of cardiomyopathic hamsters increases in stage-dependent and
organ-specific manners. Though myocyte degeneration and subsequent replacement fibrosis
do not require an ET-1 pathway, the accelerated synthesis of ET-1 in the heart may
contribute to the pathological growth of remaining myocytes in this animal model. (J Am
Coll Cardiol 1999;33:565–71) © 1999 by the American College of Cardiology
Endothelin-1 (ET-1) is a potent vasoconstrictor peptide
originally isolated from the supernants of cultured endothelial cells (1). It exerts its effect through the stimulation of
specific receptors (ETA and ETB receptors) widely distributed in the cardiovascular system (2,3). The plasma levels of
ET-1 are elevated in various pathophysiological states
including congestive heart failure (4,5). The elevated level of
ET-1 in heart failure is closely associated with the extent of
pulmonary hypertension (5). In this state, an acute administration of endothelin receptor blocker reduced vascular
resistance and improved an overall left ventricular performance (6,7). These findings suggest that ET-1 has a
From the *Department of Cardiovascular Medicine, Kyoto University, Graduate
School of Medicine, Kyoto 606-8507, Japan; †Lead Optimization Research Laboratory, Tanabe Seiyaku, Saitama, Japan; and ‡Department of Food Science, Kyoto
Women’s University, Kyoto, Japan.
Manuscript received March 20, 1998; revised manuscript received August 24, 1998,
accepted October 2, 1998.
pathophysiological role in heart failure as a circulating
hormone. In addition to its action as a systemic hormone,
ET-1 has a number of actions as a local factor (8).
Endothelin-1 is sufficient to induce the myocardial cell
hypertrophy associated with the reactivation of the fetal
gene program; i.e., the induction of atrial natriuretic factor
and b-myosin heavy chain expression (9,10). Endothelin-1
mediates load-induced hypertrophy in cultured neonatal
cardiac myocytes as an autocrine factor (11). Previous
studies demonstrated that the expression of ET-1 in the
heart is accelerated following pressure overload and myocardial infarction, and that the chronic administration of an
ET receptor antagonist blocks the development of hypertrophy in animal models (12,13). These findings demonstrate that ET-1 is involved in the development of cardiac
hypertrophy secondary to hemodynamic overload. Little is
known regarding a role of ET-1 in primary myocardial
disease, however.
566
Inada et al.
Endothelin-1 in Cardiomyopathy
Abbreviations and Acronyms
Bio 5 Bio 14.6 Syrian cardiomyopathic hamsters
ET-1 5 endothelin-1
EIA 5 enzyme-linked immunosorbent assay
F1B 5 F1B hamsters
We previously reported that in hypertrophic cardiomyopathy patients with normal pulmonary arterial pressure,
plasma ET-1 levels are significantly elevated (14). The
elevated ET-1 levels closely correlated with the myocardial
cell diameter in endomyocardial biopsy specimens. These
findings raise the possibility that ET-1 has a role in primary
myocardial disease. However, it is not known whether the
elevated plasma levels of ET-1 are derived from hearts with
hypertrophic cardiomyopathy, or whether endogenous
ET-1 directly contributes to the development of hypertrophy in primary myocardial disease. To identify the role of
endogenous ET-1 in the development of hypertrophy in
primary myocardial disease, we investigated the cardiac
synthesis of ET-1 in Bio14.6 Syrian cardiomyopathic hamsters (Bio) and examined the effect of an oral ETA receptor
antagonist on the pathogenesis of this animal model.
METHODS
Experimental animals. Male cardiomyopathic hamsters of
the Bio 14.6 Syrian strain (Bio) and control F1B hamsters
(F1B) aged 5 weeks old were purchased from Charles River
Japan (Atsugi, Japan). Hamsters were individually housed in
our animal room maintained at a temperature of 24 6 1°C
and with a 12-hour light/dark cycle (0600-1800 hours). The
animals received powdered diet (CE-7, Nihon Clea, Tokyo,
Japan) and tap water ad libitum. The following experiments
were approved by the ethical committee of the Graduate
School of Medicine, Kyoto University.
