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(Circulation. 1996;93:2068-2079.)
© 1996 American Heart Association, Inc.

Articles

Endothelin-1 Is Involved in Norepinephrine-Induced Ventricular Hypertrophy In Vivo

Acute Effects of Bosentan, an Orally Active, Mixed Endothelin ET_A and ET_B Receptor Antagonist

Samer Kaddoura, BSc, BM BCh, MRCP; John D. Firth, MA, DM, MRCP; Kenneth R. Boheler, PhD; Peter H. Sugden, MA, DPhil; Philip A. Poole-Wilson, MA, MD, FRCP

From the National Heart and Lung Institute, Imperial College, London (S.K., K.R.B., P.H.S., P.A.P.-W.); Royal Brompton Hospital, London (S.K., P.A.P.-W.); and the Institute of Molecular Medicine, John Radcliffe Hospital, Oxford (J.D.F.), England.

Correspondence to Dr Samer Kaddoura, Department of Cardiac Medicine, National Heart and Lung Institute, Imperial College, London SW3 6LY, England, UK. E-mail s.kaddoura@ic.ac.uk.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background Endothelin-1 (ET-1) has potent effects on cell growth and induces hypertrophy of cultured ventricular myocytes. Catecholamines increase expression of ET-1 mRNA by cultured myocytes. We investigated the role of endogenous ET-1 in catecholamine-induced hypertrophy in vivo by studying the effects of continuous norepinephrine infusion on physical and molecular markers of ventricular hypertrophy, ventricular and noncardiac expression of ET-1 mRNA, and the acute effects of bosentan, an orally active ET_A and ET_B receptor antagonist.

Methods and Results Seventy male Sprague-Dawley rats (175 to 200 g) were divided into four groups: (1) sham-operated rats, (2) norepinephrine-infused rats (600 µg·kg^-1·h^-1 by subcutaneous osmotic pump, up to 7 days), (3) sham-operated rats given bosentan, and (4) norepinephrine-infused rats given bosentan. Bosentan (100 mg/kg once daily) was administered by gavage for 6 days starting 1 day before operation. Norepinephrine caused increases in absolute ventricular weight and ratios of ventricular weight to body weight and ventricular RNA to protein. Ventricular expression of mRNAs for atrial natriuretic factor, skeletal -actin, and ß-myosin heavy chain, which in adult rat ventricle are indicators of hypertrophy, also increased. Ventricular expression of ET-1 mRNA was elevated in the norepinephrine group at 1, 2, and 3 days. By 5 days, this had fallen to control levels. In lung, kidney, and skeletal muscle, norepinephrine did not significantly increase expression of ET-1 mRNA. Bosentan attenuated norepinephrine-induced increases in ventricular weight, ratio of RNA to protein, and expression of skeletal -actin mRNA and ß-myosin heavy chain mRNA at 5 days, but it did not attenuate increased ventricular expression of atrial natriuretic factor mRNA.

Conclusions These data suggest that endogenous ET-1 plays a direct role in mediating norepinephrine-induced ventricular hypertrophy in vivo.

Key Words: endothelin • hypertrophy • RNA • norepinephrine • bosentan

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
The 21-amino-acid vasoconstrictor peptide ET-1 has potent effects on cell growth and induces hypertrophy of cultured cardiac myocytes in vitro.¹ Although the precise physiological and pathophysiological roles played by ET-1 in vivo remain uncertain, evidence suggests that ET-1 acts as a local autocrine and paracrine factor rather than as a circulating hormone. Circulating plasma concentrations of ET-1 peptide are usually lower than those reported to be biologically active,² and secretion of ET-1 by endothelial cells is polar, being directed toward the interstitial (basal) region rather than the vascular lumen.³ ET-1 has been suggested to mediate the effects of hypertrophic stimuli such as angiotensin II in vitro⁴ and left ventricular pressure overload secondary to aortic banding in vivo.^{5 6} Such evidence has arisen from a combination of measurements of ET-1 peptide, tissue expression of mRNA encoding prepro-ET-1 (referred to henceforth as ET-1 mRNA), and the effects of newly available ET receptor antagonists, notably BQ123. The role, if any, played by endogenous ET-1 in the development of catecholamine-induced ventricular hypertrophy in vivo is unknown.

Yanagisawa et al⁷ demonstrated that ET-1 mRNA is induced in endothelial cells through exposure to adrenaline. This effect is apparently mediated through ₁-adrenergic receptors, since it is inhibited by the selective ₁-adrenergic receptor antagonist prazosin but not by the ₂-adrenergic receptor antagonist yohimbine⁸ or the ß-adrenergic receptor antagonist propranolol.⁹ Cultured neonatal rat ventricular cardiomyocytes express a low level of ET-1 mRNA in the unstimulated state, but ET-1 mRNA expression increases in response to the catecholamine -adrenergic receptor agonist phenylephrine.¹⁰

We hypothesized that endogenous myocardial production of ET-1 plays a functional role in catecholamine-induced ventricular hypertrophy in vivo. Such hypertrophy was induced in a rat model by continuous infusion of NE over several days, administered by subcutaneous osmotic pump. The aims of this study were, first, to assess the effects of continuous NE infusion on physical indexes and molecular markers of the ventricular hypertrophic phenotype, including ventricular weight, ratios of ventricular to body weight and ventricular RNA to protein, and ventricular expression of mRNAs for ANF, - and ß-MHC, and skeletal and cardiac -actin. ANF, ß-MHC, and skeletal -actin mRNAs are expressed only at very low levels in nonhypertrophied adult rat ventricle, but their expression increases with hypertrophy.^{11 12 13 14 15 16 17} As such, their induction is frequently used as a marker of the hypertrophic response. Second, we examined the expression of ET-1 mRNA in ventricular and noncardiac tissues (lung, kidney, and skeletal muscle) in response to NE infusion. Since ET-1 peptide is not stored intracellularly and its expression seems to be modulated by regulation of the mRNA level,¹⁸ we measured tissue expression of ET-1 mRNA as an indicator of local production of ET-1. Finally, to assess whether endogenous ET-1 production was playing a causal role in this model, we examined the effects of the orally active ET_A and ET_B receptor antagonist bosentan on NE-induced hypertrophy. Bosentan is a nonpeptide, competitive antagonist whose pharmacological properties have been well described.¹⁹ In particular, bosentan does not exert any - or ß-adrenergic receptor antagonism, and its specificity as an endothelin receptor antagonist has been assessed by testing and excluding any possible interference with receptor binding of more than 40 other agents, including catecholamines.¹⁹

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Drugs and Chemicals
(-)Norepinephrine bitartrate was obtained from Sigma Chemical Co, as were all other chemicals unless otherwise stated. Bosentan was a generous gift from Dr M. Clozel, Hoffmann-La Roche, Ltd (Basel, Switzerland).

