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Cardiovascular Research 1999 44(1):197-206; doi:10.1016/S0008-6363(99)00155-8
© 1999 by European Society of Cardiology
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Copyright © 1999, European Society of Cardiology

Cardiac endothelin and big endothelin in right-heart hypertrophy due to monocrotaline-induced pulmonary hypertension in rat{star}

Friedrich Brunner*

Institut für Pharmakologie und Toxikologie, Karl-Franzens-Universität Graz, Universitätsplatz 2, A-8010 Graz, Austria

* Corresponding author. Tel.: +43-316-380-5559; fax: +43-316-380-9890 friedrich.brunner{at}kfunigraz.ac.at

Received 20 January 1999; accepted 20 April 1999


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: Recent observations suggest the existence of a myocardial endothelin (ET) system and its possible involvement in left-ventricular myocardial hypertrophy and failure. However, nothing is known about the role of myocardial ET in right-ventricular hypertrophy. Methods: Rats (80–100 g) were given an intraperitoneal injection of saline (controls) or monocrotaline (50 mg/kg) resulting in pulmonary hypertension-induced myocardial hypertrophy (n=11 in both groups). After 10 weeks, the animals were sacrificed and hearts perfused in vitro to determine levels of big ET-1 and ET-1 in coronary effluent, interstitial fluid and ventricular tissue homogenates; plasma levels were also determined. Results: In monocrotaline-treated animals, weights of right ventricles were 1.5- and of right atria 1.8-fold higher than in controls (p<0.05), indicating substantial right-ventricular hypertrophy as also evident from greatly increased myocardial production of atrial natriuretic peptide. Left-ventricular weights were not different. Release of big ET-1 in coronary effluent, and of ET-1 in coronary effluent and interstitial transudate were similar in control and hypertrophic hearts (p>0.05). Disruption of endothelium with collagenase reduced release of both peptides close to zero, indicating endothelial (not myocardial) origin of the peptides. Levels of big ET-1 and ET-1 were similar in left ventricles of both experimental groups, but lower in right ventricles of hypertrophic than control hearts (p<0.05), reflecting increased tissue mass rather than reduced peptide production. On the other hand, plasma levels of both peptides and of ANP were twofold and levels of angiotensin II 1.3-fold higher in rats with right-heart hypertrophy than in controls (p<0.05 in each case). Conclusion: These data do not support a role for local cardiac ET-1 and/or big ET-1 in right-ventricular hypertrophy, but point to blood-borne endothelins as possible mediators.

KEYWORDS Endothelin; Hypertrophy


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Although cardiac hypertrophy is an adaptive reaction of cardiac myocytes to an increased work load, ventricular hypertrophy is associated with a higher incidence of heart failure. Experimental and clinical studies have demonstrated the activation of the renin-angiotensin and endothelin systems in myocardial hypertrophy and congestive heart failure, but the mechanisms involved in these pathologies are not fully understood. Previous studies have implicated a potential role for angiotensin II in pressure overload cardiac hypertrophy by using angiotensin converting enzyme (ACE) inhibitors [1] and angiotensin II receptor antagonists [2]. More recently, interest has focused on the role of endothelins, particularly endothelin-1 (ET-1) which stimulates growth of cultured cardiomyocytes [3,4] and cardiac fibroblasts [5]. It was shown that angiotensin II induces the expression and secretion of ET-1 in cultured cardiomyocytes [6] and that in myocytes subjected to mechanical stress, secretion of both ET-1 and angiotensin II is enhanced and results in a synergistic hypertrophic response [7]. From these data the authors inferred that endogenous ET-1 acts as a local myocardial hypertrophic factor. However, the active concentrations of ET-1 in all of the above studies are much higher than ET-1 plasma levels (<1 to ~10 pg/ml [<0.4 to ~4 pmol/l]), and the interstitial levels in hypertrophy are not known.

Several recent observations suggest that ET-1 is involved in the aetiology of congestive heart failure as well. First, ET-1 is a potent vasoconstrictor and positive inotrope [8] that may affect the loading conditions of the infarcted heart and contribute to ventricular remodelling; second, levels of plasma ET-1 are increased 2–3-fold in various animal models of heart failure [9,10] and in cardiac patients [11–13]; third, up-regulation of the myocardial ET system may contribute to the progression of heart failure because in vivo antagonism of ET receptors improves survival of animals with chronic heart failure [10], decreases left-ventricular hypertrophy and cardiac fibrosis [14], partially prevents left-ventricular dilatation, and improves haemodynamics [15,16]. Finally, antagonism of ET receptors in patients with low-output heart failure improved the haemodynamic profile [17].