Administration of an ETA receptor-specific antagonist,
T0201. Five week-old Bio and F1B were randomly subjected to either treatment with T0201, an ETA receptorspecific antagonist, at a daily dose of 1.5 mg/kg body weight
(F1B: n 5 17, Bio: n 5 22) or vehicle treatment (F1B: n 5
17, Bio: n 5 22). T0201 was mixed in the diet, and the
quantity of the diet eaten by these animals was checked
weekly. These animals were sacrificed at the age of 20 weeks
or 35 weeks.
Tissue preparation. After the heart was excised and
weighed, the atria and the right ventricles were trimmed off,
and the left ventricle was rinsed in cold physiological saline.
The basal one-third of the left ventricle was cut into two
slices, one of which was embedded in OCT compound
(Miles Laboratories, Elkhart, Indiana), frozen in dry ice
acetone and stored at 270°C until use for imunohistochemistry. The other slice was fixed with 10% formalin for
histological examination with hematoxylin-eosin and
JACC Vol. 33, No. 2, 1999
February 1999:565–71
Masson-trichrome staining. For the measurement of cardiac
ET-1 levels, the apical two-thirds of the left ventricle was
immediately homogenized with a Polytron homogenizer for
30 s in 9 volumes of 1 mol/L acetic acid containing 0.1%
Triton-X, boiled for 7 min and centrifuged at 20,000g for
30 min at 4°C. Following the measurement of the protein
concentrations (pg/g), the supernatant was stored at 280°C
until use.
Measurement of myocardial ET-1 levels. ET-1 was extracted from the supernatant of homogenized ventricular
tissues according to the method of Kitamura et al. (15).
Briefly, 1 milliliter of the supernatant was acidified with
3 ml of 4% acetic acid and applied to a Sep-Pak C18
cartridge (Waters, Milford, Massachusetts). The absorbed
peptides were eluted with 86% ethanol containing 4% acetic
acid, dried under nitrogen gas and reconstituted with 250 ml
of assay buffer. The ET-1 levels were measured by means of
a sandwich enzyme-linked immunosorbent assay (EIA) kit
(Wako Pure Chemical, Osaka, Japan) as previously described (14). This sandwich EIA utilized two antibodies
that recognize different portions of ET-1: a mouse monoclonal antibody against the NH2-terminal portion of rat
ET-1 immobilized on a 96-well plate and a horseradish
peroxidase-labeled rabbit antibody against the COOHterminal portion of rat ET-1. After color development with
o-phenylenediamine as a substrate, the absorbance of each
well was measured at 492 nm. This EIA for ET-1 could
detect as little as 0.5 pg/ml of ET-1. The cross-reactivity
with ET-3 or big ET-1 was less than 0.1%. The values by
EIA presented as (pg/ml) were standardized by the protein
concentrations of the supernatant (g/ml) and expressed as
pg/g.
Immunohistochemical staining for ET-1. Sections
(4 mm) were cut on a cryostat at 220°C and mounted on
glass slides. After rinsing in PBS, the hydrated sections were
fixed in 10% phosphate-buffered formalin solution for
10 min. Then they were immunostained for ET-1 using an
indirect immunoperoxidase method as described previously
(16). Briefly, the sections were incubated with 0.3% hydrogen peroxide for 20 min to block endogenous peroxidase
activity, followed by further incubation with normal goat
serum for 30 min to block nonspecific bindings. They were
then incubated with ET-1 antiserum (Peptide Institute,
Osaka, Japan) at a final dilution of 1:40 for 16 hr at 4°C. In
the second step, they were treated with a 1:200 dilution of
peroxidase-conjugated goat antirabbit IgG (Jackson
Immuno-Research Laboratories, West Grove, Pennsylvania) for 45 min. Peroxidase activity was visualized by use of
diaminobenzidine and hydrogen peroxide. The sections
were counterstained with hematoxylin, dehydrated through
alcohol, and cleared in xylene before coverslipping. The
slides were evaluated microscopically. As a control to check
the specificity, the primary antibody was preincubated with
1 mg ET-1 peptide (Peptide Institute, Osaka, Japan) for 3 h
before application to the slides.
