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Am J Physiol Regul Integr Comp Physiol 277: R1321-R1330, 1999;
0363-6119/99 $5.00
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Vol. 277, Issue 5, R1321-R1330, November 1999

Effects of physiological or pathological pressure load in vivo on myocardial expression of ET-1 and receptors

Michihiko Ueno1, Takashi Miyauchi1,2, Satoshi Sakai1, Tsutomu Kobayashi1, Katsutoshi Goto2,3, and Iwao Yamaguchi1

1 Cardiovascular Division, Department of Internal Medicine, Institute of Clinical Medicine, 3 Department of Pharmacology, Institute of Basic Medical Sciences, and 2 Center for Tsukuba Advanced Research Alliance, University of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Endothelin (ET)-1 has potent positive inotropic and chronotropic activity in the heart and induces cardiac hypertrophy. The production of ET-1 in the heart is reported to be increased under some conditions. In normal circulation, the pressure load to the left ventricle (LV) is much greater than that to the right ventricle (RV). In this study, we investigated the gene expression of the myocardial ET-1 system (ET-1, ETA receptor, and ETB receptor) in the RV and LV of normal rats and also investigated these genes in hypertrophied RV due to pathological pulmonary hypertension (PH). Normal rats showed no differences between the RV and LV in the gene expression of either ET-1, ETA receptor, or ETB receptor in either the adult stage (11 wk old) or the neonatal stage (1 and 8 days old). On the other hand, the expression of both atrial natriuretic peptide (ANP) mRNA and B-type natriuretic peptide (BNP) mRNA was significantly greater in the LV than in the RV in adult rats. Gene expression of ET-1, ETA receptor, and ETB receptor in the RV was markedly higher in rats with monocrotaline-induced (pathological) PH than that in control rats. The expression of ANP mRNA and BNP mRNA in the RV was also markedly higher in the rats with PH. In conclusion, the data suggest that gene expression of the ET-1 system in the myocardium is not affected by physiological pressure load in either the adult stage or neonatal stage; however, it is enhanced by pathological pressure overload such as that in PH.

myocardial endothelin-1 system; pressure overload; pulmonary hypertension; cardiac hypertrophy; atrial natriuretic peptide; B-type natriuretic peptide


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ENDOTHELIN (ET)-1, a potent vasoconstrictor peptide derived from cultured endothelial cells (30, 33, 34, 53), is also produced by cardiac myocytes (48). ET-1 has potent positive inotropic (increase in myocardial contractility) and chronotropic (increase in heart rate) effects on isolated heart muscle (16, 17) and induces myocardial cell hypertrophy (46). These actions are mediated by the receptors for ET-1 [ETA receptor (3) and ETB receptor (44)] on the cardiac myocytes (for reviews, see Refs. 10, 11, and 31). We previously reported that the production of ET-1 in the heart was increased under some pathophysiological conditions, such as hypertrophied heart due to pressure overload (35, 54) and failing heart due to chronic heart failure (CHF) caused by myocardial infarction (39, 41). These data suggest that the ET-1 system (ET-1, ETA receptor, and ETB receptor), which is regarded as one of the cardiac hormone systems, is altered in the heart in these pathophysiological states.

Atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP), both vasodilators (4, 5, 55), are other cardiac hormones with potent diuretic and natriuretic activities (6, 20, 47) that have antihypertrophic action (2, 14). We and other researchers reported that the expression of both ANP mRNA and BNP mRNA was increased in ventricles subjected to pathological pressure overload or volume overload in experimental animal models (1, 25, 42).

In normal circulation, the pressure load to the left ventricle (LV) is several times greater than that to the right ventricle (RV). The RV, which controls pulmonary circulation, is a low-pressure chamber, whereas the LV, which controls systemic circulation, is a high-pressure chamber. Thus it is thought that some qualitative and functional differences exist between the RV and LV. On the other hand, pulmonary arterial pressure in neonates at birth is significantly higher than that in adults (9), indicating that neonates have physiological pulmonary hypertension (PH) that is attenuated at the later phase of the neonatal stage (9). Moreover, various pathological conditions such as lung diseases and heart diseases produce an increase in pulmonary arterial pressure, i.e., a condition of pathological PH (9, 36), and the RV suffers pressure overload from the pathological PH (36). It is not known whether the ET-1 pathway contributes differently to myocardial physiology/pathophysiology between the RV and LV under normal (physiological) and pathological conditions, and there is no report of a comparison of gene expression of the ET-1 system (ET-1, ETA receptor, and ETB receptor) between the RV and LV under these conditions.