In spite of this evidence the respective roles of the cardiac and circulating ET systems in the development of myocardial hypertrophy and heart failure are far from established. Thus, little is known about the role of ET-1 in right heart hypertrophy and failure and the interaction of the cardiac ET system with other potential mediators like the local renin-angiotensin system and atrial natriuretic peptide (ANP). Therefore, several aims were pursued in this study: i) to determine the release into coronary effluent of ET-1, its precursor big ET-1, angiotensin II, and ANP, an established marker of load-associated ventricular remodelling; ii) to measure ET-1 and big ET-1 levels in interstitial fluid and ventricular homogenates to account for possible changes in the direction of mediator release in hypertrophic hearts, and iii) to determine the cellular origin of ET-1 and big ET-1 in isolated perfused rat hearts with right-ventricular hypertrophy. The pathology was induced by injecting monocrotaline, a pyrrolizidine alkaloid that causes epithelial proliferation of small pulmonary arteries and results in pulmonary hypertension, right-ventricular hypertrophy or heart failure [18,19].


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Materials
Monocrotaline and collagenase (535 U/mg) were purchased from Sigma (Vienna, Austria). Spermine/NO was obtained from Alexis Corporation (Läufelfingen, Switzerland). [125I]big ET-1 was purchased from ANAWA (Wangen, Switzerland), [125I]ET-1, 3-[125I]-iodotyrosyl-angiotensin II ([125I]angiotensin II), and (3-[125I]-iodotyrosyl28) ANP(1-28, rat) ([125I]ANP) were from Amersham (purchased through MedPro, Vienna, Austria). All other materials were of standard grade.

2.2 Animals
Sprague-Dawley rats weighting 80–100 g were divided into two groups: One group of animals was given a single intraperitoneal injection of monocrotaline (50 mg/kg; hypertrophy group), the second group was injected with the same volume of saline (control group). The treatment group soon showed a lack of appetite, indicating general illness as was observed previously [20]. Hence, the control group was given each day only the amount of nutrition that had been consumed the previous day by the treated rats which had a free supply of food. Altogether 24 rats were treated with monocrotaline. Three weeks after injection the first rats started to die due to the toxic effects of the alkaloid; 4 weeks after injection 13 rats (54%) had died, but afterwards no more cases of death were observed. Those rats that survived this period of mortality were sacrificed 10 weeks after monocrotaline injection and their hearts used for the Langendorff experiments in this study. Although no pleural or peritoneal effusions were observed, monocrotaline-treated animals had substantial right-ventricular hypertrophy as described in Results. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No. 85-23, revised 1985).

2.3 Heart perfusions
Rats were anaesthetized with diethyl ether, the hearts arrested in ice-cold Krebs-Henseleit perfusion medium and mounted within 2 min of thoracotomy. Retrograde perfusion (Langendorff mode) was established at a rate of 10.0 ml/min per g heart wet weight with a modified Krebs-Henseleit bicarbonate buffer (composition in mmol/l: NaCl 118, NaHCO3 25, KH2PO4 1.2, KCl 4.8, MgSO4 1.2, CaCl2 2.25, glucose 11, EDTA 1.0) using the ISOHEART perfusion system (Hugo Sachs Elektronik, March-Hugstetten, Germany). To collect interstitial transudate hearts were mounted in an inverted upside-down position as described previously [21]. The perfusion solution (pH 7.4) was filtered before use to remove particulate matter (5 µm pore size) and continuously gassed with carbogen (95% O2, 5% CO2) in an oxygen chamber resulting in a pO2 of >450 mm Hg. Heart temperature was measured with a physitemp probe (Physitemp Instruments, Clifton, NJ) and was maintained at 37–38°C during the whole experiment. Cardiac parameters were monitored continuously and included heart rate, coronary perfusion pressure (CPP), left-ventricular developed pressure (LVDevP; difference between left-ventricular peak systolic pressure and end-diastolic pressure), and left-ventricular end-diastolic pressure (LVEDP). The latter two pressure readings were obtained from a fluid-filled latex balloon inserted into the left ventricle and connected to a pressure transducer via a 5 F biluminal monitoring catheter (American Edwards Laboratories, purchased through Baxter GmbH, Unterschleissheim, Germany). LVEDP was set at 0 mm Hg at start of experiment and remained at this level up to collagenase perfusion.