JACC Vol. 33, No. 2, 1999
February 1999:565–71
Inada et al.
Endothelin-1 in Cardiomyopathy
567
Histological assessment. Myocyte diameters were measured separately in the inner, middle and outer thirds of the
left ventricular free wall. Two investigators (M.A., T.I.),
who were unaware of the treatment protocol, independently
reviewed the sections and randomly selected a total of 100
myocardial fibers from each area. The myocyte diameters
were measured semiautomatically with the aid of an image
analyzer (LUZEX 3U, Nikon, Tokyo) in sections stained
with hematoxylin-eosin (17–19). The shortest diameters of
transversely cut fibers were measured at the level of the
nucleus. Myocardial fibers were excluded if their boundaries
were not clear, if their nuclei were not located centrally or if
degenerative changes were prominent. The myocardial cell
diameter of an animal was expressed by averaging the data
determined by these two investigators.
The investigator (M.A.) who was unaware of the treatment protocol measured all of the fibrotic areas in the
sections. The areas of fibrosis stained blue were quantified
semiautomatically using the LUZEX 3U image analyzer in
sections stained with Masson’s trichrome (19 –21). The
ratio of the fibrotic area to the total tissue area was
calculated as the percent area of fibrosis.
The left ventricular inner and outer cavity areas were
measured using the LUZEX 3U image analyzer. The
myocardial area was expressed as the outer cavity area
subtracted by the inner cavity area.
Statistical analysis. The results are expressed as mean
value 6 SEM. Statistical comparisons of ET-1 levels at
specific time points between Bio and F1B were assessed
using unpaired Student t tests. Otherwise, multiple comparisons were performed using ANOVA with Scheffe’s test.
In all tests, a p value ,0.05 was considered significant.
RESULTS
The cardiac expression of ET-1 in Bio is markedly
upregulated in state-dependent and organ-specific manners. To investigate the possible contribution of cardiac
ET-1 synthesis to the elevation of plasma ET-1 levels in
cardiomyopathy, we determined the left ventricular ET-1
levels in Bio and F1B using a specific sandwich EIA. At 5
weeks of age the left ventricular ET-1 levels of the Bio did
not differ from those of the F1B. At the age of 20 weeks, the
heart weight/body weight ratio was 25.5% higher in the Bio
compared with the age-matched F1B. The left ventricular
inner cavity area of Bio (12.6 6 0.20 mm2) did not differ
significantly from that of F1B (13.8 6 0.21 mm2). However, the myocardial area of Bio (48.3 6 0.70 mm2) was
significantly (p , 0.01) larger than that of F1B (38.9 6
0.69 mm2). The histological examination revealed focal
myocyte degeneration with fibrosis and inhomogenous
zones of hypertrophy. In these cardiomyopathic hamsters,
the ventricular ET-1 levels were mildly (1.8-fold) elevated
(Fig. 1A). At the age of 35 weeks, the left ventricular inner
cavity area of Bio (30.4 6 0.33 mm2) was much larger (p ,
0.0001) than that of F1B (16.3 6 0.29 mm2) while the
Figure 1. Cardiac (A) and pulmonary (B) ET-1 levels in Bio 14.6
Syrian cardiomyopathic hamsters (Bio-CTL) compared with F1B
controls (F1B-CTL). Cardiac and pulmonary ET-1 levels were
measured by a specific sandwich EIA as described in Materials and
Methods. Values are mean 6 SEM. (A) #p , 0.0001 vs.
age-matched F1B-CTL, 5-week old Bio and 20-week old BioCTL. *p , 0.0005 vs. age-matched F1B-CTL. (B) **p , 0.005 vs.
age-matched F1B-CTL, 5-week old Bio-CTL and 20-week old
Bio-CTL. Open squares 5 F1B; solid squares 5 Bio.
myocardial area of Bio (42.1 6 0.47 mm2) was similar to
that of F1B (44.5 6 0.58 mm2). The histological examination revealed the replacement of degenerative myocytes with
fibrosis. The heart weight/body weight ratio increase
(22.9%) was similar compared to that at the age of 20 weeks.