Next, we hypothesized that the ET-1 system contributes differently to myocardium under physiological conditions and pathological conditions. In this study, to address this hypothesis, we experimented as follows. We investigated gene expression of the myocardial ET-1 system in the RV and LV in normal rats in the adult stage and in the neonatal stage when physiological PH exists. Because a single subcutaneous injection of monocrotaline, a pyrrolizidine alkaloid, has been reported to cause severe pathological PH in rats (35), we used monocrotaline-induced PH and RV hypertrophy as a model of pathological PH to investigate the above-mentioned gene expression. Furthermore, we compared the expression pattern of these genes with that of ANP and BNP genes in the heart under normal (physiological) and pathological conditions.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Study protocols. In the present study, three series of experiments were performed. The purpose of the first series of experiments was to compare prepro-ET-1, ETA receptor, and ETB receptor with respect to gene expression in the RV and LV in normal adult rats. We also investigated the expression of ANP and BNP genes in the RV and LV in these rats. The purpose of the second series of experiments was to investigate whether the expression of these genes was altered during the neonatal term when physiological PH exists. The purpose of the third series of experiments was to investigate whether the expression of these genes in the heart was altered under pathological pressure overload due to severe PH induced by monocrotaline.

First series of experiments. We used 11-wk-old normal male Sprague-Dawley (SD) rats weighing 420-450 g. The hemodynamic parameters were measured according to our previous papers (35, 39, 41, 54) with minor modifications. In brief, on the day of the experiment, the rats were anesthetized with pentobarbital sodium (50 mg/kg ip). A polyethylene catheter was inserted in the right carotid artery. After arterial blood pressure and heart rate were monitored, the catheter was advanced in the LV for the evaluation of LV pressure. Next, a polyethylene catheter was inserted in the right jugular vein and advanced in the RV for the evaluation of RV pressure. These hemodynamic measurements were recorded with a polygraph system (AP-601G amplifier and WT-687G thermal pen recorder; Nihon Koden, Tokyo, Japan). In addition, LV + dP/dtmax, LV - dP/dtmax, RV + dP/dtmax, and RV - dP/dtmax were derived by an active analog differentiation of the pressure signal differentiation amplifier (model EQ-601G; Nihon Koden).

After hemodynamic measurement, the heart was excised and divided into the RV, LV, and intraventricular septum (IVS). Each ventricle (without the IVS) was weighed and rapidly frozen in liquid nitrogen. The tissue samples were stored at -80°C until determination of the mRNA expression of prepro-ET-1, ETA receptor, ETB receptor, ANP, and BNP by RT-PCR.

Second series of experiments. We used 1-day-old rats weighing 5.57-6.53 g and 8-day-old rats weighing 20.2-21.6 g born from an identical female SD rat. On the experimental day, the neonatal rats were anesthetized with diethyl ether. The heart was excised and divided into the RV and LV. Each ventricle was weighed, frozen in liquid nitrogen, and stored at -80°C until evaluation of the expression of prepro-ET-1 mRNA, ANP mRNA, and BNP mRNA by RT-PCR.

Third series of experiments. Four-week-old male SD rats were used. Treatment with monocrotaline (Wako Pure Chemical, Osaka, Japan) was performed as we previously described (35); the rats were given a single subcutaneous injection of 60 mg/kg monocrotaline (pulmonary hypertensive rats) or saline (vehicle; control rats) and were killed 3 wk after the injection. On the experimental day, the rats were anesthetized, and hemodynamics were measured by the above-mentioned methods. In brief, the rats were anesthetized with pentobarbital sodium (50 mg/kg ip). A polyethylene catheter was inserted in the right carotid artery. After arterial blood pressure and heart rate were monitored, the catheter was advanced in the LV for the evaluation of LV pressure. Next, a polyethylene catheter was inserted in the right jugular vein and was advanced in the RV for the evaluation of RV pressure. These hemodynamic measurements were recorded with a polygraph system (AP-601G amplifier and WT-687G thermal pen recorder; Nihon Koden). After hemodynamic measurement, the heart was excised and divided into the RV and LV. Each ventricle was weighed and frozen in liquid nitrogen. The tissue samples were stored at -80°C until determination of the mRNA expression of prepro-ET-1, ETA receptor, ETB receptor, ANP, and BNP by RT-PCR.