2.4 Experimental protocol
After equilibration (15 min) hearts were perfused for 120 min under normoxic conditions with Krebs-Henseleit buffer at constant flow and haemodynamic parameters were recorded and effluent and transudate samples collected as described below. Thereafter, endothelial cells were disrupted through perfusion with collagenase (0.1% dissolved in perfusion buffer) for 2 min, followed by washout of collagenase for 8 min with normal perfusion buffer. Because the aortic valve is closed in retrograde perfusion, no collagenase entered the ventricular lumen, leaving endocardial endothelium intact. To validate coronary endothelium disruption, the effects of the endothelium-dependent agonist acetylcholine (10 nmol/l) and the endothelium-independent agonist spermine/NO (100 µmol/l) were tested (at 130–150 min after equilibration). As expected, collagenase treatment completely abolished the relaxant effect of acetylcholine, while relaxation by spermine/NO was only little affected, as reported previously [22]. Hearts were then perfused for another 120 min, haemodynamic parameters recorded, and effluent and transudate samples collected as described below (end of experiment at 270 min after equilibration; compare Fig. 1). After the experiment the left and right atria were removed from the hearts and the free wall of the right ventricle and the left ventricle plus interventricular septum were separated. All specimens were weighted and frozen at –20°C.


Figure 1
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  		Fig. 1 Functional parameters in hearts from rats treated with saline (control; solid circles) or monocrotaline (hypertrophic; open circles). Hearts were equilibrated (15 min, not shown) and perfused in vitro with crystalloid perfusion medium for 120 min, followed by disruption of the endothelium with collagenase and testing for endothelial function. Hearts were then perfused for another 120 min with endothelium disrupted. Left-ventricular developed pressure (LVDevP), left-ventricular end-diastolic pressure (LVEDP) and coronary perfusion pressure (CPP) are shown. Results are means±SEM, n=11 hearts in each case. There were no significant differences between experimental groups.

 
2.5 Sample collection
Coronary effluent which left the heart through the pulmonary artery was collected quantitatively in intervals of 7.5 min in exchangeable vials. Interstitial fluid (transudate) produced by ventricles and appearing on their surface was collected under a latex cap using slight suction and sampled quantitatively in intervals of 30 min. In this model all irrigated parts, i.e. myocardium and vascular tissues of both right and left heart, contribute to generation of transudate according to their surface area. Transudate flow rate was 50–78 µl/min prior to endothelium disruption and about 4 times higher after disruption. All details of the procedure were reported previously [21,23]. Samples were collected on ice and quickly frozen at –20°C pending analysis. After removal of the heart, blood was collected from the thoracic cavity into plastic tubes spiked with EDTA, aprotinine and heparin, giving approximate final concentrations of 2 mmol/l, 40 µg/ml, and 100 U/ml heparin, respectively. Plasma was allowed to separate from cells (1 h, 4°C), centrifuged and stored at –70°C pending analysis.

2.6 Determination of endothelins, angiotensin II and ANP
2.6.1 ET-1
ET-1 was concentrated by solid-phase extraction from individual effluent samples (~75 ml), the combined transudates from 2 hearts (3–4.5 ml), or 1 ml of plasma (diluted with 9 ml of RIA buffer), followed by quantitative radioimmunoassay (RIA) as described previously [21]. Briefly, the samples were loaded onto pre-conditioned C2 Spe-edTM cartridges (Inovec, Vienna, Austria), ET-1 was eluted with acetonitrile (70%), the eluate freeze-dried and the ET-1 contained in the sediment was dissolved in buffer and determined by RIA using an antibody specific for ET-1 (RAS 6901; Peninsula, Belmont, CA, USA). Standards and samples were incubated with antibody for 48 h, followed by a second incubation over 20 h with [125I]ET-1 at 4°C. Bound radioactivity was separated using polyethylene glycol and counted in a {gamma}-counter. The IC50 value of the standard curve was 1.1±0.04 pg/tube, and the detection limit was 0.08 pg/tube. The intra- and inter-assay coefficients of variation for the determination of 1 pg ET-1 dissolved in 100 µl assay buffer were 4.5% and 5.4%, respectively (n=5). Incubation of ET-1 at 10 pg/ml and of big ET-1 at 1 ng/ml (i.e., respective IC50 values) with collagenase (0.05%, 37°C, 2 h) did not in the least affect breakdown of either peptide (n=5).