At this stage, the ventricular ET-1 levels were markedly
elevated (6.4-fold) in the Bio compared with the F1B (Fig.
1A). Thus, the left ventricular ET-1 levels increased in the
Bio in a stage-dependent manner. We also determined the
568
Inada et al.
Endothelin-1 in Cardiomyopathy
Figure 2. Immunohistochemical detection of ET-1 in the left
ventricles of 35 week-old Bio14.6 cardiomyopathic (A and B) and
age-matched control F1B (C) hamsters. Immuno-reactive ET-1
was detected with an indirect peroxidase method as described in
Materials and Methods. Preincubating an excess of synthetic ET-1
with a primary antibody resulted in no staining (B). Original
magnification 3 200.
ET-1 levels in the lungs (Fig. 1B). The pulmonary ET-1
levels did not differ between the F1B and Bio at the ages of
5 weeks and 20 weeks. At 35 weeks, the levels were 1.3-fold
higher in the Bio than in the F1B. Thus, the elevation of
ET-1 levels in the Bio compared with the F1B was much
more prominent in the heart than in the lung, suggesting
the organ-specific activation of ET-1 synthesis.
Immunohistochemical detection of ET-1 in the left
ventricular myocardium. In addition to endothelial cells,
various cell types including cardiac myocytes have the ability
to synthesize ET-1 (11,22). To determine which cell type is
responsible for the elevated levels of ET-1 in the Bio heart,
we performed immunohistochemical staining. Strong positive brown signals for ET-1 were widely distributed within
the left ventricular myocardium of the Bio (Fig. 2A). The
specificity of the immunostaining was confirmed by the
finding that the signals were completely abolished by
preincubating the primary antibody with an excess of
synthetic ET-1 (Fig. 2B). The ET-1 staining intensity in
cardiac myocytes was much higher in the Bio (Fig. 2A) than
in the F1B (Fig. 2C). The ET-1 expression was homogenous, suggesting that systemic hormonal factors are involved
in the ET-1 expression in cardiac myocytes. The interstitial
cells and the endothelial and smooth muscle cells of the
intramyocardial coronary arteries showed modest staining
for ET-1, and the intensity did not differ between the Bio
and F1B. Thus, although various kinds of cells including
fibroblasts, endothelial and smooth muscle cells and cardiac
myocytes expressed ET-1 in the hearts of both F1B and
Bio, the elevated levels of ET-1 in the Bio hearts may be
attributable to ET-1 synthesis in cardiac myocytes.
The effect of T0201 on organ weights and histological
changes. To determine whether the accelerated synthesis
of ET-1 in the heart participates in the pathogenesis of
cardiomyopathy, we randomly assigned five-week-old Bio
and F1B hamsters to treatment with vehicle or an oral ET
type A receptor-specific antagonist, T0201. No deaths
JACC Vol. 33, No. 2, 1999
February 1999:565–71
Figure 3. The effects of ET type A receptor antagonist T0201 on
heart/body weight ratio in Bio and F1B. An oral administration of
the ET type A receptor antagonist T0201 was started at five weeks
of age in Bio and F1B hamsters. Values are mean 6 SEM.
occurred in the vehicle- or T0201-treated Bio and F1B until
sacrifice at the ages of 20 weeks and 35 weeks. Organ
weights were compared between the T0201- and vehicletreated groups. As shown in Figure 3, T0201 did not affect
the heart weight/body weight ratio in the F1B. However, in
the Bio, the heart weight/body weight ratio was significantly
lower in the T0201-treated group than in the vehicletreated group at both 20 weeks and 35 weeks. T0201 did
not affect the lung weight/body weight ratio or the kidney
weight/body weight ratio in the F1B or Bio (data not
shown).