RT-PCR to evaluate the expression of prepro-ET-1 mRNA, ETA receptor mRNA, ETB receptor mRNA, ANP mRNA, and BNP mRNA in the heart. The expression of prepro-ET-1 mRNA, ETA receptor mRNA, ETB receptor mRNA, ANP mRNA, and BNP mRNA was analyzed by RT-PCR. The expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was also determined as an internal control.

Total tissue RNA was isolated by acid guanidinium thiocyanate-phenol-chloroform extraction with ISOGEN (Nippon Gene, Tokyo, Japan) according to methods described in our previous papers (22, 23, 29, 40). The tissue was homogenized in ISOGEN (100 mg tissue/1 ml ISOGEN) with a Polytron tissue homogenizer (PT10SK/35; Kinematica, Lucerne, Switzerland). Homogenization was followed by chloroform extraction, isopropanol precipitation, and 80% (vol/vol) ethanol washing of precipitated RNA. The obtained RNA was resolved in diethyl pyrocarbonate-treated water, treated with DNase I (TaKaRa, Otsu, Japan), and extracted again using ISOGEN to eliminate the genomic DNA. The RNA concentration was determined spectrophotometrically at 260 nm.

Total RNA (5 µg) was primed with 0.05 µg oligo(dT)12-18 and was reverse transcribed using avian myelloblastosis virus reverse transcriptase using a First-Strand cDNA Synthesis kit (Life Sciences). The reaction was performed at 43°C for 60 min.

The cDNA was diluted in a 1:10 ratio, and 1 µl was used for PCR. Each PCR reaction contained 10 mM Tris · HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 200 µM of each dNTP, 0.5 µM of each gene-specific primer, and 0.025 U/µl Taq polymerase (TaKaRa). The gene-specific primers were synthesized according to the published cDNA sequences for each of the following: prepro-ET-1 (43), ETA receptor (28), ETB receptor (44), ANP (21), BNP (24), and GAPDH (51). The sequences of the oligonucleotides were as follows: prepro-ET-1 (sense), 5'-TCTTCTCTCTGCTGTTTGTG-3' (nucleotide 20-39); prepro-ET-1 (antisense), 5'-TTAGTTTTCTTCCCTCCACC-3' (nucleotide 483-502); ETA receptor (sense), 5'-ATCGCTGACAATGCTGAGAG-3' (nucleotide 55-74); ETA receptor (antisense), 5'-CCACGATGAAAATGGTACAG-3' (nucleotide 261-280); ETB receptor (sense), 5'-GAAAAGAGGATTCCCACCTG-3' (nucleotide 81-100); ETB receptor (antisense), 5'-ACGAACACGAGGCATGATAC-3' (nucleotide 316-335); ANP (sense), 5'-ATGGGCTCCTTCTCCATCACC-3' (nucleotide 1-21); ANP (antisense), 5'-TCCGCTCTGGGCTCCAATCCTGT-3' (nucleotide 404-426); BNP (sense), 5'-TAATCTGTCGCCGCTGGGAGG-3' (nucleotide 51-71); BNP (antisense), 5'-GAGCTGGGGAAAGAAGAGCCG-3' (nucleotide 409-429); GAPDH (sense), 5'-GCCATCAACGACCCCTTCATTG-3' (nucleotide 88-109); GAPDH (antisense), 5'-TGCCAGTGAGCTTCCCGTTC-3' (nucleotide 666-685).

PCR was carried out using a PCR thermal cycler (TP-3000; TaKaRa). The cycle profile included denaturation for 15 s at 94°C, annealing for 15 s at each suitable temperature, and extension for 45 s at 72°C. The annealing temperatures were set as follows: prepro-ET-1, 54°C; ETA receptor, 58°C; ETB receptor, 58°C; ANP, 65°C; BNP, 70°C; and GAPDH, 62°C. The reaction cycles of PCR were performed in the range that demonstrates a linear correlation between the amount of cDNA and the yield of PCR products (29). The PCR products were found to be of the expected size as shown by 1.2% agarose gel electrophoresis. In addition, the specificity of the amplified sequences was confirmed by restriction enzyme analysis and DNA sequencing.