2.6.2 Big ET-1
For the determination of big ET-1, a similar procedure was used: pooled effluents from 2 hearts (~150 ml) or 1 ml of plasma diluted with 9 ml RIA buffer were loaded onto C2 cartridges and big ET-1 was eluted and processed as described above for ET-1. The concentration of big ET-1 in transudate was below the detection limit even when transudates of 4 hearts were pooled. Big ET-1 was determined by RIA using a polyclonal antiserum that we had raised against big ET-1 (1–39; rat) in rabbits. Standard solutions of big ET-1 (1–39; rat) (2–512 pg/assay) were prepared in RIA buffer and incubated with antiserum (dilution: 1:10 000) for 48 h at 4°C, followed by addition of [125I]big ET-1 (1–39; rat) for another 20 h (total volume: 300 µl). Bound radioactivity was separated as described for ET-1 and counted. The detection limit was ~2 pg per assay tube (~10% displacement of the radioactive tracer) and the IC50 value of the standard curve was 45 pg/tube. Non-specific binding was determined with 10 ng of big ET-1 (1–39; rat) and was typically 4% of total binding. Cross-reaction of the antiserum was 9% with big ET-1 (1–38; human), 47% with big ET-1 (1–39; porcine), 16% with big ET-1 (22–39; bovine), and zero with ET-1, ANP (1–28; rat) and angiotensin II. The intra- and inter-assay coefficients of variation for the determination of 100 pg big ET-1 in 100 µl assay buffer were 2.9 and 4.7%, respectively (n=4).

2.6.3 Angiotensin II
Plasma (1 ml) was diluted 1:10 with RIA buffer and applied to C2 cartridges, angiotensin II was eluted from the cartridges with acetonitrile (70%), eluted angiotensin II was dried and reconstituted in 250 µl buffer for RIA using commercial materials as described previously [24]. Standards (0.0625–256 fmol/tube) and samples were incubated ~24 h at 4°C with anti-angiotensin II antiserum (Amersham; Vienna, Austria), followed by addition of [125I]angiotensin II for ~20 h. Precipitation of bound angiotensin II was achieved through addition of 100 µl {gamma}-globulin (10 mg/ml) and 750 µl polyethylene glycol 6000 (20%). The IC50 value for the standard curve was 15±1.1 fmol/tube; the detection limit was 0.08±0.02 fmol/tube (n=4). The intra- and inter-assay coefficients of variation for the determination of 20 fmol angiotensin II in 100 µl assay buffer were 6.3 and 7.8%, respectively (n=3). No angiotensin II was detected in coronary effluents or transudates pooled from 4 hearts.

2.6.4 ANP
The extraction and assay of ANP in plasma and coronary effluents was reported in detail previously [25]. Briefly, plasma (0.25 ml) was acidified with 0.25 ml trifluoroacetic acid (TFA; 0.5%), loaded on C18 columns, ANP was eluted with 2 ml of 80% acetonitrile in 0.1% TFA, the eluate freeze-dried and the ANP contained in the sediment was dissolved in buffer and determined by RIA. The recovery of the extraction procedure was tested with [125I]ANP and was 74±1% (n=6). Effluent samples were appropriately diluted with RIA buffer (usually 3-fold) and used in the assay without prior extraction. Samples and standards (0.0625–256 fmol/tube) were incubated with anti-atrial natriuretic factor (rat) serum (Phoenix Pharmaceuticals, Belmont, CA, USA; diluted 1:2 with RIA buffer) for 24 h, followed by addition of ~10 000 cpm of [125I]ANP and incubation for another 20 h at 4°C. Precipitation of bound ANP was achieved through addition of 100 µl {gamma}-globulin (10 mg/ml) and 750 µl polyethylene glycol 6000 (20%). The IC50 value of the standard curve was 1.6±0.08 fmol/tube and the detection limit was 0.031±0.001 fmol/tube (n=3). The intra- and inter-assay coefficients of variation with 2 fmol ANP in 100 µl buffer were 4.6 and 6.9%, respectively (n=3).

