Endogenous Endothelin-1 Participates in the Maintenance of Cardiac Function in Rats With Congestive Heart Failure

Cardiac myocytes¹ as well as vascular endothelial cells² produce ET-1. In addition to its potent vasocontractile effects,² ET-1 has potent positive inotropic and chronotropic effects on isolated heart muscles in vitro³⁴ and induces myocardial cell hypertrophy.⁵ Although it has been reported that plasma ET-1 concentrations are elevated in CHF in both humans⁶⁷⁸ and experimental animal models,⁹¹⁰ the production of ET-1 in the failing heart is not known. Furthermore, it is not known whether myocardial ET-1 participates in the modulation of cardiac function in CHF.

It has been reported that the production of ET-1 in vascular endothelial cells is increased by some humoral factors (eg, angiotensin II,¹¹ vasopressin,¹¹ and norepinephrine² ) and mechanical factors (eg, shear stress¹² and endothelial stretching¹³ ). In cultured ventricular myocytes, it has also been shown that the expression of prepro–ET-1 mRNA is increased by angiotensin II.¹⁴ Therefore, it is thought that the production of ET-1 in the heart may be also regulated by several stimuli in vitro. Using an in vivo model of rats with cardiac hypertrophy, we previously reported that the production of ET-1 was markedly increased in the hypertrophied heart because of hemodynamic pressure overload due to either aortic banding¹⁵ or pulmonary hypertension.¹⁶ These results suggested that the production of ET-1 in the heart is altered in some pathological conditions in vivo. However, because these rats did not have CHF, it could not be determined whether the production of ET-1 is altered in the failing heart.

In the present study, we investigated the production of ET-1 in the heart and the density of myocardial ET receptors in rats with CHF. Furthermore, to study the pathophysiological roles of myocardial ET-1 in CHF, we investigated the effects of intravenously infused BQ-123, an ET_A receptor antagonist,¹⁷ on both heart rate and myocardial contractility in rats with CHF and in sham-operated rats. In the present study, we used rats with coronary artery ligation as a model of experimental CHF because this is a well-established model representing pathophysiological alterations similar to those seen in the most common contemporary cause of heart failure in humans, ischemic heart disease.¹⁸¹⁹

Methods

Experimental Heart Failure

We used the left coronary artery–ligated rat model of CHF, which is well established.¹⁸ Left ventricular free wall myocardial infarction was induced in male Sprague-Dawley rats weighing 170 to 200 g by the method of Pfeffer et al.¹⁸ In brief, each rat was anesthetized with ether. A thoracotomy was performed, the heart was rapidly exteriorized, and the proximal portion of the left coronary artery was ligated with a 5-0 silk suture. The heart was then returned to its normal position, and the thorax was closed. Except for coronary arterial ligation, sham-operated rats were produced by an identical procedure. Mortality in the animals with myocardial infarction was ≈67% within the first 24 hours. The surviving rats (CHF rats) were maintained on standard rat chow and water ad libitum for 3 weeks.

Study Protocols

In the present study, two series of experiments were performed. The purpose of the first series of experiments was to investigate the production of ET-1 in the heart and the density of myocardial ET receptors in the CHF rats. The purpose of the second series of experiments was to investigate the effects of an ET receptor antagonist on the hemodynamics of the CHF rats in vivo.

First Series of Experiments

Hemodynamic Measurement and Tissue Sampling

On the day of the experiment, the rats were anesthetized with sodium pentobarbital (50 mg/kg IP). A microtip pressure transducer catheter (model SPC-320, Millar Instruments) was inserted into the right carotid artery. After arterial blood pressure was monitored, the catheter was advanced into the left ventricle for evaluation of left ventricular pressure. These hemodynamic measurements were recorded with the use of a polygraph system (AP-601G amplifier and WT-687G thermal pen recorder). In addition, LV+dP/dt_max and LV[+dP/dt/Pi]_max were derived by active analogue differentiation of the pressure signal differentiation amplifier (model EQ-601G, Nihon Koden). Subsequently, a polyethylene catheter was inserted into the right jugular vein to measure CVP and right ventricular pressure. Sham-operated rats were randomly selected as control animals. Only rats that underwent ligation with LVEDP ≥15 mm Hg were considered to have CHF. After hemodynamic measurement, a blood sample was collected from the right carotid artery. The heart was subsequently excised and divided into the right ventricle and left ventricle, including septum, in cold Krebs-Ringer solution. Each ventricle was weighed before and after removal of the scarred area and was frozen in liquid nitrogen. Some of the left ventricles were immersion-fixed in 10% buffered formalin. The plasma and tissue samples were stored at −80°C for mature ET-1 peptide assay by an EIA, for determination of prepro–ET-1 mRNA expression by Northern blot analysis, and for binding assay of ET receptors. The lungs and kidneys were also weighed and frozen in liquid nitrogen.