Quantitative analysis of PCR products. The amplified PCR products were electrophoresed on 1.2% agarose gels, stained with ethidium bromide, visualized by an ultraviolet transilluminator, and photographed. The PCR products were running on the same gel in each comparison study. The photographs were scanned by a scanner (CanoScan 600; Canon, Tokyo Japan), and quantification was performed by a personal computer with MacBAS software (FUJI Film, Tokyo, Japan).

Preparation of the positive-control cDNAs of prepro-ET-1, ETA receptor, ETB receptor, ANP, BNP, and GAPDH. The cDNAs for the verification of the semiquantitative PCR analysis were performed from each gene PCR product of rat cDNA. Each PCR product was purified, quantified, and used as positive-control cDNAs.

Verification of the semiquantitative PCR analysis. We performed quantitative PCR analysis to evaluate the expression level of prepro-ET-1 mRNA, ETA receptor mRNA, ETB receptor mRNA, ANP mRNA, BNP mRNA, and GAPDH mRNA. To demonstrate that our quantitative PCR strategy was valid, serial dilutions of the each positive-control cDNA were amplified by PCR and quantified by scanner.

Statistical analysis. All data are presented as means ± SE. All statistical comparisons were performed with a commercially available statistical package for the Macintosh personal computer (STAT VIEW, version 4.5; Abacus Concepts, Berkeley, CA). Differences were calculated using the unpaired Student's t-test. The results were considered statistically significant at the level of P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

First series of experiments. The mean weights of the hearts used in this series are shown in Table 1: LV, 0.58 ± 0.02 g (n = 10); RV, 0.27 ± 0.01 g (n = 10). All of the hemodynamic parameters were within the normal range. Systolic pressure, +dP/dtmax, and -dP/dtmax of the RV and LV are also shown in Table 1. The values for the LV were significantly higher than those for the RV (Table 1). The end-diastolic pressure did not differ between the RV and LV (Table 1). These results indicate that the rats used in the experiments were normal.

                              
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  		Table 1.   Wet weight and hemodynamic parameter of right ventricle and left ventricle of adult normal rats

The relationships between the amount of cDNA and the yield of PCR products are shown in Fig. 1. As indicated in Fig. 1A, there was a linear correlation between the initial amount of prepro-ET-1 cDNA and the yield of PCR products. In the cases of ETA receptor, ETB receptor, ANP, BNP, and GAPDH, the yield of PCR products was also in proportion to the initial amount of cDNA (Fig. 1, B-F). The expression of prepro-ET-1 mRNA did not differ between the RV and LV (Fig. 2, A and B), nor was there any difference in the expression of mRNA of either ETA receptor or ETB receptor between the RV and LV (Fig. 2, A, C, and D). On the other hand, the expression of ANP mRNA and BNP mRNA was 2-fold and 2.6-fold higher, respectively, in the LV than in the RV (Fig. 2, A, E, and F).


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  		Fig. 1.   Verification of semiquantitative PCR analysis for prepro-endothelin (ET)-1 mRNA, endothelin-A (ETA) receptor mRNA, endothelin-B (ETB) receptor mRNA, atrial natriuretic peptide (ANP) mRNA, B-type natriuretic peptide (BNP) mRNA, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. Serial dilutions of positive-control cDNA of prepro-ET-1 (A), ETA receptor mRNA (B), ETB receptor mRNA (C), ANP mRNA (D), BNP mRNA (E), and GAPDH mRNA (F) were amplified for each suitable cycle of PCR. PCR products were electrophoresed, and the images of the PCR products were quantified by a scanner. Results are expressed as densitometric values. Each value was determined in duplicate.



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  		Fig. 2.   Comparison of gene expression of the ET-1 system (ET-1, ETA receptor, and ETB receptor) and natriuretic peptides between the right ventricle (RV) and left ventricle (LV) in normal adult rats. In A, representative gels of PCR analysis are shown for prepro-ET-1 mRNA, ETA receptor mRNA, ETB receptor mRNA, ANP mRNA, and BNP mRNA. The expression of GAPDH mRNA is shown as an internal control. B-F show the results of statistical analysis of the expression of these genes. PCR products were scanned by a densitometer, and the ratios of prepro-ET-1 mRNA (B), ETA receptor mRNA (C), ETB receptor mRNA (D), ANP mRNA (E), and BNP mRNA (F) to GAPDH mRNA were calculated. Thus the level of expression of each gene was normalized by that of GAPDH. Open bars indicate the RV, and hatched bars indicate the LV. Each column and bar represent the means ± SE of 10 normal adult rats. NS, not significant.

