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(Hypertension. 1996;27:259-264.)
© 1996 American Heart Association, Inc.

Articles

Endothelin-1 and Its Receptor in Hypertrophic Cardiomyopathy

Koji Hasegawa; Hisayoshi Fujiwara; Masatoshi Koshiji; Tsukasa Inada; Seiji Ohtani; Kiyoshi Doyama; Masaru Tanaka; Akira Matsumori; Takako Fujiwara; Gotaro Shirakami; Kiminori Hosoda; Kazuwa Nakao; Sigetake Sasayama

From The Third (K. Hasegawa, T.I., S.O., K.D., M.T., A.M., S.S.) and Second (G.S., K. Hosoda, K.N.) Divisions, Department of Medicine, Kyoto (Japan) University School of Medicine; The Second Division, Department of Medicine, Gifu (Japan) University School of Medicine (H.F., M.K.); and the Kyoto (Japan) Women's University (T.F.).

Correspondence to Hisayoshi Fujiwara, MD, The Second Division, Department of Medicine, Gifu University School of Medicine, 40 Tsukasa-machi, Gifu, 500, Japan.

	Abstract

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Abstract Endothelin-1, a potent vasoconstrictor produced by vascular endothelial cells, activates the hypertrophic program in cultured heart muscle cells. However, the role of endothelin-1 in cardiac hypertrophy in humans is unknown. Therefore, we studied hypertrophic cardiomyopathy patients with normal pulmonary arterial pressure, in whom cardiac hypertrophy is a specific feature of the disease. Radioimmunoassay with a monoclonal antibody to human endothelin-1 showed that the plasma level of immunoreactive endothelin was more than twofold higher in hypertrophic cardiomyopathy patients than in control subjects (P<.005). In situ hybridization analysis of endomyocardial biopsy specimens showed positive signals of endothelin-1 type A receptor mRNA in ventricular myocytes of all specimens. The receptor expression in ventricular myocytes was similar between hypertrophic cardiomyopathy patients and control subjects. We propose that endothelin-1 might represent an important factor involved in hypertrophic cardiomyopathy. Whether endothelin-1 plays a causal role in cardiac hypertrophy or is a marker of its occurrence needs to be clarified.

Key Words: heart hypertrophy • receptors, endothelin • cardiomyopathy, hypertrophic

	Introduction

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Endothelin was initially identified in porcine vascular endothelial cells as a potent vasoconstrictor peptide with 21 amino acid residues.¹ Cloning and sequencing of endothelin genes has revealed three isopeptides, ET-1, ET-2, and ET-3, that have subsequently been found in a wide variety of vascular and nonvascular tissues.^{2 3} In humans, substantial increases in circulating ET-1 levels have been demonstrated in various pathological states that are associated with low blood pressure,^{4 5} pulmonary hypertension,^{6 7 8} and renal failure.^{9 10} Two functionally distinct endothelin receptors, ET_A and ET_B, have been identified.^{11 12 13 14} The rank order of binding to the ET_A receptor is ET-1>ET-2>>ET-3,^{11 13} and that for the ET_B receptor is ET-1=ET-2=ET-3.^{12 14} In vessel walls, medial smooth muscles express both ET_A and ET_B receptors, which are responsible for the vasoconstrictive action of ET-1.^{11 13 15}

ET-1 not only has contractile effects but also has growth effects on both smooth muscle and heart muscle cells in vitro. In cultured heart muscle cells, ET-1 induces cardiac cell hypertrophy, concomitantly activating the reexpression of cardiac-specific fetal genes.^{16 17} Furthermore, not only endothelial cells but also cardiac myocytes are capable of producing ET-1.¹⁸ These findings prompted us to investigate a possible role of ET-1 in cardiac hypertrophy in vivo. The elevation of ET-1 levels in congestive heart failure specifically correlate with the extent of pulmonary hypertension.⁷ Therefore, we measured IR-ET plasma levels in HCM patients with normal pulmonary arterial pressure, in whom cardiac hypertrophy is a specific feature of the disease. We also examined the expression of the ET_A receptor gene in the ventricular myocardium.