Experimental Animals and Surgical Procedures
Seventy male Sprague-Dawley rats weighing 175 to 200 g (Charles River, UK) were divided into four experimental groups: (1) sham-operated (group S) without NE infusion, (2) NE-infused rats (group NE), (3) sham-operated rats given oral bosentan (group B+S), and (4) NE-infused rats given oral bosentan (group B+NE). Saline (0.9%) with ascorbate (1 mmol/L) vehicle or (-)norepinephrine bitartrate dissolved in 0.9% saline with 1 mmol/L ascorbate was administered continuously by subcutaneously implanted osmotic pumps (Alzet mini osmotic pump, model 2001; Alza Corp). Rats were sedated with a mixture of fentanyl citrate 0.16 mg/kg and fluanisone 5 mg/kg IM (Hypnorm; Janssen), given 30 minutes before implantation, and lidocaine hydrochloride (1 mL, 1%, SC) was administered at the site of implantation. NE was infused at a rate of 600 µg·kg^-1·h^-1 for up to 7 days. Bosentan (25 mg/mL) was prepared fresh daily as a microsuspension in 5% gum arabic and administered by once-daily gavage at a dose of 100 mg·kg^-1·d^-1 for 6 days commencing 1 day before implantation of osmotic pumps. Rats were sedated with a mixture of fentanyl citrate 0.27 mg/kg with fluanisone 8.3 mg/kg (Hypnorm; Janssen, UK) and midazolam 8.3 mg/kg IP (Hypnovel; Roche), given 30 minutes before gavage. All sham-operated rats had pumps implanted and were gavaged with 5% gum arabic. At 1, 2, 3, 5, and 7 days after implantation of osmotic pumps, rats were killed by cervical dislocation. The ventricles (left ventricular free wall, interventricular septum, and right ventricle) were dissected free of atria, weighed, and immediately frozen in liquid nitrogen. Samples of lung, kidney, and skeletal muscle were also rapidly removed and immediately frozen in liquid nitrogen. Tissues were stored at -70°C.

RNA Extraction
Total RNA from ventricular tissue, lung, kidney, and skeletal muscle was extracted by a modification of the method of Chomczynski and Sacchi²⁰ using RNAzol B (Biotecx Laboratories Inc, Biogenesis Ltd) according to the manufacturer's instructions. Tissues frozen at -70°C were homogenized in RNAzol B (2 mL RNAzol B per 100 mg tissue) with a Polytron tissue homogenizer (Brinkmann Instruments), followed by phenol-chloroform extraction, 2-propanol precipitation, and 75% vol/vol ethanol washing of precipitated RNA. RNA was stored in ethanol at -70°C.

Templates for Antisense Riboprobes to ET-1, ANF, and GAPDH mRNAs
As previously described,²¹ standard PCR and cloning techniques were used to subclone a fragment of the gene for rat ET-1, producing a 0.6-kb fragment containing 154 bp of exon 2 coding sequence for ET-1 and adjacent 5' intron into the Asp718/HindII sites of the riboprobe vector pAM19 (Amersham International). Sequencing of the coding region by the deoxy chain termination method (Sequenase; United States Biochemical Corp) confirmed the identity of ET-1. An intronic Bgl II site 242 bp from the 3' end of the insert was used to linearize the riboprobe template.²¹ To assay levels of ANF mRNA, a rat ANF cDNA representing the entire ANF coding region cloned into the Pst I site of pGEM-1 was used (generous gift of Dr K.R. Chien, San Diego, Calif).²² Digestion with Xho I linearized the template. For analysis of GAPDH mRNA levels, a 316-bp fragment of the rat GAPDH gene derived from exons 5 through 8²³ inserted into the Sac I/BamHI sites of the pTRIPLEscript transcription vector was used (Ambion, AMS Biotechnology). The plasmid was linearized by digestion with Sty I.

Quantitative RNase Protection Assays for ET-1, ANF, and GAPDH mRNAs
Continuously labeled antisense RNA transcripts to ET-1 were generated by in vitro transcription of the linearized DNA template described above using SP6 DNA-dependent RNA polymerase (Amersham International) and [-³²P]GTP (specific activity, 410 Ci/mmol; Amersham International). This produced a 272-nt riboprobe that protected 154 nt of ET-1 mRNA (nt 249 through 402; GenBank accession No. M64711).^{21 24} The riboprobe had previously been shown to be specific for ET-1 mRNA and not to cross-react with ET-2 or ET-3 mRNAs.²¹ The same technique, but using T7 RNA polymerase (Amersham International), was used to produce a 141-nt antisense probe, protecting a 95-nt fragment of rat ANF mRNA²² (nt 539 through 633; GenBank accession No. M27498),²⁵ and a 194-nt probe that protected 134 nt of constitutive rat GAPDH mRNA (nt 552 through 685; GenBank accession No. X00972).²³ For analysis of mRNA, precipitated total RNA was dissolved in an aliquot of hybridization buffer (80% formamide, 40 mmol/L PIPES, 400 mmol/L NaCl, 1 mmol/L EDTA, pH 8.0), and RNA concentration was determined by absorbance measurements at 260 nm (model 2600 spectrophotometer; Gilford Instruments). The concentrations were adjusted to yield 50-µL samples containing 10 µg of total RNA. After denaturation at 90°C for 15 minutes, hybridization was performed overnight at 60°C with 2.5x10⁵ cpm of the ET-1 probe, 2.5x10⁵ cpm of the ANF probe, and 2.5x10⁵ cpm of the GAPDH probe. After hybridization, RNase digestion was carried out for 30 minutes at 37°C by the addition of 350 µL of a solution containing 40 mg/mL RNase A (Boehringer Mannheim), 2 mg/mL RNase T1 (Boehringer Mannheim), 10 mmol/L Tris, pH 7.5, 5 mmol/L EDTA, and 300 mmol/L NaCl. The reaction was terminated by the addition of 60 µL of proteinase K (1 mg/mL) with 3% SDS and further incubation at 37°C for 30 minutes. Phenol-chloroform and chloroform extractions were performed, and the RNA fragments were precipitated with 2.5 vol absolute alcohol. The precipitated RNA was dissolved in 7 µL of 80% formamide loading buffer and electrophoresed on a denaturing 8% polyacrylamide gel. After electrophoresis, the gels were dried and subjected to autoradiography at -70°C. The autoradiographs were quantified with a laser densitometer (Howtek) linked to a computer analysis system (PDI). Preliminary experiments showed that this method produced a linear relationship between radioactivity loaded and absorbance. The results were expressed as ratios of ET-1 mRNA to GAPDH mRNA and of ANF mRNA to GAPDH mRNA.