Second series of experiments. In neonatal rats used in this study, the weight of the heart was as follows: at age 1 day, the RV weighed 10.25 ± 0.74 mg (n = 6), and the LV weighed 17.25 ± 1.60 mg (n = 6); and at age of 8 days, the RV weighed 29.9 ± 2.50 mg (n = 6), and the LV weighed 69.9 ± 2.79 mg (n = 6). A comparison of RV weights between neonatal rats and adult rats is shown in Table 2. From these data, it is considered that in neonatal rats physiological PH exists at age 1 day and it is attenuating at age 8 days.

                              
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  		Table 2.   Wet weight of right ventricle: comparison between neonatal rats and adult rat

The relationships between the amount of cDNA and the yield of PCR products are shown in Fig. 1. The expression of prepro-ET-1 mRNA did not differ between the RV and LV at either age 1 day or age 8 days (Fig. 3, A, B, E, and F). These findings suggest that the expression of prepro-ET-1 mRNA was not affected by physiological PH during the neonatal stage. On the other hand, the expression of ANP mRNA and BNP mRNA did not differ between the RV and LV at age 1 day (Fig. 3, A, C, and D); however, the expression of both ANP mRNA and BNP mRNA was significantly lower in the RV than in the LV at age 8 days (Fig. 3, E, G, and H). By the direct comparison, the expression of ANP mRNA and BNP mRNA was significantly decreased from RV at age 1 day to RV at age 8 days (one-third and one-fourth, respectively). These findings suggest that the expression of both ANP mRNA and BNP mRNA in RV was affected by the degree of physiological PH.


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  		Fig. 3.   Comparison of gene expression of ET-1, ANP, and BNP between the RV and LV in neonatal rats. Physiological pulmonary hypertension (PH) exists at age 1 day and diminishes at age 8 days. Representative gels of RT-PCR analysis are shown for prepro-ET-1 mRNA at age 1 day (A) and at age 8 days (E), ANP mRNA at age 1 day (A) and at age 8 days (E), BNP mRNA at age 1 day (A) and at age 8 days (E). Expression of GAPDH mRNA is shown as an internal control. B-D and F-H show the results of statistical analysis of the expression of these genes. PCR products were scanned by a densitometer, and the ratios of prepro-ET-1 mRNA to GAPDH mRNA at age 1 day (B) and at age 8 days (F), ANP mRNA to GAPDH mRNA at age 1 day (C) and at age 8 days (G), BNP mRNA to GAPDH mRNA at age 1 day (D) and at age 8 days (H) were calculated. Thus the level of expression of each gene was normalized by that of GAPDH. Open bars indicate the RV, and hatched bars indicate the LV. Each column and bar represent the mean ± SE of 6 neonatal rats.

Third series of experiments. Hemodynamic parameter and wet weight of control rats and pulmonary hypertensive rats are shown in Table 3. The body weight of pulmonary hypertensive rats was significantly lower than that of control rats (n = 8). The mean arterial blood pressure was significantly lower in pulmonary hypertensive rats than in control rats (Table 3). The RV systolic pressure was markedly higher in pulmonary hypertensive rats than in control rats (Table 3), suggesting that pulmonary hypertensive rats developed severe PH. Both RV weight-to-body weight ratio and RV weight-to-LV weight ratio were markedly higher in pulmonary hypertensive rats than in control rats (Table 3), indicating that the RV developed marked cardiac hypertrophy due to severe PH in pulmonary hypertensive rats. The relationships between the amount of cDNA and the yield of PCR products are shown in Fig. 1. The expression of prepro-ET-1 mRNA, ETA receptor mRNA, and ETB receptor mRNA in the RV was markedly higher in pulmonary hypertensive rats than in control rats (Fig. 4, A-D). The expression of ANP mRNA and BNP mRNA in the RV was also markedly higher in pulmonary hypertensive rats than in control rats (Fig. 4, A, E, and F). In pulmonary hypertensive rats, the expression of prepro-ET-1 mRNA, ETA receptor mRNA, and ETB receptor mRNA was 2.2-fold, 1.6-fold, and 2.0-fold higher, respectively, in the RV than in the LV. Furthermore, the expression of ANP mRNA was 1.3-fold higher in the RV than in the LV, and the expression of BNP mRNA did not differ between the RV and LV in pulmonary hypertensive rats.