To measure the infarct size, the following experiments were performed. After being weighed, the left ventricle was immersion-fixed in 10% buffered formalin. At a later time, the ventricle was cut into four transverse sections from apex to base. These sections were processed in standard fashion and embedded in paraffin. A Masson-trichrome–stained thin section from each level was projected, and the perimeters of the infarcted and noninfarcted epicardial and endocardial surfaces were traced and digitized. The infarcted portion (proportion of the infarcted left ventricle) was calculated from these measurements.¹⁸

EIA for Determination of Plasma and Heart ET-1 Levels

Each blood sample was placed into a chilled tube containing aprotinin (300 kallikrein inhibiting units/mL) and EDTA (2 mg/mL) and centrifuged at 3000g for 15 minutes at 4°C. The plasma was stored at −80°C until use. Plasma ET-1 concentration was measured by an EIA as previously described.¹⁶²⁰²¹ In brief, plasma (1 mL) was acidified with 3 mL of 4% acetic acid, and immunoreactive ET-1 was extracted with a Sep-Pak C-18 cartridge (Waters Associates). The elutes were reconstituted with 0.25 mL of assay buffer and subjected to EIA for ET-1. EIA for ET-1 was carried out as previously described with immobilized mouse monoclonal antibody AwETN40, which recognizes the amino-terminal portion of ET-1, and peroxidase-labeled rabbit anti–ET-1 carboxyl-terminal peptide(15-21) Fab′.¹⁵²⁰²¹ The assay for ET-1 did not cross-react with ET-3 or big ET-1 (cross-reactivity, <0.1%). The detection limit of this EIA was 0.4 pg/mL.²⁰²¹

The left ventricular ET-1 level was determined as previously described.¹⁵ Briefly, the left ventricle without scar tissue, which was frozen in liquid nitrogen and stored at −80°C, was homogenized with a Polytron homogenizer for 60 seconds in 10 vol of 1 mol/L acetic acid containing 10 μg/mL pepstatin (Peptide Institute) and immediately boiled for 10 minutes. After being chilled, the homogenate was centrifuged at 20 000g for 30 minutes at 4°C and the supernatant was stored at −80°C until use. The supernatant was subjected to an EIA for ET-1.

The samples of the plasma and heart were stored at −80°C, and the measurements of ET-1 levels were performed within 1 month. Under these conditions, we have confirmed that ET-1 levels of fresh samples of the plasma (1.1±0.1 pg/mL, n=6) and heart (107±7 pg/g tissue, n=4) are not different than those of stored frozen samples (plasma, 1.0±0.1 pg/mL, n=6; heart, 112±9 pg/g tissue, n=4). We reported that general anesthesia (isoflurane, nitrous oxide/oxygen, pentobarbital) does not affect plasma ET-1 levels in humans.²² In the present study, the plasma of the rats was collected with the animals under pentobarbital anesthesia. We have also confirmed that pentobarbital anesthesia does not affect plasma ET-1 levels in rats.¹⁶

Northern Blot Analysis for Prepro–ET-1 mRNA in Heart and Kidney

Total RNA was prepared from tissues by selective precipitation in 3 mol/L LiCl and 6 mol/L urea.¹⁶²³ Total RNA (15 μg per lane) from the left and right ventricles, excluding scar tissue or kidney, was separated with the use of formamide/1.1% agarose gel electrophoresis and transferred onto nylon membranes (Hybond N, Amersham). The membranes were prehybridized at 42°C for 3 hours in a solution containing 5× standard saline citrate, 50% formamide, 1% sodium dodecyl sulfate, and 150 μg/mL fragmented salmon sperm DNA and were then hybridized with a ³²P-labeled cDNA probe in the same solution at 42°C for 24 hours. After hybridization, the filter was finally washed in 0.1× standard saline citrate/0.1% sodium dodecyl sulfate at 50°C and autoradiographed with intensifying screens at −80°C for 5 days. The prepro–ET-1 cDNA used as a probe in the present study was a previously described full-length insert of λ rET1-2²³ that was labeled by random priming with [α-³²P]dCTP (≈3000 Ci/mmol, Amersham). To normalize the prepro–ET-1 signals for the loaded amounts and transfer efficiencies, the same membranes were rehybridized with a GAPDH cDNA or ethidium bromide staining of 18S ribosomal RNA as the internal control.