"Hot" RT-PCR and Differential Enzymatic Digestion for Simultaneous Analysis of the - and ß-MHC and Skeletal and Cardiac -Actin Iso-mRNAs
The relative proportions of the cardiac iso-mRNAs of MHC were determined by a modification of a rapid "hot" (radioactively labeled) RT-PCR amplification technique as described recently.²⁶ The principle underlying this technique is the assumption that one set of oligonucleotide primers will anneal to identical sequences in the - and ß-MHC transcripts with equal efficiencies, and since the lengths of elongation are identical, the amplified fragments should be representative of endogenous levels of mRNA for these two transcripts. This assumption has been tested and verified in total cardiac RNA from neonatal and adult rat heart through comparisons with the results generated by standard hybridization techniques and through mixing experiments.²⁶ The relative proportions of the amplified DNA fragments are determined by differential restriction endonuclease digestion with an enzyme for which a restriction site is found only in one of the fragments, followed by polyacrylamide gel electrophoresis. cDNA was synthesized from 1 µg total RNA extracted from ventricles of sham, NE-infused, bosentan-treated, and bosentan plus NE–infused rats using a first-strand cDNA synthesis kit and, as primer, oligo (dT)₁₈ according to the manufacturer's instructions (Pharmacia). Incubations were at 37°C for 1 hour, followed by denaturation at 95°C for 10 minutes before amplifications. PCR amplifications were achieved with the following oligonucleotides identical to sequences for both - and ß-MHC: forward primer, 5'-GCA GAC CAT CAA GGA CCT (nt 5371 through 5388; GenBank accession No. X15939)²⁷ and reverse primer, 5'-GTT GGC CTG TTC CTC CGC C (nt 5662 through 5680, complementary strand). The reaction mix contained 50 mmol/L KCl, 10 mmol/L Tris-HCl, pH 9.0, 0.1% Triton X-100, 1.5 mmol/L MgCl₂, 18 pmol forward primer, 20 pmol reverse primer, 0.8 mmol/L dNTPs, and 2.5 U Taq DNA polymerase (Promega). The reaction was supplemented with 2 pmol radioactively labeled forward primer (synthesized by use of T4 polynucleotide kinase [Promega] in the presence of [³²P]ATP, 5000 Ci/mmol, Amersham International). Amplifications were as follows: program 1, 95°C, 3 minutes; 63°C, 30 seconds; and 72°C, 35 seconds (1 cycle); program 2, 95°C, 45 seconds; 63°C, 30 seconds; and 72°C, 35 seconds (25 cycles); and program 3, 95°C, 45 seconds; 63°C, 30 seconds; and 72°C, 5 minutes (1 cycle). Distinction between the - and ß-MHC iso-mRNAs was achieved by digestion of 10 µL of the PCR reaction mixture with 10 U Tru 91 (10 U/µL, Boehringer) in a standard reaction buffer at 65°C for 90 minutes. This yielded fragments of 309 bp for -MHC and 259+50 bp for ß-MHC. Fragments were separated by electrophoresis on an 8% polyacrylamide gel after addition of 5 µL 80% formamide loading buffer. After electrophoresis, the gels were dried and subjected to autoradiography, and the resultant bands were quantified by laser densitometry as described above. The results were expressed as ratios of ß-MHC mRNA to -MHC mRNA.

A similar technique, using the same cDNAs as generated for the MHC analyses above, was used for analysis of the relative proportions of skeletal and cardiac -actin iso-mRNAs.^{26 28} PCR amplifications were achieved in the reaction mixture described above by use of the following oligonucleotides: forward primer, 5'-ACC AGG GTG TCA TGG (nt 203 through 217; GenBank accession No. X80130) and reverse primer, 5'-GTG AGC AGG GTC GGG (nt 387 through 401, complementary strand). Amplifications were as follows: program 1, 95°C, 5 minutes; 60°C, 30 seconds; and 72°C, 35 seconds (1 cycle); program 2, 95°C, 45 seconds; 60°C, 30 seconds; and 72°C, 35 seconds (25 cycles); and program 3, 95°C, 45 seconds; 60°C, 30 seconds; and 72°C, 5 minutes (1 cycle). Distinction between the -actin iso-mRNAs was achieved by digestion of 10 µL of the PCR reaction mixture with 15 U Sac I (10 U/µL, Boehringer) to yield fragments of 202 bp for skeletal -actin and 161+37 bp for cardiac -actin. Fragments were separated by electrophoresis and quantified as described above. Results were expressed as ratios of skeletal -actin mRNA to cardiac -actin mRNA.

Estimation of Ventricular RNA and Protein Content
Assays based on the standard spectrophotometric assays of Munro and Fleck²⁹ and Gornall et al³⁰ were used to estimate ventricular RNA and protein contents, respectively. Ventricular samples (100 to 150 mg) were homogenized in 2 mL 0.56 mol/L perchloric acid in a Polytron homogenizer (Brinkmann Instruments) and centrifuged, and the supernatant was discarded. The precipitates were dissolved in distilled water, and 1 mol/L NaOH was added to a final NaOH concentration of 0.1 mol/L. Samples were incubated at 37°C for 1 hour. A series of protein standards (0 to 5 mg BSA, 10 mg/mL) and the ventricular protein NaOH digests were each made up to a final volume of 0.5 mL with distilled water. Biuret reagent³⁰ (3 mL) was added to all samples, and the mixtures were heated at 100°C for 2 minutes. After cooling, absorbance at 540 nm was measured, and the protein contents were derived from the standard curve as milligrams BSA. This value was corrected to give milligrams rat ventricular protein by multiplying results by 1.076, since rat ventricular protein gives a lower absorbance at 540 nm than BSA.³¹ For assay of ventricular total RNA, 0.5-mL samples of ventricular NaOH digests were precipitated with 5 mL 0.56 mol/L perchloric acid and cooled to 4°C for 15 minutes. Samples were centrifuged, the absorbance at 260 nm of the supernatants was measured, and total RNA was calculated. The results were expressed as ratios of ventricular RNA to protein.