The hearts of cardiomyopathic hamsters spontaneously
develop focal necrosis followed by fibrotic replacement and
secondary hypertrophy (23). We examined the effect of
T0201 on these histological changes in Bio hamsters as
shown in Figure 4. The extent of fibrosis in the Bio did not
differ between the T0201-treated group and the vehicletreated group (Fig. 4A). However, the cardiac myocyte
diameter in the Bio was significantly lower in the T0201treated group than in the vehicle-treated group at 20 weeks
and 35 weeks (Fig. 4B). Administration of T0201 reduced
the cell diameter of Bio to a level similar to that of F1B. By
contrast, T0201 did not affect the myocyte diameter in F1B.
Thus, T0201 did not inhibit replacement fibrosis but
suppressed the growth of remaining myocytes in the Bio
hamsters.
DISCUSSION
To clarify the role of endogenous ET-1 in cardiac growth in
primary myocardial disease, we have studied Syrian cardiomyopathic hamsters of the Bio14.6 strain. The results of the
present study demonstrated that cardiac ET-1 levels in
these animals increased in stage-dependent and organspecific manners. The elevated levels of ET-1 were localized
mainly in cardiac myocytes. In addition, a chronic ETA
receptor blockade with T0201 inhibited myocardial cell
growth in the Bio14.6 hamsters but not in the control F1B
JACC Vol. 33, No. 2, 1999
February 1999:565–71
Figure 4. The effects of ET type A receptor antagonist T0201 on
fibrosis (A) and myocyte diameter (B) in the left ventricles of Bio
and F1B. Values are mean 6 SEM.
animals. These findings suggest that the accelerated cardiac
synthesis of ET-1 contributes to the pathological myocardial growth in this animal model.
Cardiac expression of ET-1 in the Bio14.6 cardiomyopathic hamsters. Plasma ET-1 levels are reported to be
high in various kinds of cardiovascular diseases. In patients
with chronic heart failure, the increase of circulating ET-1
levels correlates with functional class and alterations in
hemodynamics (4,5). Although plasma ET-1 levels are
increased in patients with hypertrophic cardiomyopathy
(14), the increase in these patients is only 2- to 3-fold. The
present study demonstrated that the myocardial ET-1 levels
in cardiomyopathic hamsters increased in a stage-specific
manner. At 35 weeks, the ET-1 levels in the left ventricles
were markedly (6.4-fold) increased in the cardiomyopathic
hamsters compared with the controls. The prominent elevation of ET-1 levels in the heart is compatible with the
idea that ET-1 plays a pathophysiological role as a local
factor rather than a systemic hormone (8,24). In contrast,
Inada et al.
Endothelin-1 in Cardiomyopathy
569
the increase of ET-1 levels in the lungs of the Bio hamsters
was only 1.3-fold. Although these data do not rule out the
possible contribution of other organs to the increased levels
of ET-1 in plasma, these findings suggest that the heart is
one of the main sources of plasma ET-1 in cardiomyopathy.
ET-1 is primarily identified in the supernant of cultured
endothelial cells (1). However, the expression of ET-1 is not
confined to these cells but is also observed in various cell
types including cardiac myocytes (11,22). Stimulation with
angiotensin II or stretch induces the expression of ET-1 in
cultured neonatal cardiac myocytes, and an ETA receptor
antagonist can block the hypertrophy evoked by these
stimuli (11,22). Using an immunohistochemical technique,
the present study demonstrated that the elevated level of
ET-1 in the heart of cardiomyopathic hamsters is localized
mainly in cardiac myocytes. In addition, the chronic administration of an ETA receptor antagonist reduced the myocardial cell diameter of Bio to a level similar to that of
control F1B. Since vasodilative agents such as hydralazine
do not improve the pathology of cardiomyopathic hamsters
(25), the complete blockade of myocardial cell size by an
ETA receptor in cardiomyopathy may not be explained by
hemodynamic unloading. In fact, the dosage of T0201 used
in the present study was not sufficient to decrease the blood
pressure in the animals (data not shown). Our findings are
compatible with in vitro data (11,22) and suggest that ET-1
plays a role as an autocrine factor for hypertrophy in
cardiomyopathy. The triggers that stimulate the cardiac
synthesis of ET-1 in these animals are unclear at present. It
has been reported that sympathetic nervous systems are
activated in these cardiomyopathic hamsters (26). Since an
a1-adrenergic agonist is a potent inducer of ET-1 expression in cultured neonatal cardiac myocytes (27), such neurohormonal factors might contribute to the accelerated
synthesis of cardiac ET-1 in this animal model. However,
further studies are needed to clarify the precise mechanisms
that induce ET-1 expression in cardiac myocytes.