	Methods

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Patient Profile
Twenty-six patients with HCM were evaluated clinically by both noninvasive and invasive methods. The diagnosis of HCM was made according to the definition and classification proposed by the World Health Organization International Society and Federation of Cardiology task force.¹⁹ All patients had normal sinus rhythms and normal ejection fractions and were categorized as New York Heart Association functional class I. All had normal serum creatinine levels less than or equal to 0.11 mmol/L. No patient had an apparent history or clinical findings of congestive heart failure. Eight of the patients had a significant systolic intraventricular pressure gradient of greater than 20 mm Hg at a basal level or after provocation by postextrasystole, isoproterenol, amyl nitrate, or the Valsalva maneuver. The other 18 HCM patients showed no signs of intraventricular pressure gradient. As healthy control subjects, we evaluated six subjects in whom cardiac disease was clinically suspected because of chest pain, minimal electrocardiographic changes, or arrhythmia but in whom invasive and noninvasive examinations had revealed no specific abnormalities. The characteristics of all patients are listed in the Table.

View this table: [in this window] [in a new window]   Table 1. Hemodynamic, Angiographic, Echocardiographic, and Histopathological Data

All patients studied gave their informed consent. The study protocol was approved by the ethics committee on human research of Kyoto University.

Endomyocardial Biopsy Procedure
Ventricular specimens were obtained by endomyocardial biopsy during cardiac catheterization in 20 HCM patients and 4 healthy control subjects. They were obtained from both the right ventricular side of the ventricular septum (RVB) and the left ventricular free wall (LVB). At least two RVB and LVB specimens were obtained from every patient. One of the two specimens was used for the evaluation of histological parameters such as myocyte diameter (mean of 30 to 50 myocytes per specimen), fibrosis, and disarray. The other specimens were stored at -70°C until used for in situ hybridization.

In Situ Hybridization Histochemistry
We analyzed the expression and distribution of ET_A receptor mRNA in a total of 24 ventricular specimens (12 RVB and 12 LVB) by in situ hybridization. With human ET_A receptor cDNA (nucleotides 256 to 1081)¹³ used as a template, both antisense and sense RNA probes with digoxigenin-labeled dUTP (Boehringer Mannheim) were generated by in vitro transcription. The in situ hybridization procedure was performed as previously described.²⁰

To ensure the specificity of the in situ hybridization signals, we performed the following control studies: (1) Negative control probe, sections were hybridized with the corresponding concentrations of digoxigenin-labeled sense cRNA probe, and (2) RNase digestion, sections were incubated with RNase A (1 Kunitz unit per liter) for 1 hour at 37°C before hybridization.

Tissue Section Analysis
In situ hybridization staining was performed at the same time for all specimens and at least twice on serial sections in each specimen. The presence of ET_A receptor mRNA signals was assessed by light microscopy at x200 magnification. The staining was judged to be positive when purple hybridization signals were visible at this magnification. Since ET_A receptor mRNA is expressed in the medial smooth muscle cells of vessel walls,²⁰ we used internal mammary artery specimens, obtained during cardiovascular surgery, as positive controls. We graded the intensity of the positive signals in ventricular myocytes of endomyocardial biopsy specimens from +1 to +3 as follows: +1, mild: signal positive but intensity is lower than the intermediate between negative and positive controls; +2, moderate: signal intensity is higher than the intermediate between negative and positive controls but lower than positive controls; and +3, severe: same as the positive control. Two observers (T.I., S.O.), who were unaware of the patients' data, reviewed the sections and assessed the signal intensity in ventricular myocytes. Unanimity on the intensity was acquired for the serial sections of all specimens and between the two observers for all sections.

Plasma Sampling
Plasma was sampled in 19 HCM patients and 5 healthy control subjects. After the drugs had been discontinued overnight, blood was withdrawn from the antecubital vein at 9 AM while the subjects were recumbent. The samples were immediately transferred to chilled siliconized glass tubes containing Na₂EDTA (3 mmol/L) and aprotinin (1x10⁶ trypsin inhibitor units per liter, Ohkura Pharmaceutical) and centrifuged at 4°C. Plasma was frozen immediately and stored at -70°C until assay.