Double-Label Immunohistochemistry Using Antibodies to ET-1 and ß-MHC
To corroborate ET-1 expression at the protein level and to attempt to localize the cell types involved in its production in the myocardium, indirect immunohistochemistry was carried out both on sections of left ventricle from sham and NE-infused rats and on cultured ventricular myocytes in culture after challenge with NE, as described below, with primary antibodies to ET-1 and ß-MHC. Cryostat sections (6 µm) of left ventricle from sham and NE-infused groups were cut at -24°C and placed on poly-L-lysine–coated glass slides. Sections were fixed in freshly prepared 3% paraformaldehyde in PBS, pH 7.4, for 10 minutes, washed three times in PBS, and permeabilized in 0.3% Triton X-100 in PBS for a further 10 minutes at room temperature. Nonspecific binding sites were blocked by application of 1% BSA/0.3% Triton X-100 in PBS for 10 minutes. Slides were incubated with a rabbit polyclonal ET-1 antiserum (dilution, 1 in 250; Peninsula) for 2 hours at 37°C in a humidified chamber. The specificity of this antibody has been extensively validated and used in previous studies.^{32 33 34} After washing with PBS and reblocking of nonspecific sites with 1% BSA/0.3% Triton X-100, we then incubated all treated slides with biotinylated F(ab')₂ fragment of affinity-isolated swine immunoglobulins to rabbit immunoglobulins (dilution, 1 in 200; Dakopatts) for 30 minutes at 37°C, followed by incubation with streptavidin–Texas Red (dilution, 1 in 200; Amersham) for a further 30 minutes. After washing with PBS and reblocking, the sections were incubated with a mouse monoclonal antibody to ß-MHC (dilution, 1 in 40; Novocastra Laboratories) for 1 hour at 37°C, followed by incubation with fluorescein-conjugated rabbit immunoglobulin to mouse immunoglobulins (dilution, 1 in 200; Dakopatts). To provide a negative control, some slides were not exposed to primary antibodies (anti–ET-1 and anti–ß-MHC) but were otherwise treated in the same way as other slides. Cell nuclei were counterstained with 10 µg/mL Hoechst dye 33342 (Sigma) for 10 minutes at room temperature. Coverslips were mounted with Univert mountant (Merck), and slides were viewed by epi-illumination on a Zeiss Axioskop fluorescence microscope. Sections from sham and NE-infused groups were photographed with a Zeiss camera and Kodak Ektachrome 400x color reversal film under identical lighting and timing conditions, the camera electronically controlled by a Zeiss timer.

Isolation, Culture, and Treatment of Rat Ventricular Cardiomyocytes
Primary cultures of ventricular cardiomyocytes from neonatal (1- to 2-day-old) Sprague-Dawley rats were established by a technique based on that of Sen et al.³⁵ This method yields 4x10⁶ million cells per heart, 85% of which are beating myocytes.³⁵ Neonatal rats were decapitated and the hearts removed immediately. Ventricles were separated from atria, opened, and washed in sterile Ads buffer (in mmol/L: NaCl 116, HEPES 20, Na₂HPO₄ 0.8, glucose 5.6, KCl 5.4, MgSO₄ 0.8, pH 7.35). Cardiac myocytes were dissociated from the ventricles by serial digestion with 0.4 mg/mL collagenase and 0.6 mg/mL pancreatin in Ads buffer. After each digestion, the enzymes in the cell suspension were inactivated by the addition of one-fifth volume of neonatal calf serum. Cells were retrieved by centrifugation, and the pellet was resuspended in plating medium (DMEM/medium 199 [4:1 vol/vol] supplemented with 10% horse serum, 5% fetal calf serum, and 100 U/mL of both penicillin and streptomycin). The resulting cell suspension was enriched for myocytes by preplating for 30 minutes on uncoated 60-mm culture dishes (Primaria, Falcon) to remove nonmyocytes, which attached to the culture plates. Viable cells were identified by trypan blue exclusion and counted in a counting chamber. Nonadhering myocytes were plated at a density of 1.4x10³ cells/mm² on 60-mm plates that had been precoated with 1% gelatin in sterile PBS. Cells were maintained in a 5% CO₂ atmosphere at 37°C. After 18 hours, myocytes were confluent and beating spontaneously. Maintenance medium (DMEM/medium 199, 4:1 vol/vol, containing 100 U/mL of both penicillin and streptomycin) was used for subsequent medium changes. Thirty-six to 48 hours after plating, serum was withdrawn for 18 hours before the cells were further treated with maintenance medium with ascorbate 100 µmol/L vehicle alone (control cells) or NE 10 or 100 µmol/L in maintenance medium/ascorbate. Extraction of myocyte total RNA was by the RNAzol B method described above. Myocytes were scraped into 1 mL RNAzol B placed directly into each 60-mm plate after the culture medium had been removed.

For double-labeling immunofluorescence using primary antibodies to ET-1 and ß-MHC, myocytes were plated at 5x10⁴ cells/cm² on eight-well chamber slides (Labtek) that had been precoated with 1% gelatin and 20 µg/mL laminin in sterile PBS. After fixation in 3% paraformaldehyde, the chamber slides were treated as described above for tissue sections.

Statistical Analyses
Results were reported as mean±SEM, with n being the number of rats. Statistical comparisons between sham and NE-infused groups at each day were made by two-tailed, unpaired Student's t tests to assess the effect of NE infusion on physical indexes and molecular markers of ventricular hypertrophy. One-way ANOVA with Bonferroni multiple comparisons test was used to test the hypothesis that production of ET-1 was playing a causal role in NE-induced hypertrophy by examining the effects of the ET_A and ET_B receptor antagonist bosentan, comparing sham, NE-infused, bosentan, and bosentan plus NE–infused groups at 5 days after operation.