The effect of ETA receptor blocker in the cardiomyopathic hamsters. The Bio 14.6 Syrian hamster has been
pharmacologically well-studied as an experimental model of
cardiomyopathy. In this model, focal myocyte degeneration
occurs at an early stage and is followed by replacement with
fibrosis (23). The number of calcium channels is increased
early in the course of the cardiomyopathy in these hamsters
(28). Calcium-channel blockers such as verapamil have been
shown to reduce the extent of fibrosis and collagen content
in the hearts of these hamsters (25,28). These findings
demonstrate that voltage-dependent calcium uptake and
subsequent calcium accumulation play a significant role in
the development of myocardial cell injury in this animal
model. ET-1 has been shown to increase cytosolic calcium
levels in cardiac myocytes (29). However, while focal myocardial degeneration starts as early as 5 weeks of age in Bio
hamsters, the cardiac ET-1 levels were not elevated at this
stage. In addition, the present findings demonstrate that the
570
Inada et al.
Endothelin-1 in Cardiomyopathy
chronic ETA receptor blockade with T0201 reduced the
myocardial cell diameter in the Bio but not the extent of
fibrosis in cardiomyopathy. These findings suggest that
genetically determined myocyte degeneration occurs
through ET-1-independent pathways while myocardial
ET-1 plays a role in the secondary hypertrophy of the
remaining myocytes. As pharmacological intervention to
prevent the hypertrophic process (for example, angiotensinconverting enzyme inhibitor treatment) has been shown to
be beneficial in this hamster model of cardiomyopathy (30),
blockade of hypertrophy by T0201 would be beneficial in
this animal model.
Hypertrophic cardiomyopathy is a cardiac disease characterized by a hypertrophied, nondilated left ventricle in the
absence of other cardiac disease. Some patients have a left
ventricular outflow obstruction with a relatively high incidence of sudden death, and others display dilatation of the
left ventricle and heart failure in the later stage. Although
missense mutations in the cardiac b-myosin heavy chain
gene have been identified in patients with familial hypertrophic cardiomyopathy (31), the precise mechanisms that
lead to cardiac hypertrophy are still unclear. The present
findings demonstrate that an ET-1 receptor blocker can
inhibit the development of myocellular hypertrophy in the
Syrian hamster model of cardiomyopathy. This model has
focal zones of necrosis and scarring, while hypertrophic
cardiomyopathy in humans shows diffuse abnormalities.
Therefore, we cannot apply the data of the present study to
cardiomyopathy in humans. Nevertheless, taken together
with our previous report that plasma ET-1 levels are
elevated in patients with hypertrophic cardiomyopathy, it
would be of particular interest to investigate whether ET
receptor blockers are useful as therapeutic agents for hypertrophic cardiomyopathy in humans.
Acknowledgments
We greatly appreciate Ms. T. Hoshino and Dr. S. Murata
for their scientific help. This work was supported in part by
grants from the Kanae Foundation of Research for New
Medicine, the Japanese Heart Foundation, the Japan Cardiovascular Research Foundation and the Ministry of Education, Science and Culture of Japan.