                              
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  		Table 3.   Hemodynamic parameter and wet weight of control rats and pulmonary hypertensive rats



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  		Fig. 4.   Comparison of gene expression of the ET-1 system (ET-1, ETA receptor, and ETB receptor) and natriuretic peptides in the RV between control rats and rats with monocrotaline-induced PH (pulmonary hypertensive rats). A: representative gels of RT-PCR analysis are shown for prepro-ET-1 mRNA, ETA receptor mRNA, ETB receptor mRNA, ANP mRNA, and BNP mRNA. Expression of GAPDH mRNA is shown as an internal control. B-F show the results of statistical analysis of the expression of these genes. PCR products were scanned by a densitometer, and the ratios of prepro-ET-1 mRNA (B), ETA receptor mRNA (C), ETB receptor mRNA (D), ANP mRNA (E), and BNP mRNA (F) to GAPDH mRNA were calculated. Thus the level of expression of each gene was normalized by that of GAPDH. Open bars indicate the RV in control rats, and filled bars indicate the RV in pulmonary hypertensive rats. Each column and bar represent the mean ± SE of 8 control rats and 8 pulmonary hypertensive rats.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The present study showed that gene expression of the ET-1 system (ET-1, ETA receptor, and ETB receptor) did not differ between the RV and LV in normal adult rats, although the physiological pressure load to the LV was much greater than that to the RV. In neonatal rats, the expression of the ET-1 gene (prepro-ET-1 mRNA) did not differ between the RV and LV at either age 1 day when physiological PH exists or at age 8 days when physiological PH diminishes. These findings suggest that the regulation of the gene expression of the myocardial ET-1 system is not affected by physiological pressure load at either the adult stage or at the neonatal stage. On the other hand, the expression of ANP mRNA and BNP mRNA was significantly higher in the LV than in the RV in normal adult rats, suggesting that the physiological pressure load to the myocardium caused an increase in the expression of ANP mRNA and BNP mRNA at the adult stage. In neonatal rats, the expression of ANP mRNA and BNP mRNA did not differ between the RV and LV at age 1 day but was significantly lower in the RV than in the LV at age 8 days, suggesting that the expression of both ANP mRNA and BNP mRNA in the RV decreases with the attenuation of physiological PH in the later stage of the neonatal term. On the contrary, under pathological conditions, gene expression of the ET-1 system (ET-1, ETA receptor, and ETB receptor) was markedly higher in hypertrophied RV due to pathological PH induced by monocrotaline than in the RV of normal control rats. Furthermore, the expression of ANP mRNA and BNP mRNA was also markedly increased in the RV of pulmonary hypertensive rats. These findings suggest that the pattern of gene expression in the heart differs between the ET-1 system and natriuretic peptides; the gene expression of the ET-1 system is increased only when the heart is exposed to a pathological pressure load but not when exposed to a physiological pressure load, whereas the gene expression of ANP and BNP in the heart is affected by pressure load, whether it is physiological or pathological.

It is considered that the major stimulus for myocardial hypertrophy and the induction of growth signals is an increase in wall stress. The basic formula for wall stress consists of systolic pressure and wall thickness, so it is likely that there is not so much difference in wall stress between the RV and LV. However, under the pathological pressure overload, the wall stress to the myocardium is increased. It was previously reported that cardiac c-fos and c-jun protooncogene are induced by increased systolic wall stress to the ventricle (45). It has been demonstrated that the 5' flanking region of the prepro-ET-1 mRNA gene has three octanucleotide sequences that conform with a consensus of AP-1/Jun-binding elements (15). Accordingly, it is likely that increased wall stress may induce prepro-ET-1 mRNA expression via the expression of the trans-acting transcription factors Fos and Jun. Therefore, it is speculated that the gene expression of the ET-1 system does not differ between the RV and LV in physiological conditions because wall stress to the RV and LV is similar, and, under pathological pressure overload, increased wall stress induces this gene expression.