Measurement of Plasma IR-ET
Plasma IR-ET concentration was measured by RIA with a monoclonal antibody (KY-ET-1-IV), as previously reported.^{8 21 22} This antibody has a high affinity for ET-1 (association constant, 4.8x10¹¹ L/mol). The 50% inhibitory concentration of the RIA with this antibody was 0.68 fmol per tube. Cross-reactivity with ET-2, ET-3, and human big ET-1 was 80%, 20%, and 80%, respectively.^{8 21 22} Standard ET-1 was purchased from the Peptide Institute Inc. The intra-assay and interassay variations of the RIA with KY-ET-1-IV were 4.0% (n=10) and 6.4% (n=10), respectively. Extraction of endothelin from plasma was performed with polystyrene beads coated with the purified monoclonal antibody (KY-ET-1-IV), as previously reported.^{8 21 22} The plasma volume used for the extraction was 0.5 mL.

In a subset of the population (12 HCM patients and 5 healthy control subjects), we performed an ELISA (Wako Chemical Co) according to the manufacturer's instructions. This ELISA is a two-step sandwich method using a monoclonal antibody that recognizes the N-terminal of ET-1 and a peroxidase-conjugated polyclonal antibody that recognizes the C-terminal of ET-1. In this system, cross-reactivity with ET-3 or big ET-1 is less than 0.4%.

Statistical Analysis
Values are expressed as mean±SD. Statistical comparisons were performed with ² analysis, Student's t test, or one-way ANOVA with multiple comparisons, as appropriate. Statistical significance was designated at a probability value less than .05.

	Results

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ET_A Receptor mRNA in Ventricular Myocardium
By in situ hybridization, we observed purple hybridization signals of ET_A receptor mRNA in the medial smooth muscle cells of the internal mammary artery but not in the endothelial cells (Fig 1a). In the endomyocardial specimens, ET_A receptor mRNA signals were distributed not only in small vessels but also in the ventricular myocytes of HCM patients and control subjects. The signals in ventricular myocytes were detected in all of the specimens (Fig 1b through 1d). The signal intensity in ventricular myocytes did not differ between HCM patients (1.6±0.9) and control subjects (1.3±0.3). The intensity was not correlated with any hemodynamic, angiographic, or histological parameters and showed no significant difference between the RVB (1.6±0.9) and LVB (1.4±0.8) specimens.

View larger version (0K): [in this window] [in a new window]   Figure 1. Photomicrographs show expression of ETA receptor mRNA in internal mammary artery specimens (a) and endomyocardial biopsy specimens (b through f) that were analyzed by in situ hybridization with a digoxigenin labeled cRNA probe. The signals were visualized using nitroblue tetrazolium as the substrate. The nuclear counterstaining was performed by methylgreen. In the arteries (a), positive ETA mRNA signals were observed in medial smooth muscle cells (between thin arrows) but not in endothelial cells (thick arrow). Arrow head, asterisk, and square indicate internal elastic lamina, neointima, and adventitia, respectively. In the endomyocardial biopsy specimens of healthy subjects (b) and HCM patients (c and d, respectively), the signals were detected in ventricular myocytes. b, c, and d show representative cases of signal intensity grades 1, 2, and 3, respectively. Signals were not detected in negative controls of RNAase treatment before hybridization (e) or hybridization with the sense cRNA probe (f). Original magnification x200.

In situ hybridization control studies showed that positive staining in the endomyocardial biopsy specimens was abolished by digestion with RNase before hybridization (Fig 1e). Parallel in situ hybridization procedures with the sense cRNA probe were performed on all sections. None of the control sections hybridized with the corresponding concentrations of the sense cRNA probe labeled with digoxigenin showed positive staining (Fig 1f).

Plasma Levels of IR-ET
Fig 2 shows plasma levels of IR-ET in HCM patients and healthy control subjects. The plasma IR-ET level in control subjects was 2.0±0.95 fmol/L (n=5); the level was significantly increased in HCM patients (n=19, 4.8±1.7 fmol/L, P<.005) compared with control subjects. There were no significant differences in IR-ET levels in HCM patients with (n=5, 4.3±1.1 fmol/L) or without (n=14, 5.0±1.9 fmol/L) obstruction. IR-ET levels did not differ among HCM patients receiving no medication (n=4, 4.6±1.7 fmol/L), those receiving calcium antagonists (n=9, 4.9±2.1 fmol/L), those receiving ß-blockers (n=3, 5.6±1.5 fmol/L), and those receiving both (n=3, 4.1±0.65 fmol/L). IR-ET levels were not correlated with hemodynamic and angiographic parameters. However, the IR-ET level had a significant linear correlation with myocyte diameter in LVB specimens (Fig 3a). In RVB specimens, the IR-ET level was grossly correlated with myocyte diameter, but the relationship was not significant (Fig 3b). At present, it is not clear whether this difference in the findings between LVB and RVB specimens represents a significant meaning or is attributed to the low number of data analyzed.