For the in vitro experiments with cultured myocytes, comparisons between control and NE-treated groups were made by unpaired two-tailed Student's t tests. A value of P<.05 was considered significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Changes in Ventricular Weight and Ratios of Ventricular Weight to Body Weight and Ventricular RNA to Protein
There were significant increases in absolute ventricular weights and in ratios of ventricular weight to body weight and of ventricular RNA to protein in the NE-infused group compared with the sham group (Fig 1). Some increases were evident within 1 day of the commencement of the NE infusion and were maintained throughout the 7-day infusion period. Both ventricular total RNA and protein increased in this model of hypertrophy, the former to a greater extent. Total RNA (µg/g ventricular wet wt) at 1 day was 1131±68 for sham, n=6, versus 1460±29 for NE-infused, n=8, P<.001, and at 5 days was 1386±77 for sham, n=5, versus 2094±89 for NE-infused, n=5, P<.001. Ventricular protein (mg/g ventricular wet wt) at 1 day was 151.0±9.0 for sham, n=6, versus 157.0±3.4 for NE-infused, n=8, P=NS, and at 5 days was 154.0±7.6 for sham, n=5, versus 179.0±7.4 for NE-infused, n=5, P<.05. There was a significant decrease in body weight with NE infusion relative to shams throughout the time period examined.

View larger version (21K): [in this window] [in a new window]   Figure 1. The effects of NE infusion and oral bosentan on changes in (A) body weight (g), (B) ventricular weight (mg), (C) ratio of ventricular to body weight (mg/g), and (D) ratio of ventricular RNA to protein (mg/g) with time after implantation of subcutaneous osmotic pumps delivering vehicle alone (sham) or NE (600 µg·kg-1·h-1). Values are mean±SEM, n=3 to 8 for each group, total n=70. LV indicates left ventricle; RV, right ventricle; NE, NE-infused; Bos, sham-operated rats given bosentan (100 mg/kg orally once daily); and Bos+NE, NE-infused rats given bosentan. *P<.05, P<.01, P<.001, vs sham. ¶P<.001 vs NE. §P<.001 vs bosentan.

Changes in Tissue Expression of ANF mRNA and in Ratios of ß-MHC to -MHC mRNA and Skeletal to Cardiac -Actin mRNA
As molecular markers of the hypertrophic response, ventricular expression of ANF, ß-MHC, and skeletal -actin mRNAs was examined. Expression of ANF mRNA increased significantly in the NE-infused group compared with the sham group, reaching a maximum 25-fold increase by day 7 (ratio of ANF mRNA to GAPDH mRNA, 0.38±0.06 for sham, n=5, versus 9.86±0.87 for NE-infused, n=6, at 7 days, P<.0001). There was a significant fall in the ratio of sham ANF mRNA to GAPDH mRNA between day 1 and days 5 and 7; Fig 2 shows a representative autoradiograph demonstrating changes in ventricular ANF mRNA expression with time during NE infusion, and Fig 3A shows quantitative changes in ventricular ANF mRNA levels. Lung, kidney, and skeletal muscle did not express ANF mRNA in either the sham or NE-infused group (data not shown).

View larger version (43K): [in this window] [in a new window]   Figure 2. Representative quantitative RNase protection assay (3 days' exposure of autoradiograph) showing the effect of NE infusion at 1, 2, and 3 days on ventricular expression of mRNAs for ANF, ET-1, and constitutive GAPDH. Ventricular total RNA (10 µg per lane) was hybridized with 32P-labeled antisense riboprobes to fragments of ANF, GAPDH, and ET-1 mRNAs; the protected mRNA fragments are 95, 134, and 154 bp, respectively. Each lane represents ventricular expression of mRNAs from a separate rat (n=3 for each group except at 2 days sham, for which n=4). NE indicates NE-infused rats.

View larger version (21K): [in this window] [in a new window]   Figure 3. Bar graphs showing changes in ventricular expression of mRNAs, measured by quantitative RNase protection assays. A, Quantitative expression of ANF mRNA normalized for constitutive GAPDH mRNA. B, Quantitative expression of ET-1 mRNA normalized for GAPDH mRNA. Data are expressed as mean ratios of ANF mRNA to GAPDH mRNA and ET-1 mRNA to GAPDH mRNA. Bars indicate SEM. n=3 to 8 for each group. *P<.01, P<.0001 vs sham-operated group. ¶P<.05 vs 1-day sham group.

NE infusion also led to a significant increase in the ratios of ventricular ß-MHC to -MHC mRNA (0.08±0.01 for sham, n=5, versus 1.73±0.19 for NE-infused, n=7, at 5 days, P<.001) and skeletal to cardiac -actin (0.24±0.02 for sham, n=5, versus 0.60±0.07 for NE-infused, n=7, at 5 days, P<.001) (see Figs 6 and 7).

View larger version (28K): [in this window] [in a new window]   Figure 6. Autoradiographs showing the effects of NE infusion and bosentan on ventricular expression of the iso-mRNAs for - and ß-MHC (top) and skeletal and cardiac -actin (bottom). The iso-mRNAs were assayed by RT-PCR followed by restriction endonuclease digestion with Tru 91 (MHC iso-mRNAs) or Sac I (-actin iso-mRNAs). - and ß-MHC bands are 309 and 259 bp, and the skeletal and cardiac -actin bands are 202 and 161 bp, respectively. NE (NE group) or vehicle (sham group) was administered by subcutaneous osmotic pump for 5 days. Bosentan was given for 6 days, commencing 1 day before operation, to sham-operated (bosentan group) or NE-infused (bosentan+NE group) animals. Two representative lanes are shown for each group, each from a separate rat.

View larger version (22K): [in this window] [in a new window]   Figure 7. Bar graphs showing the effects of acute oral administration of bosentan on NE-induced expression of molecular markers of ventricular hypertrophy at 5 days after osmotic pump implantation. Bosentan (100 mg/kg once daily) was given by gavage for 6 days commencing 1 day before operation. A, Effect on ratio of ß-MHC mRNA to -MHC mRNA. B, Effect on ratio of skeletal -actin mRNA to cardiac -actin mRNA. C, Effect on ANF mRNA expression, measured by RNase protection assay and expressed as ratio of ANF mRNA to GAPDH mRNA. All results are means of number of rats as indicated in the figure by n. Bars represent SEM. ANOVA *P<.001, P<.01 vs sham. #P<.001, P<.05 vs NE-infused.

Changes in Tissue Expression of ET-1 mRNA and ET-1 Peptide With NE Infusion
Ventricular expression of ET-1 mRNA was low in the sham-operated group (ratio of ET-1 mRNA to GAPDH mRNA, 0.01±0.01 at 1 day, n=6). In the NE-infused group, there was a significant increase in expression of ET-1 mRNA, peaking at 1 day (ratio of ET-1 mRNA to GAPDH mRNA, 0.35±0.08, n=8, P<.01 relative to sham), remaining elevated at 2 and 3 days (ratios of ET-1 mRNA to GAPDH mRNA, 0.14±0.01, n=3, and 0.09±0.01, n=7, respectively, each P<.001 relative to sham), and falling to control levels by 5 days (Figs 2 and 3B).