2.7 Tissue extraction of ET-1 and big ET-1
ET-1 and big ET-1 were extracted from ventricular tissue following published methods [10]. Right ventricles (pooled from two hearts) or left ventricles were homogenized at 4°C with a Polytron homogenizer (Ultra Turrax) in 2 ml of ice-cooled acetic acid (0.2 mol/l) containing 0.5 mol/l NaCl. The suspension was centrifuged at 2000xg for 15 min at 4°C and the supernatant solution was applied to solid phase extraction and quantitative RIA as described above for ET-1 and big ET-1. Recovery of added [125I]ET-1 and [125I]big ET-1 added to the tissue before homogenization was 88±2% and 92±2%, respectively (n=4).

2.8 Presentation of data and statistical analysis
Group data are presented as arithmetic mean values±SEM. A one-way analysis of variance (ANOVA) followed by the Scheffe test was performed for comparisons of functional parameters measured over time. The secretion data for ET-1 and big ET-1 are presented as secretion rates (pg/min per g wet heart weight) and respective concentrations (pg/ml coronary effluent or interstitial transudate) to account for a possible dependence of peptide secretion on fluid flow. A probability of <0.05 was considered as significant and is denoted by an asterisk. P values <0.01 were not indicated separately.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Weights of organs
Although body weights did not differ between the two groups (301±27 g and 294±20 g, respectively) right heart weight was significantly higher in monocrotaline-treated rats indicating substantial right-ventricular hypertrophy. Wet weight of right ventricles was 169±10 mg in controls and 254±13 mg in the hypertrophy group (1.5-fold increase; n=11); the corresponding weights of right auricles were 38±2 mg and 67±4 mg, respectively (1.8-fold increase; n=11; p<0.05 in both cases). The weights of left ventricles plus interventricular septum (724±49 and 701±41 mg), left auricles (22±2 vs. 22±2 mg) and lungs (1.66±0.07 and 1.82±0.05 g) were not different between groups (p>0.05; n=11).

3.2 Cardiac functional parameters
The functional parameters LVDevP, CPP, and LVEDP were remarkably stable and not different between hearts from controls and monocrotaline-treated rats (compare time-courses in Fig. 1). Spontaneous heart rate was 303±5 min–1 in control and 304±8 min–1 in treated animals (4 measurements at 30 min-intervals; P>0.05, n=11 hearts). Disruption of endothelium with collagenase led to similar functional changes in the control and monocrotaline-treated groups, i.e. a decline of LVDevP (–42%), a rise in LVEDP (from zero to a maximum of 21±2 mm Hg) and an increase in CPP (2.5-fold) (n=11, p<0.05 disrupted vs. intact endothelium). Heart rate was not different between groups or from pre-collagenase values (288±2 and 290±7 min–1, n=11).

3.3 Mediator levels in plasma
The concentrations of ET-1, big ET-1, ANP and angiotensin II in plasma were significantly elevated in monocrotaline-treated rats (Fig. 2). ET-1 rose from 10.7±0.5 to 16.6±0.6 pg/ml (1.5-fold), big ET-1 from 469.4±40.3 to 1046.1±42.0 pg/ml (2.2-fold), ANP from 51.3±2.6 to 97.7±6.5 pmol/l (1.9-fold increase) and levels of angiotensin II from 63.5±2.0 to 84.0±2.1 pmol/l (1.3-fold) (n=10, p<0.05 in each case).


Figure 2
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  		Fig. 2 Plasma concentrations of ET-1, big ET-1, atrial natriuretic peptide (ANP) and angiotensin II in control and monocrotaline-treated (hypertrophy, HTR) rats. The peptides were extracted and quantified by RIA as described in Methods. Results are means±SEM, n=10 (ET-1, big ET-1, ANP) or 6 (angiotensin II). *P<0.05 vs. respective control.

