- Email: [email protected]
Upregulated expression of cardiac endothelin-1 participates in myocardial cell growth in Bio14.6 Syrian cardiomyopathic hamsters
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.
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.