The NE-induced increase in ET-1 mRNA expression in ventricular tissue was not seen in the other tissues examined (Fig 4). The highest level of tissue ET-1 mRNA expression in the sham group was observed in lung (ratio of ET-1 mRNA to GAPDH mRNA, 1.11±0.14, n=5), but NE infusion did not cause this to increase significantly (ratio of ET-1 mRNA to GAPDH mRNA, 1.59±0.18 at 1 day, n=5, P=NS). Expression of ET-1 mRNA was low in kidney, with no significant difference between sham and NE groups at 1 day (0.04±0.00 for sham, n=5, versus 0.05±0.01 for NE-infused, n=5, P=NS). ET-1 mRNA was not detected in sham or NE-infused skeletal muscle.

View larger version (28K): [in this window] [in a new window]   Figure 4. Differential effects of NE infusion on tissue expression of ET-1 mRNA. A, Representative autoradiographs showing the effect of NE infusion on tissue expression of ET-1 mRNA at 1 day. Total RNA (10 µg/lane) from ventricle, lung, kidney, or skeletal muscle from sham-operated or NE-infused rats was hybridized with 32P-labeled antisense riboprobes to fragments of ET-1 and GAPDH mRNAs; protected fragments are 154 and 134 bp, respectively. Expression of mRNAs from two separate (representative) rats is shown. B, Bar graph showing the effect of NE infusion at 1 day on expression of ET-1 mRNA in ventricle, lung, kidney, and skeletal muscle. Data are expressed as mean ratio of ET-1 mRNA to GAPDH mRNA. Bars show SEM. n=6 and n=8 for sham and NE-infused ventricle groups, respectively. n=5 for all other groups. *P<.01 vs sham.

To determine the cellular localization of ET-1 production with corroboration at the protein level, double-staining immunohistochemistry was carried out on frozen sections of left ventricular tissue from sham and NE-infused groups by use of antibodies to ET-1 and ß-MHC. Fig 5 shows representative sections from sham-operated and NE-infused rats at 1 day. In the sham group, there was very weak immunostaining for ET-1 in myocytes (identified as weakly ß-MHC positive) but stronger staining at the endothelial surface of blood vessels penetrating the ventricular myocardium (ß-MHC negative) (Fig 5A and 5B). NE infusion was associated with greatly increased ET-1 immunostaining in association with myocytes (5D), and endothelial staining remained present. NE infusion also increased myocyte staining for ß-MHC, as one would expect in the hypertrophic response (5C). This suggests that there is increased ET-1 peptide production by the myocardium in response to NE infusion and that myocytes contribute to this increase, in addition to production by other cell types such as vascular endothelial cells.

View larger version (82K): [in this window] [in a new window]   Figure 5. Indirect double-label immunohistochemical staining of representative left ventricular sections from sham (A and B) and NE-infused (C and D) rats at 1 day using antibodies to ß-MHC (A and C) and ET-1 (B and D). The ß-MHC antibody has been localized with a fluorescein-conjugated secondary (green fluorescence) and the ET-1 antibody with a Texas Red–conjugated secondary (red fluorescence). In left ventricle from sham rats, there is very weak immunostaining for ET-1 peptide in association with myocytes, identified as weakly ß-MHC positive (A and B). There is stronger ET-1 immunostaining at the endothelial surface of blood vessels penetrating the ventricular myocardium, which are ß-MHC negative (arrows in A and B). In the NE-infused group, there is increased ET-1 immunostaining in association with myocytes, which show staining for ß-MHC (C and D). Endothelial ET-1 immunostaining remains present in penetrating vessels (C and D). Magnification x200.

Acute Effects of Oral Bosentan on NE-Induced Hypertrophy
To assess whether endogenous production of ET-1 was playing a role in NE-induced hypertrophy, the effects of the ET_A and ET_B receptor antagonist bosentan were examined. Oral administration of bosentan attenuated the NE-induced increases in absolute ventricular weight and in ratios of ventricular to body weight and of RNA to protein (Fig 1). There was still significant NE-induced hypertrophy at 5 days in the presence of bosentan (ratio of ventricular to body weight, 3.18±0.07 mg/g for bosentan, n=7, versus 4.07±0.13 for bosentan+NE, n=7, P<.001). Bosentan attenuated NE-induced increases in ratios of ß-MHC mRNA to -MHC mRNA by 67±12% (P<.001 versus NE) and of skeletal -actin mRNA to cardiac -actin mRNA by 33±13% (P<.05 versus NE) at 5 days (Figs 6 and 7) but did not significantly affect the increase in ventricular ANF mRNA (an increase of 7±14% of the NE effect, P=NS) (Fig 7). Bosentan reduced the ratio of RNA to protein, but no other indexes, versus sham (Figs 1, 6, and 7). It did not affect ventricular expression of ET-1 mRNA, which remained at low levels, in the sham or in the NE-infused group at 5 days (data not shown).

In Vitro Experiments With Cultured Ventricular Myocytes
To further determine the cellular localization of ET-1 production, ET-1 mRNA was measured and immunohistochemical staining for ET-1 and ß-MHC proteins was performed on cultured rat ventricular cardiomyocytes in vitro. Fig 8 shows the effect of NE 100 µmol/L in the culture medium on expression of ET-1 mRNA by cultured cardiomyocytes. There was a significant increase relative to controls within 1 hour, an effect maintained for the 24-hour time period of the experiment. Immunohistochemical staining (Fig 9) shows weak staining for ET-1 and ß-MHC in control cells (Fig 9A through 9C). With NE 10 µmol/L challenge for 24 hours, cell size and striation increase, as does staining for ET-1 and ß-MHC (Fig 9D through 9F). In noncardiomyocytes (ß-MHC–negative cells), there is also positive staining with anti–ET-1 antibody (example shown by arrow in Fig 9D). The specificity of ET-1 immunostaining was demonstrated by its abolition by the addition of excess synthetic ET-1. At a higher concentration of NE (100 µmol/L), the effects on cell size and antibody staining are more pronounced (Fig 9G through 9I). This suggests that both cardiomyocytes and noncardiomyocytes are producing ET-1 in response to stimulation with NE in vitro.