 
3.4 Release of ET peptides in coronary effluent and interstitial fluid
The effect of monocrotaline treatment on the release of ET-1 in coronary effluent and the effect of endothelium disruption are shown in Fig. 3A. Both in control and hypertrophic hearts, release of ET-1 was stable and similar over 2 h, and disruption of endothelium reduced it to ~30% in both cases. Accordingly, the respective ET-1 concentrations in coronary effluent were similar (Fig. 3B). The abluminal release of ET-1 was measured in interstitial transudate as described previously [21,22] and is depicted in Fig. 4. Much less ET-1 (~4%) appeared in interstitial transudate than coronary effluent, and after disruption of endothelium, release fell to below detection limit. However, due to the large differences in transudate and coronary perfusate flow, the ET-1 concentration was ~5-fold higher in interstitial transudate (Fig. 4B) than the coronary effluent (Fig. 3B; n=8 hearts in each case).


Figure 3
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  		Fig. 3 Rate of ET-1 release in coronary effluent (A) and resulting ET-1 concentrations (B) in hearts from rats treated with saline (control; solid circles) or monocrotaline (hypertrophy; open circles). There was no difference between experimental groups neither before nor after collagenase treatment, but the latter significantly reduced ET-1 formation. Results are means±SEM, n=6 hearts in each case.

 

Figure 4
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  		Fig. 4 Rate of ET-1 release in interstitial transudate (A) and resulting ET-1 concentrations (B) in hearts from rats treated with saline (control; solid circles) or monocrotaline (hypertrophy; open circles). There was no difference between experimental groups. After endothelium disruption with collagenase, ET-1 formation fell below the detection limit. Transudates from 4 hearts were pooled into one sample. Results are means from 2 such samples (8 hearts).

 
Release of big ET-1 into coronary effluent and the resulting concentrations are shown is Figs. 5A and B. Monocrotaline treatment had no effect on release rate, and endothelium disruption reduced the production of big ET-1 to about one half (Fig. 5A). The resulting big ET-1 concentration (Fig. 5B) was about ten-fold higher than the corresponding concentration of mature ET-1 (Fig. 3B).


Figure 5
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  		Fig. 5 Rate of big ET-1 release in coronary effluent (A) and resulting big ET-1 concentrations (B) in hearts from rats treated with saline (control; solid circles) or monocrotaline (hypertrophy; open circles). There was no difference between experimental groups neither before nor after collagenase treatment, but the latter significantly reduced ET-1 formation. Effluents from 2 hearts were pooled into one sample. Results are means±SEM from 4 such samples (8 hearts).

 
3.5 ET-1 and big ET-1 levels in myocardial homogenate
The levels of ET-1 and big ET-1 in ventricles are depicted in Fig. 6. Monocrotaline treatment had no influence on ET-1 or big ET-1 content in left ventricles (ET-1: 7.37±0.75 [control] and 7.75±0.47 [monocrotaline] pg/g; big ET-1: 103.7±13.5 [control] and 107.0±14.8 [monocrotaline] pg/g; n=6; p>0.05). However, ET-1 and big ET-1 content of right ventricles was lower in hypertrophic than control hearts (7.31±0.64 pg ET-1/g in control and 5.68±0.22 pg ET-1/g in hypertrophy group [n=6]; 44.7±6.6 pg big ET-1/g in control and 26.9±5.4 pg big ET-1/g in hypertrophy group [n=8]; p<0.05 in both cases).


Figure 6
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  		Fig. 6 Levels of ET-1 and big ET-1 in ventricles of control rats and rats treated with monocrotaline (hypertrophy, HTR). ET-1 (A,B) and big ET-1 (C,D) were extracted from right and left ventricles as described in Methods. The data are means±SEM from 6 left ventricles analyzed individually and 6 right ventricles pooled into 3 samples of two ventricles each and are expressed per unit tissue wet weight. *P<0.05 vs. respective control.

 
3.6 ANP
The effect of monocrotaline treatment on the release of ANP in coronary effluent is depicted in Fig. 7. In the treatment group, ANP release was increased ~5-fold, and disruption of endothelium had no effect (n=9; p<0.05).