View larger version (13K): [in this window] [in a new window]   Figure 2. Graph shows plasma levels of IR-ET in healthy control subjects (n=5) and HCM patients (n=19).

View larger version (12K): [in this window] [in a new window]   Figure 3. Line graphs show relationship between plasma levels of IR-ET and myocyte diameter in LVB and RVB specimens from HCM patients.

To examine whether mature ET-1 is increased in the plasma of HCM patients, we performed sandwich ELISA, in which cross-reactivity with big ET-1 is less than 0.4%. The result of this ELISA showed that plasma ET-1 levels were significantly higher in the HCM group (n=12, 0.85±0.12 fmol/L) than the healthy group (n=5, 0.54±0.14 fmol/L) (P<.005).

	Discussion

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HCM is a cardiac disease characterized by a hypertrophied, nondilated left ventricle in the absence of other cardiac diseases. Although missense mutations in the cardiac ß-myosin heavy chain gene have been identified in patients with familial HCM,²³ the precise mechanism of cardiac hypertrophy in HCM is still unclear. We demonstrated in the present study that plasma IR-ET levels were higher in HCM patients than in healthy control subjects. However, physiological effects of ET-1 as increases in systemic vascular resistance or decreases in cardiac output²⁴ were not observed in the HCM patients studied here. In a previous report,²⁵ we showed that plasma levels of atrial and brain natriuretic peptides were elevated in HCM patients. Thus, it is conceivable that these peptides may counteract the hemodynamic effects of ET-1.

ET-1 is considered to function more importantly as a local regulator than as a systemic hormone.⁵ Systemic elevation of IR-ET observed in this study may only partly reflect local synthesis. The precise mechanism responsible for the elevation of IR-ET levels in HCM is not clear at present. In congestive heart failure, elevated levels of IR-ET are specifically correlated with the extent of pulmonary hypertension.⁷ In this condition, ET-1 is synthesized in endothelial cells in the pulmonary circulation.²⁶ However, all of the patients studied here had normal pulmonary arterial pressure. Therefore, it is highly unlikely that the elevated level of IR-ET in HCM is attributed to increased synthesis in the lung. A recent report showed that ET-1 mRNA synthesis in the heart is upregulated in hypertrophied hearts by pressure overload.^{27 28} More recently, we have shown that the cardiac content of IR-ET is six times higher in cardiomyopathic hamsters without heart failure compared with age-matched controls.²⁹ In contrast, there was no difference in the pulmonary content of IR-ET between cardiomyopathic hamsters and controls. These findings suggest that the synthesis of ET-1 in hypertrophied hearts is specifically accelerated. However, no direct tissue analysis of endothelin levels has been performed in this study. This is the limitation of an endomyocardial biopsy study, in which specimens are too small to allow us to perform an exact quantitative analysis. Recently, a transgenic animal model of HCM has been reported.³⁰ Study in such an animal model will enable us to clarify the source of elevated endothelin levels in HCM.

Growing evidence suggests that local endocrine factors play an important role in cardiac hypertrophy. ET-1 induces cardiac cell hypertrophy, concomitantly activating the reexpression of cardiac-specific fetal genes.^{16 17} It has been shown that mechanical stretch of cardiac myocytes causes release of angiotensin II from myocytes.³¹ Angiotensin II not only induces cardiac hypertrophy but also upregulates the synthesis and secretion of ET-1 in cardiac myocytes.¹⁸ These findings suggest that angiotensin II and ET-1 constitute a complex positive circuit acting on heart muscle cells in an autocrine/paracrine fashion. Our findings showed that the elevation of IR-ET levels in HCM was associated with cardiac cell hypertrophy. We cannot determine from the current study whether the increase of plasma IR-ET represents a biological marker for the occurrence of cardiac hypertrophy in HCM or whether ET-1 contributes to the pathophysiology of HCM as a specific mechanism of cardiac hypertrophy. However, a recent report showed that an ET_A receptor antagonist blocked left ventricular hypertrophy by pressure overload in vivo.²⁷ These findings suggest a role of ET-1 in the development of cardiac hypertrophy in vivo.