The marked increase in the expression of prepro-ET-1 mRNA observed in this study in the RV of pulmonary hypertensive rats at severe PH stage was in accordance with findings in our previous report (35). Moreover, this study demonstrated for the first time that the expression of ETA receptor mRNA and ETB receptor mRNA was also significantly increased in the RV of pulmonary hypertensive rats, suggesting that the ET-1 system was markedly activated in the hypertrophied RV due to pathological pressure load in pulmonary hypertensive rats. Therefore, it is likely that the endogenously activated ET-1 pathway plays some role in the heart of pulmonary hypertensive rats. We previously reported that the ET-1 pathway was markedly activated in the failing myocardium of rats with CHF due to myocardial infarction; a marked increase in ET-1 production was observed in the failing heart (39, 41). In that study, we investigated the role of the activated ET-1 pathway in cardiac function by administration of an ETA-selective receptor antagonist (41). The administration of an ETA-selective receptor antagonist for 120 min significantly decreased both heart rate and LV + dP/dtmax, a parameter of myocardial contractility, in rats with CHF but did not alter these hemodynamic parameters in control sham-operated rats (41). These findings suggested that endogenous ET-1 produced in the myocardium was not involved in the increase in heart rate and myocardial contractility under normal conditions in the rats, whereas the endogenously activated ET-1 pathway led to the increase in heart rate and myocardial contractility in the failing myocardium of rats with CHF where myocardial ET-1 production was greatly enhanced. These findings suggest that the level of ET-1 in the myocardium under normal conditions is not sufficient to drive pharmacological actions; however, the level of myocardial ET-1 under pathological conditions, such as pathological PH and CHF, is sufficient to exert actions such as positive inotropic (increase in myocardial contractility) and chronotropic (increase in heart rate) effects. Thus it is probable that the effect of ET-1 in the heart is exerted only in certain pathological conditions in which the ET-1 production is increased, but not under normal conditions.

It has been clarified that the synthesis of ANP and BNP is increased in the ventricle subjected to various pathological stressors (1, 12, 13, 25). It was reported that the expression of ANP mRNA and BNP mRNA (37) and the transcription of ANP gene (8) were more prevalent in the LV than in the RV. Also, in human tissues from the fetal stage, when the pulmonary vascular resistance is higher than the systemic vascular resistance and the RV pressure is very high (9), it was reported that the tissue content of ANP was higher in the RV than in the LV (52), suggesting that the expression of the ANP gene in human ventricles is developmentally regulated from the early gestational stage (52). In the present study, the gene expression of ANP and BNP in the heart is augmented by pressure load whether it is physiological or pathological. The findings of this study concerning the gene expression of ANP and BNP are in accordance with those of previous reports (8, 13, 37, 52). The present study demonstrated for the first time that the gene expression of ANP and BNP in the myocardium is regulated differently from that of the ET-1 system (ET-1, ETA receptor, and ETB receptor).

The present study has the following study limitation. In this study, we supposed that the gene expression of the ET system and natriuretic peptides represents the protein level and receptor activation of the gene expression of the ET system and natriuretic peptides. However, it is considerable that the mRNA level of each gene and protein level or receptor activation is not always changing similarly. Concerning the ET-1 system, the expression of ET-converting enzyme mRNA did not differ between the RV and LV in normal adult rats in our unpublished observation. Therefore, we guess that the protein level and receptor activation of the ET system do not differ between the RV and LV in normal adult rats; however, it still remains a study limitation. In summary, the pattern of the gene expression of the ET-1 system in the heart differed under physiological and pathological pressure loads. The gene expression of the ET-1 system, including ET-1, ETA receptor, and ETB receptor in the myocardium, was not affected by physiological pressure load either at the adult stage or neonatal stage; however, it was increased by pathological pressure load due to pathological PH. This pattern of alternation of the ET-1 system is not in accordance with the pattern of the gene expression of natriuretic peptides, i.e., the gene expression of ANP and BNP was enhanced by pressure load not only under pathological load but also under physiological load. Therefore, it is suggested that pressure overload-induced alteration of gene expression in the heart under physiological conditions and pathological conditions differs between the ET-1 system and the natriuretic peptide system.