Reprint requests and correspondence: Koji Hasegawa, MD,
Department of Cardiovascular Medicine, Kyoto University, Graduate School of Medicine, 54 Shogoin Kawahara-cho, Sakyo-ku,
Kyoto 606-8507, Japan. E-mail: [email protected].
JACC Vol. 33, No. 2, 1999
February 1999:565–71
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
REFERENCES
19.
1. Yanagisawa M, Kurihara H, Kimura S, et al. A noble potent
vasoconstrictor peptide produced by vascular endothelial cells.
Nature 1988;332:411–5.
2. Arai H, Hori S, Aramori I, Ohkubo H, Nakanishi S. Cloning
and expression of a cDNA encoding an endothelin receptor.
Nature 1990;348:730 –2.
3. Sakurai T, Yanagisawa M, Takuwa Y, Miyazaki H, Kimura S,
20.
Goto K, et al. Cloning of a cDNA encoding a nonisopeptideselective subtype of the endothelin receptor. Nature 1990;348:
732–5.
Wei CM, Lerman A, Rodeheffer RJ, et al. Endothelin in
human congestive heart failure. Circulation 1994;89:1580 – 6.
Cody RJ, Hass GJ, Binkley PF, Capers Q, Kelley R. Plasma
endothelin correlates with extent of pulmonary hypertension
in patients with chronic congestive heart failure. Circulation
1992;85:504 –7.
Shimoyama H, Sabbah HN, Borzak S, et al. Short-term
hemodynamic effects of endothelin receptor blockade in dogs
with chronic heart failure. Circulation 1996;94:779 – 84.
Kiowski W, Sutsch G, Hunziker P, Muller P, Kim J, Oechslin
E, Schmitt R, Jones R, Bertel O. Evidence for endothelin-1mediated vasoconstriction severe chronic heart failure. Lancet
1995;346:732– 6.
Haynes WG, Webb DJ. The endothelin family of peptides:
local hormones with diverse roles in health and disease. Clin
Sci 1993;84:485–500.
Shubeita HE, McDonough PM, Harris AN, et al. Endothelin
induction of inositol phospholipid hydrolysis, sarcomere assembly, and cardiac gene expression in ventricular myocytes: a
paracrine mechanism for myocardial cell hypertrophy. J Biol
Chem 1990;25:20,555– 62.
Ito H, Hirata Y, Hiroe M, et al. Endothelin-1 induces
hypertrophy with enhanced expression of muscle-specific
genes in cultured neonatal rat cardiomyocytes. Circ Res
1991;69:209 –15.
Yamazaki T, Komuro I, Kudoh S, Zou Y, Shiojima I, Hiroi Y,
Mizuno T, Maemura K, Kurihara H, Aikawa R, Tanaka H,
Yazaki Y. Endothelin-1 is involved in mechanical stressinduced cardiomyocyte hypertrophy. J Biol Chem 1996;271:
3221– 8.
Ito H, Hiroe M, Hirata Y, et al. Endothelin ETA receptor
antagonist blocks cardiac hypertrophy provoked by hemodynamic overload. Circulation 1994;89:2198 –203.
Sakai S, Miyauchi T, Kobayashi M, Goto K, Sugishita Y.
Inhibition of myocardial endothelin pathway improves longterm survival in heart failure. Nature 1996;384:353–5.
Hasegawa K, Fujiwara H, Koshiji M, Inada T, Ohtani S,
Doyama K, Tanaka M, Matsumori A, Fujiwara T, Shirakami
G, Hosoda K, Nakao K, Sasayama S. Endothelin-1 and its
receptor in hypertrophic cardiomyopathy. Hypertension 1996;
27:259 – 64.
Kitamura K, Tanaka J, Kato T, Ogawa T, Eto T, Tanaka K.
Immunoreactive endothelin in rat kidney inner medulla:
marked decrease in spontaneously hypertensive rats. Biochem
Biophys Res Commun 1989;156:1182– 6.
Hasegawa K, Fujiwara H, Doyama K, et al. Ventricular
expression of brain natriuretic peptide in hypertrophic cardiomyopathy. Circulation 1993;88:372– 80.