View larger version (31K): [in this window] [in a new window]   Figure 8. The effect of NE on expression of ET-1 mRNA by cultured ventricular cardiomyocytes. Cardiomyocytes were challenged with vehicle (100 µmol/L ascorbate in maintenance medium, control) or NE (100 µmol/L in vehicle) for various times up to 24 hours. A, Representative autoradiographs after an RNase protection assay for ET-1 and GAPDH mRNAs. Each lane represents RNA from a separate culture plate. Total RNA (10 µg) was used in each case. B, Bar graph showing quantitative expression of ET-1 mRNA by myocytes. Autoradiographs were quantified by laser densitometry. Data are expressed as mean ratios of ET-1 mRNA to GAPDH mRNA. Bars indicate SEM. n=4 separate cell preparations for each group. *P<.05 and P<.01 vs control.

View larger version (93K): [in this window] [in a new window]   Figure 9. Immunohistochemical staining of cultured rat ventricular cardiomyocytes double-labeled with antibodies to ET-1 (A, D, and G) and ß-MHC (B, E, and H). The primary antibodies to ET-1 and ß-MHC were stained with secondaries conjugated with Texas Red (red fluorescence) and fluorescein (green fluorescence), respectively. The nuclei were counterstained with Hoechst dye (C, F, and I). A through C represent control cardiomyocytes exposed to vehicle (100 µmol/L ascorbate in maintenance medium). Note weak staining for ET-1 and ß-MHC in these cells. D through F show cardiomyocytes exposed to NE (10 µmol/L in vehicle) for 24 hours. There is increased cell size and increased staining for ET-1 and for ß-MHC. Note that some ß-MHC–negative cells (ie, noncardiomyocytes) also stain positive for ET-1 (example shown by arrow in D). Exposure of cardiomyocytes to a higher concentration of NE (100 µmol/L) for 24 hours results in a greater increase in cell size and staining for ET-1 and ß-MHC (G through I). Magnification x400.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
These data support the hypothesis that endogenous production of ET-1 plays a role in NE-induced ventricular hypertrophy in vivo. NE causes progressive ventricular hypertrophy in this rat model, some changes commencing as early as 1 day after the start of the infusion, as shown by increases in absolute ventricular weight, ratios of ventricular to body weight and of RNA to protein, and increased expression of mRNAs for ß-MHC, skeletal -actin, and ANF. Catecholamines increase expression of ET-1 mRNA by cultured ventricular myocytes in vitro,¹⁰ and exogenous ET-1 induces hypertrophy of cultured myocytes.^{1 36 37} The present study demonstrates for the first time that NE infusion in vivo results in a transient, early, and large increase in ventricular expression of ET-1 mRNA. This is similar to the effect reported to occur secondary to pressure overload due to aortic coarctation.^{5 6} The effect of NE on ET-1 mRNA is tissue specific, not being observed in lung, kidney, or skeletal muscle. Since ET-1 is not stored intracellularly but is generated de novo and its production is controlled at the level of mRNA production,¹⁸ our demonstration of increased ventricular expression of ET-1 mRNA is likely to reflect increased local production of ET-1 peptide. This is supported by our immunohistochemical observations. We suggest that this contributes to the generation of the hypertrophic phenotype brought about by NE. While ventricular myocytes produce ET-1 mRNA in vitro in response to catecholamines,¹⁰ it is probable that a number of other cell types within the myocardium (particularly endothelial cells, also possibly smooth muscle cells and fibroblasts) are contributing to our observed increases in ventricular expression of ET-1 mRNA in vivo. Our data do not allow us to define which cell type is predominantly responsible for this increase in ET-1 mRNA, although the results from immunohistochemical staining of ventricular sections with antibodies to ET-1 and ß-MHC and from the in vitro experiments suggest that both myocytes and nonmyocytes are contributing to the observed increase.

The response to ET-1 of the different cell types within the rat ventricular myocardium differs, depending on whether the cells express ET receptors and the receptor type.^{38 39} Adult rat ventricular cardiomyocytes express only ET_A receptors.⁴⁰ These mediate the hypertrophic effects of ET-1 on cultured adult⁴⁰ and neonatal⁴ ventricular cardiomyocytes. Although adult cardiomyocytes do not appear to express ET_B receptors,⁴⁰ angiotensin II has been shown to increase expression of ET_B receptor mRNA in cultured neonatal rat cardiomyocytes.⁴¹ Endothelial cells express ET_B receptors, stimulation of which results in release of prostacyclin and nitric oxide and vascular relaxation.⁴² ET-1 has potent mitogenic effects on vascular smooth muscle cells.^{43 44} These cells express ET_A receptors, which mediate contraction in response to ET-1.⁴⁵ ET_B receptors are not restricted to the endothelium and have been demonstrated on vascular smooth muscle cells from certain tissues, including the ventricle.³⁸ ET_B receptors can mediate not only vasodilatation but also vasoconstriction⁴⁶ ; coronary vasoconstriction in the perfused rat heart in response to ET-1 has been shown to be mediated by both ET_A and ET_B receptors.^{47 48} Cultured adult rat cardiac fibroblasts express ET_A^{49 50} and ET_B⁴⁹ receptors, with ET_B numbers predominating.⁴⁹ Increased DNA synthesis induced by ET-1 in cultured fibroblasts can be abolished by the ET_A receptor antagonist BQ123,⁵⁰ whereas the increase in collagen synthesis by these cells caused by ET-1 is mediated through ET_A and ET_B receptors.⁵¹ ET_A and ET_B receptor binding sites have also been demonstrated in the atria of a number of species, in which stimulation results in positive chronotropism, release of ANF, and decreased action potential duration,³⁸ and in association with ventricular conducting tissues.⁵² Thus, with some exceptions, ET_A receptors appear to be largely responsible for mediating the mitogenic and vasoconstrictor responses to ET-1 in the ventricle, whereas ET_B receptors appear to couple to prostacyclin and nitric oxide production, contributing to vasodilatation of the microvasculature. ET_B receptors, however, may also contribute to vasoconstriction of epicardial coronary arteries.