Figure 7
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  		Fig. 7 Atrial natriuretic peptide (ANP) release in coronary effluent of hearts derived from vehicle (control; solid circles) and monocrotaline-treated animals (hypertrophy; open circles). The latter significantly (asterisks omitted) increased ANP formation irrespective of presence or absence of endothelium. Results are means±SEM, n=9 hearts in each case.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
The monocrotaline model is frequently used as a model of pulmonary hypertension and occasionally as a model of congestive heart failure [19]. In accordance with previous observations, some rats died within several weeks of monocrotaline injection, presumably of congestive heart failure as evident from copious peritoneal effusions post mortem. The surviving animals appeared quite normal and were sacrificed at 10 weeks. These animals developed no peritoneal or pleural effusions and had similar lung weights as controls, but showed marked right-ventricular hypertrophy. These features, together with the markedly increased levels of ANP in plasma and coronary effluent (Figs. 2 and 7Go) probably indicate that these animals were in a state of compensated heart failure [26]. Upon perfusion in vitro, baseline LVDevP and coronary function were indistinguishable between control hearts and hearts from treated animals.

Release of peptides was measured in the coronary effluent and interstitial transudate, and rates of release were converted to the respective concentrations in both fluids (Figs. 3–5GoGo). As in several previous studies from our laboratory using this experimental setup, we have found a considerably higher rate of release of endothelins to the luminal than to the abluminal side, but because of the much lower transudate flow rate (≤1%) than effluent flow rate, the resulting concentrations were reversed, i.e. about 5-fold higher in the transudate than the coronary effluent. Therefore, cardiac luminal release of endothelins may have contributed to the levels of ET-1 and big ET-1 measured in plasma (Fig. 2).

The present data clearly indicate no change in ET-1 and big ET-1 release in hearts of animals injected with monocrotaline compared to controls, whereas plasma levels of both peptides were increased about two-fold in treated animals. Therefore, the present data do not support a role for myocardial ET-1 in the development of right-ventricular hypertrophy in rats approaching adulthood. Rather, they point to a possible role for circulating ET-1 because plasma levels of both peptides were higher in animals with myocardial hypertrophy. A possible increase in ET-1 and big ET-1 in the hypertrophic right heart could have been missed by sampling coronary effluent at the coronary sinus which contains effluent from the right and the left heart. However, this is unlikely as evident from measurements of big ET-1 and ET-1 in right-ventricular tissue which showed a reduced, rather than increased, content of peptides per unit wet weight compared to controls. The latter finding is likely explained by the "dilution" of big ET-1 and ET-1 in hypertrophic ventricular tissue and indirectly also supports the conclusion that the production of both peptides was not increased in right ventricles as a result of right-heart haemodynamic overload. Hence, the additional ET-1 and its precursor found in the plasma of hypertrophic rats (Fig. 2) is unlikely to be of cardiac origin.

The present results extend previous observations using ET receptor antagonists. Thus, the progression of cardiopulmonary alterations following monocrotaline treatment was attenuated, albeit not reversed, by the selective ETA-receptor blockers BQ-123 [27], CI-1020 (previously, PD 156707) [28], LU 135252 [29] or FR139317 [30]. Similarly, monocrotaline-injected rats gavaged once daily with bosentan, a mixed ETA/ETB receptor antagonist, showed blunted pulmonary hypertension, less pulmonary vascular thickening, and reduced right-ventricular hypertrophy [31]. Measurements of ET-1 mRNA levels in various tissues following exposure to monocrotaline indicated two- to three-fold increased expression in ventricles and kidney [27,32], pulmonary vessels [33] and lung [34], although in the latter organ reduced levels of peptide and mRNA for ET-1 were also observed [27]. These data, together with the consistent finding of increased plasma levels in monocrotaline-treated rats and the similar rates of ET-1 and big ET-1 release in control and treated animals reported here, suggest that the right-ventricular hypertrophic response in rats with pulmonary hypertension results from an endocrine action of circulating rather than cardiac-derived ET-1. The fact that the level of ET-1 was not increased in right-ventricular hypertrophic tissue (Fig. 6) does not contradict this view, because increased in vivo plasma levels of endothelins (Fig. 2), likely reflecting enhanced synthesis outside the heart, would not translate into a measurably higher concentration in total heart homogenate.

The role of ET receptor subtypes in mediating the hypertrophic response has been the subject of few investigations. The expression of ETB receptor mRNA was perceptibly increased in ventricles of monocrotaline-treated rats compared to controls (Fig. 2 in [35]) and reduced in pulmonary arteries following ET converting enzyme inhibition [36].