Hoshino T, Fujiwara H, Kawai C, Hamashima Y. Myocardial
fiber diameter and regional distribution in the ventricular wall
of normal adult hearts, hypertensive hearts and hearts with
hypertrophic cardiomyopathy. Circulation 1983;67:1109 –16.
Fujiwara H, Hoshino T, Yamana K, et al. Number and size of
myocytes and amount of interstitial space in the ventricular
septum and in the left ventricular free wall in hypertrophic
cardiomyopathy. Am J Cardiol 1983;52:818 –23.
Uegaito T, Fujiwara H, Ishida M, et al. Hypertrophy of
surviving myocytes overlying the infarct in human old myocardial infarctions with abnormal Q waves. Int J Cardiol
1991;32:93–102.
Tanaka M, Fujiwara H, Onodera T, Wu DJ, Hamashima Y,
Kawai C. Quantitative analysis of myocardial fibrosis in
normals, hypertensive hearts, and hypertrophic cardiomyopathy. Br Heart J 1986;55:575– 81.
Inada et al.
Endothelin-1 in Cardiomyopathy
JACC Vol. 33, No. 2, 1999
February 1999:565–71
21. Onodera T, Fujiwara H, Tanaka M, Wu DJ, Hamashima Y,
Kawai C. Quantitative analysis of cardiac fibrosis in normal
hearts, hearts with secondary eccentric hypertrophy and hearts
with dilated cardiomyopathy. Card-Ang Bull 1986;3:53–9.
22. Ito H, Hirata Y, Adachi M, et al. Endothelin-1 is an
autocrine/paracrine factor in the mechanism of angiotensin
II-induced hypertrophy in cultured rat cardiomyocytes. J Clin
Invest 1993;92:398 – 403.
23. Gertz EW. Cardiomyopathic syrian hamster: a possible model
of human disease. Prog Exp Tumor Res 1972;16:242– 60.
24. Haynes WG, Webb DJ. The endothelin family of peptides:
local hormones with diverse roles in health and disease? Clin
Sci 1993;84:485–500.
25. Rouleau JL, Chuck LH, Hollosi G, et al. Verapamil preserves
myocardial contractility in the hereditary cardiomyopathy of
the Syrian hamster. Circ Res 1982;50:405–12.
26. Ikegaya T, Nishiyama T, Kobayashi A, Yamazaki N. Role of
alpha 1-adrenergic receptors and the effect of bunazosin on the
histopathology of cardiomyopathic Syrian hamsters of strain
BIO 14.6. Jpn Circ 1988;52:181–7.
27. Kaburagi S, Hasegawa K, Morimoto T, et al. The role of
28.
29.
30.
31.
571
endothelin-converting enzyme-1 in the development of a1adrenergic stimulated hypertrophy in cultured neonatal rat
cardiac myocytes. Circulation in press.
Kobayashi A, Yamashita T, Kaneko M, et al. Effects of
verapamil on experimental cardiomyopathy in the Bio 14.6
Syrian hamster. J Am Coll Cardiol 1987;10:1128 –38.
Uusimaa PA, Hassinen IE, Vuolteenaho O, Ruskoaho H.
Endothelin-induced atrial natriuretic peptide release from
cultured neonatal cardiac myocytes: the role of extracellular
calcium and protein kinase-C. Endocrinology 1992;130:
2455– 64.
Haleen SJ, Weishaar RE, Overhiser RW, et al. Effects of
quinapril, a new angiotensin converting enzyme inhibitor, on
left ventricular failure and survival in the cardiomyopathic
hamster-Hemodynamic, morphological, and biochemical correlates. Circ Res 1991;68:1302–12.
Geisterfer-Lowrance AAT, Kass S, Tanigawa G, et al. A
molecular basis for familiar hypertrophic cardiomyopathy: a b
cardiac myosin heavy chain gene missense mutation. Cell
1990;62:999 –1006.