Perspectives

The present findings suggest that the gene expression of the ET-1 system in the myocardium is not affected by physiological pressure load at either the adult stage or neonatal stage; however, it is enhanced by pathological pressure overload due to pathological PH. It was reported that ET-1 induces myocardial cell hypertrophy in cultured rat cardiomyocytes (18, 46). We reported that the production of ET-1 was significantly increased in the LV of rats with aortic banding (54) in which the LV suffered pathological pressure load due to aortic stenosis (54). In the case of pulmonary hypertensive rats, the RV suffers pressure overload from pathological PH and develops a marked cardiac hypertrophy, as shown in this study and in our previous study (35). We reported that, in the case of rats with CHF, ET-1 production was markedly increased in the failing myocardium in the LV, and the cardiac myocytes in the LV of these rats also developed cardiac hypertrophy (39). Taken together, it can be concluded that cardiac hypertrophy caused by cardiovascular diseases, i.e., pathological cardiac hypertrophy, is often accompanied by an increase in the production of myocardial ET-1.

On the other hand, various studies have been performed on the ET receptor system. In the human heart, there are ETA receptors and ETB receptors; however, only ETA receptors are of functional importance (38). Also, in end-stage CHF, the functional responsiveness of the cardiac ETA receptor system is not altered (38). In the experiment of an animal model, it was reported that ETA receptor density did not differ between normal pigs and pigs with CHF; however, electrical stimulation increased myocyte secretion of ET-1, and ET-1 increased contractility only in normal myocytes (49). Furthermore, in the studies in which the regulation of ET-1 and ET receptors was investigated concerning intracellular free calcium concentration, it was reported that ET-1-related actions and ET-1 receptor regulation are not modified in cardiac volume overload (7) and pressure overload (50). In this study, both expression of ETA receptor mRNA and ETB receptor mRNA did not differ between the RV and LV under normal conditions and were increased in the RV of pulmonary hypertensive rats. Further investigations are required on the activities and roles of the ET receptor system under physiological/pathological conditions.

It has been reported that pathological cardiac hypertrophy is one of the risk factors of unfavorable cardiac events in humans (26, 27). On the other hand, long-term exercise causes cardiac hypertrophy, which is a physiological cardiac hypertrophy called athlete's heart. Various biochemical and biomechanical properties differ between physiological cardiac hypertrophy and pathological cardiac hypertrophy: the cardiac function is augmented in physiological cardiac hypertrophy, including athlete's heart, whereas it is diminished in pathological cardiac hypertrophy. We have reported that, in rats with physiological cardiac hypertrophy due to exercise (swimming) of long duration (4 mo), the expression of prepro-ET-1 mRNA in the heart tended to be lower than in sedentary control rats (32), suggesting that the expression of ET-1 in the heart is differently altered in physiological cardiac hypertrophy and pathological cardiac hypertrophy. We also reported that, in animal models with pathological cardiac hypertrophy, the chronic inhibition of an upregulated myocardial ET-1 pathway by an ET receptor antagonist ameliorated pathological cardiac hypertrophy in rats with aortic banding (42) and pulmonary hypertensive rats (35) and improved hemodynamic parameters and long-term survival in rats with CHF (39). Thus, from a therapeutic viewpoint of cardiac diseases, it may be important to determine whether the myocardial ET-1 pathway is enhanced, because the ET-1 pathway in the myocardium appears to be specifically activated only in the pathological state. Indeed, we reported that expression of ET-1 in the heart is decreased in athlete's heart, a physiological cardiac hypertrophy, in rats (34). Therefore, it is of great interest and of importance to determine whether a hypertrophied heart with enhanced ET-1 expression becomes a therapeutic target of ET receptor antagonists for the prevention of unfavorable cardiac events.


    ACKNOWLEDGEMENTS

This work was supported by grants-in-aid for scientific research from the Ministry of Education, Science, Sports and Culture of Japan (8670757, 9770473, 10670629, 11557047), a grant from the Study Group of Molecular Cardiology, Uehara Memorial Foundation, the Japan Heart Foundation, and the Miyauchi project of the Tsukuba Advanced Research Alliance at the University of Tsukuba.


    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: T. Miyauchi, Cardiovascular Division, Dept. of Internal Medicine, Institute of Clinical Medicine, Univ. of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan (E-mail: t-miyauc{at}md.tsukuba.ac.jp).

Received 3 August 1998; accepted in final form 4 June 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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