After disruption of the coronary endothelium, release of both ET peptides was greatly reduced, indicating that the major portion of cardiac ET-1 and big ET-1 was derived from coronary endothelial cells. We have previously shown that the procedure used for disrupting the vascular endothelium leads to total abolishment of endothelium-dependent relaxation [22]. The origin of the small remaining release of ET-1 into the coronary circulation was not ascertained; however, previous studies have shown that myocytes generate the peptide in culture [37], and following ischemia, in vivo [38]. The reasons for the reduction of left-ventricular pressure development after collagenase treatment were not investigated; in part this may have been due to reduced production of ET-1 which exerts a positive inotropic effect in isolated heart preparations [8].

Although the isolated perfused rat heart is capable of producing angiotensin II in the presence of renin [39] or renin and angiotensinogen [40], we found no angiotensin II in coronary effluents or interstitial transudate in the hearts of rats subjected to vehicle or monocrotaline treatment. Similarly, van Gilst et al. found no angiotensin II in the coronary effluent of untreated hearts [41]. In a previous study using a model of left-ventricular hypertrophy due to aortic insufficiency, left-ventricular angiotensin II was also unchanged, but specific angiotensin AT1 receptor blockade with losartan antagonized the hypertrophic response and the rise in left-ventricular ET-1 [42]. These latter data as well as the data reported in this paper suggest that it is blood-borne rather than local angiotensin II that interacts with the local ET system to produce the hypertrophic response. Studies using AT1 and ET receptor blockers are necessary to further probe the role of the interaction between the angiotensin and ET systems for the development of hypertrophy in the monocrotaline model.

Endothelin is a potent secretagogue for ANP in cultured rat atrial myocytes and isolated hearts [43,44], and the concentration of ANP in plasma has been found to be increased in patients [45] and in rats with heart failure [20,26]. In agreement with the latter animal studies, we found two-fold higher plasma ANP levels in monocrotaline-treated rats (Fig. 2), all of which showed signs of right-heart hypertrophy but had no pleural or peritoneal effusions that would be indicative of heart failure. The cardiac tissue levels of ANP in hypertrophic or failing hearts, measured in extracts of homogenized tissues, have been controversial. In renal hypertensive rats, Matsubara et al. showed a two-fold increase in the level of ANP in hypertrophied right ventricle, a seven-fold increase in extracts of hypertrophied left ventricles, and a considerable decrease in left and right atria [46], whereas in cardiomyopathic hamsters, Franch et al. found a reduced level of peptide in both ventricles [47] and Comini et al., in the same model as used here, reported unchanged levels in ventricles [26]. In part, these discrepancies may be due to referencing ANP to total tissue weight (i. e., myocyte and variably increased non-myocyte tissues) which was avoided in this report by measuring ANP release rates that better reflect the myocardial ANP content. These measurements clearly showed a five-fold higher release in hypertrophied hearts than in non-hypertrophic controls (Fig. 7), and this increase is likely due to stimulated ANP synthesis as suggested by increased mRNA content in the ventricles of monocrotaline-treated rats reported previously [26]. Thus, the reduced content of ET-1 and big ET-1 in extracts of right atrial and right-ventricular tissue (Fig. 6), in the face of increased release, is most likely due to "dilution" in the hypertrophic tissue. Whether the increase in ANP release is partly due to the activity of ET-1 as recently shown in a dog model of pacing-induced heart failure [48], was not tested in this study. If this is the case, ET receptor antagonists might partly blunt the counter-regulatory activity of ANP in myocardial hypertrophy and heart failure.

In conclusion, we have shown in rat hearts with marked signs of right atrial and right-ventricular hypertrophy but not of heart failure, that the local (intramyocardial) ET system is not activated and is unlikely to contribute to the functional derangements characteristic for this experimental model. Therefore, the previously shown effectiveness of ET receptor blockers against monocrotaline-induced pulmonary hypertension and/or right heart hypertrophy more likely results from inhibition of ET-1 originating in other organs, i. e. the endocrine actions of the peptide.

Time for primary review 27 days.


   Acknowledgements
 
The author is grateful to Gerald Wölkart for his invaluable technical assistance and acknowledges the financial support by the Austrian Research Fund (FWF), projects 12934 and 13013, and the Dr. Heinrich-Jörg-Stiftung, Karl-Franzens-Universität Graz, Austria.


   Notes
 
{star} Dedicated to Professor Emeritus Walter R. Kukovetz on the occasion of his 70th birthday. Back


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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