The effects of bosentan provide further evidence that production of ET-1 in vivo is playing a causal role in NE-induced hypertrophy. Acute administration of this oral ET_A and ET_B receptor antagonist attenuated the increase in absolute ventricular weight and in ratios of ventricular to body weight and of RNA to protein seen with NE infusion at 5 days. Furthermore, increased prevalence of the ß-MHC and skeletal -actin mRNA isoforms was attenuated, supporting the notion of "cross-talk" between the ET-1 and NE systems in modulating the transcription of mRNAs for these contractile proteins. Bosentan may be exerting its effects in a number of ways. First, we speculate that bosentan acts directly on ventricular myocardium to attenuate the effects of endogenous ET-1, whose production is induced by NE. Local production of ET-1 peptide in the myocardium in response to NE is supported by our immunohistochemical findings in ventricular sections and in cultured myocytes in vitro. Second, bosentan may exert some effects peripherally by reducing systemic arterial blood pressure. Teerlink et al⁵³ showed a 5 mm Hg reduction in mean arterial pressure over a 2-week period when oral bosentan, 100 mg·kg^-1·d^-1 as in our study, was administered to sham-operated conscious rats. This is a consequence of a reduction in ET-1–associated vascular tone. This dose of bosentan is also sufficient to prevent the rise in systemic blood pressure caused by an exogenous infusion of big ET in anesthetized rats (Reference 19¹⁹ and J.D.F, unpublished data). It is likely that both these mechanisms, and possibly others, operate in our model. Whichever is the predominant mechanism, the bosentan data lend support to the involvement of ET-1 in the ventricular hypertrophic response.

Since bosentan is a combined antagonist, it is not possible to conclude whether the effect seen on NE-induced hypertrophy is exerted through the ET_A or ET_B receptor. Although both types may be involved, there is reason to believe, on the basis of existing evidence, that the ET_A receptor plays the major role: (1) adult rat ventricular cardiomyocytes have only ET_A receptors⁴⁰ ; (2) the effect seen with oral bosentan is similar to that seen with BQ123 (ET_A receptor antagonist) in a rat coarctation model of hypertrophy,⁴ although BQ123 is a peptide and was given parenterally; and (3) the hypertrophic effects of ET-1 on cardiomyocytes and the other cell types present in the ventricle (eg, vascular smooth muscle cells and fibroblasts) appear to be mediated primarily through the ET_A receptor.^{38 39 49 50 51} We can conclude that oral administration of this combined antagonist attenuates NE-induced hypertrophy in this model.

Interestingly, the effect of bosentan on expression of mRNA encoding ANF was different from its effect on the mRNAs for the contractile proteins (-actin and MHC); bosentan did not attenuate NE-induced increases in ventricular expression of ANF mRNA. Although this is difficult to explain fully, there are a number of possible explanations. First, this lack of effect on ANF compared with the attenuation of expression of ß-MHC and -actin mRNAs may suggest that alternative pathways are involved in the regulation of ventricular ANF mRNA expression and in the regulation of the production of these myocardial contractile proteins. If this is the case, our data suggest that endogenous ET-1 plays a more important role in the latter system and a lesser or absent role in the former. A second explanation relates particularly to this experimental model of hypertrophy and to the regulation of adult ventricular expression of ANF mRNA. This remains incompletely understood but appears to be under the influence of a large number of factors,¹¹ many of which can alter during the metabolic and hemodynamic changes brought about by NE infusion. Circulating humoral factors such as glucocorticoids can interact through their receptors with unique cis-acting sequences to directly promote ANF gene expression,⁵⁴ independent of the hypertrophic response. Glucocorticoids increase ANF mRNA levels in cultured ventricular and atrial myocytes by affecting ANF gene transcription^{54 55 56} and raise circulating ANF concentrations in vivo. Dexamethasone can induce ventricular expression of the ANF gene by a mechanism that is additive to that produced by ventricular hypertrophy.⁵⁶ Ventricular expression of ANF mRNA in our NE-infused model may therefore not be a specific marker of the hypertrophic response but may additionally reflect the direct influence of other factors on the ventricle. It is noteworthy that ANF mRNA expression fell significantly with time after operation in the sham group. This may support the idea that induction of ANF is more nonspecific than the other cardiac genes examined. That an endothelin receptor antagonist such as bosentan is unable to block the effects of the other factors that may be operating to augment ANF gene expression in this model is perhaps unsurprising. A further, and possibly the most important, explanation for the difference between the effect of bosentan on ANF induction and the other cardiac genes examined is that this may be merely due to quantitative (kinetic, dose-response, or threshold) effects, without the need to implicate alternative pathways.

It is interesting to note that Ito et al⁵ found that ventricular ANF mRNA expression due to aortic coarctation was only partially blocked at 1 week by subcutaneous administration of the peptide ET_A receptor antagonist BQ123, and this effect was completely lost by 2 weeks. They speculated that cardiac ET-1 may act as an "initiating" hypertropic factor during the early phase of pressure overload but that other factors, such as the local renin-angiotensin system and several growth factors, might take over as "maintaining" factors during the late phase of pressure overload. Our data are consistent with such an effect occurring in the NE model, with early local ET-1 production acting as a "triggering factor" to hypertrophy.

In conclusion, our study suggests that ET-1 plays a direct role in NE-induced hypertrophy in vivo. It is clearly not the single factor by which NE exerts its effects. There was still significant NE-induced hypertrophy in the presence of bosentan. We suggest that early (within the first day) local production of ET-1 sets in motion a further cascade of events that contributes to the development of the hypertrophic phenotype. This has several important implications for our understanding of the signaling pathways involved in producing the hypertrophic phenotype, the pathophysiological role of ET-1, and the potential role of endothelin receptor antagonists in the management of hypertrophy, particularly when associated with states of catecholamine excess (eg, pheochromocytoma). If ET-1 is indeed acting as an early "triggering factor," with further additional maintaining factors, endothelin receptor antagonists may be of value in preventing the development of hypertrophy if given very early in the process.

	Selected Abbreviations and Acronyms

 

ANF = atrial natriuretic factor ET-1 = endothelin-1 MHC = myosin heavy chain NE = norepinephrine nt = nucleotides RNase = ribonuclease RT-PCR = reverse transcriptase–polymerase chain reaction

	Acknowledgments

 
This research was supported by an MRC clinical training fellowship to Dr Kaddoura (fellowship G84/3114) from the Medical Research Council, UK. Dr Firth is supported by the Wellcome Trust. Dr Boheler is supported by the British Heart Foundation (project grants PG/94132 and PG/93148). We thank Professor A.R. Kaddoura for helpful advice during the course of this study.

Received October 23, 1995; revision received December 19, 1995; accepted December 21, 1995.

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