Previous Northern blot studies have shown that human ventricles express ET_A receptor mRNA more abundantly than ET_B receptor mRNA.¹³ However, which cell types express the receptor is still unknown. Therefore, we examined localization of ET_A receptor mRNA in endomyocardial biopsy specimens by in situ hybridization. We have demonstrated that the ET_A receptor gene is expressed in ventricular myocytes in both HCM patients and healthy control subjects. These findings suggest that the physiological action of ET-1 on ventricular myocytes is mediated at least in part through ET_A receptors. It is well known that the prolonged stimulation of the receptor by its agonist results in a decrease in receptor density (homologous downregulation). The expression of ET_A receptor mRNA in ventricular myocytes was similar between healthy subjects and HCM patients. The analysis of ET_A receptor is only semiquantitative. There is a small possibility that we could not detect the difference in the receptor expression between healthy subjects and HCM patients because of the low sensitivity of this semiquantitative method. In hearts with heart failure, in which ET-1 levels are reported to be high, Northern blots showed that the receptor expression was similar to that in normal hearts (unpublished data, 1995). The quantitative Northern blot study for receptor expression in HCM patients without pulmonary hypertension is now ongoing in our laboratory. Recently, we have shown that the ET_A receptor gene is expressed in the thickened arterial intima of hypertensive patients but not in the intima of normotensive patients.²¹ These findings suggest that the regulated expression of the ET_A receptor affects cell growth activity. However, further studies are needed to elucidate the precise mechanism that underlies endothelin receptor regulation.

The monoclonal antibody used in our study recognizes ET-1, big ET-1, and another precursor form of endothelin (6K).^{8 22 23} In patients with acute myocardial infarction, ET-1 and big ET-1 in plasma are almost equally elevated.⁵ However, Wei et al³² have suggested that the increase of endothelin measured in heart failure is due to big ET-1 rather than mature ET-1. To examine whether mature ET-1 is increased in plasma from HCM patients, we performed sandwich ELISA, in which cross-reactivity with big ET-1 is less than 0.4%. The result obtained by this ELISA demonstrated that mature ET-1 levels are higher in HCM patients compared with healthy subjects. The values obtained by this ELISA were much lower compared with those obtained by RIA with KY-ET-1-IV. This may be attributed to the fact that the antibody used in the ELISA has lower cross-reactivities with big ET-1 than that used in the RIA. The values by RIA minus those by ELISA were also higher in HCM patients compared with healthy subjects, suggesting that not only ET-1 levels but also big ET-1 levels are increased in HCM.

Finally, some of the HCM patients studied had received calcium antagonists or ß-blockers, although all drugs were discontinued overnight before blood sampling. It has not yet been clarified whether these drugs have any effect on ET-1 production. However, IR-ET levels did not differ among HCM patients receiving no medication, calcium antagonists, ß-blockers, or both. Therefore, the elevation of IR-ET levels in HCM may not be explained by these drug effects.

In summary, we have shown that IR-ET levels are elevated in HCM patients with normal pulmonary arterial pressure, in whom cardiac hypertrophy is a specific feature of the disease. ET_A receptor is expressed in ventricular myocytes of HCM patients and healthy control subjects. The receptor expression was similar between healthy subjects and HCM patients. Whether ET-1 plays a causal role in cardiac hypertrophy or is a marker of its occurrence is not clear at present. However, given our results and those of a recent report on effective block of cardiac hypertrophy by an ET_A receptor antagonist,²⁷ we propose that the endothelin system might play an important role in HCM.

	Selected Abbreviations and Acronyms

 

ELISA = enzyme-linked immunosorbent assay ET-1, -2, -3 = endothelin-1, -2, -3 ETA, ETB = endothelin-A, endothelin-B HCM = hypertrophic cardiomyopathy IR-ET = immunoreactive endothelin LVB = left ventricular biopsy RIA = radioimmunoassay RVB = right ventricular biopsy

	Acknowledgments

 
This work was supported in part by research grants No. 04670563 (1992) and No. 06454289 (1994) from the Ministry of Education, Science, and Culture of Japan. We greatly thank Akiko Miyashita and Mami Kohno for their technical assistance.

Received September 14, 1995; first decision October 23, 1995; accepted October 23, 1995.

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