Cardiac Membrane Preparation and Binding Experiments for ET Receptors

The left ventricles of both sham-operated rats and CHF rats, which were stored at −80°C until use, were placed in 3-(N-morpholino)propanesulfonic acid buffer containing 20% (wt/vol) sucrose at 4°C, cut into small pieces, and homogenized for 60 seconds with a Polytron homogenizer. The homogenates were centrifuged at 1000g for 15 minutes at 4°C. The supernatants were centrifuged at 10 000g for 15 minutes at 4°C. Finally, the resulting supernatants were centrifuged at 105 000g for 40 minutes at 4°C. Then, the pellets were suspended in 5 mmol/L HEPES-Tris buffer (pH 7.4) and stored at −80°C until use. Protein concentration was determined with the use of bicinchoninic protein assay.²⁴

Experiments regarding the binding of [¹²⁵I]ET-1 to the membranes of the left ventricle of CHF rats and sham-operated rats were performed according to a previously described method.²⁵ The membranes were incubated with 10 pmol/L [¹²⁵I]ET-1 and unlabeled ET-1 at concentrations ranging from 900 fmol/L to 200 μmol/L in triplicate at 25°C in 50 mmol/L Tris-HCl, pH 7.4, containing 0.1 mmol/L phenylmethylsulfonyl fluoride, 2 μmol/L leupeptin, 1 mmol/L 1,10,-phenanthroline, 1 mmol/L EDTA, and 0.1% BSA. After 4 hours of incubation, buffer A (cold 5 mmol/L HEPES-Tris, pH 7.4, containing 0.3% BSA) was added to the mixture. Free and bound [¹²⁵I]ET-1 were separated with the use of a cell harvester (model M-24, Brandel) by rapid filtration through glass fiber filters (GF/C, Whatman) that had been presoaked in buffer A. After the filters were washed with buffer A, radioactivity was measured with a γ counter (ARC-1000M, Aloka). Specific [¹²⁵I]ET-1 binding was defined as the difference between total binding and nonspecific binding in the presence of 200 nmol/L ET-1. The B_max and K_d values were determined by regression analysis of displacement curves with the use of the LIGAND program.²⁶ Furthermore, to study the ratio of ET_A and ET_B receptor subtypes in the normal rat heart, we conducted competitive displacement experiments of [¹²⁵I]ET-1 binding to rat cardiac membranes with BQ-123 (an ET_A receptor antagonist) in the sham-operated rats.

Determination of Plasma Renin Activity

Plasma renin activity was assessed by a standard technique²⁷ with a commercial radioimmunoassay kit (Travenol Corp) to quantify the amount of angiotensin I generated from angiotensinogen.

Second Series of Experiments

Effects of Intravenously Infused BQ-123, an ET_A Receptor Antagonist, on Hemodynamics in Rats

Rats were anesthetized with urethane (750 mg/kg IP, Wako Pure Chemical) and α-chloralose (80 mg/kg IP, Wako). BQ-123, which was a gift from Dr Masaru Nishikibe (Tsukuba Research Institute, Banyu Pharmaceutical Co, Tsukuba, Japan), was dissolved in saline (2 mg/mL) and infused into the rats by syringe pump (CFV-2100, Nihon Koden) through the right femoral vein for 120 minutes. The rats were divided into the following three groups: (1) sham-operated rats infused with BQ-123 (0.1 mg·kg⁻¹·min⁻¹) (n=7), (2) CHF rats infused with saline (0.05 mL·kg⁻¹·min⁻¹) (n=7), and (3) CHF rats infused with BQ-123 (0.1 mg·kg⁻¹·min⁻¹) (n=8).

The total volume of infused saline alone or saline containing BQ-123 was 6 mL/kg for 120 minutes. Heart rate and arterial blood pressure were measured continuously before and during BQ-123 infusion. LVSP, LV+dP/dt_max, LV[+dP/dt/Pi]_max, and LVEDP were measured at two time points: before and at the end of the 120-minute infusion of BQ-123.

In the sham-operated rats infused with saline (3 mL·kg⁻¹·h⁻¹), a bolus injection of ET-1 (1 nmol/kg) produced a marked increase in blood pressure (56.1±4.7 mm Hg [n=7]). However, in the sham-operated rats infused with BQ-123 (0.1 mg·kg⁻¹·min⁻¹), a potent pressor effect of ET-1 (1 nmol/kg) was greatly attenuated (increase in blood pressure by ET-1, 3.8±1.4 mm Hg [n=6]). Therefore, the present data indicated that the dosage of BQ-123 (0.1 mg·kg⁻¹·min⁻¹) used in the present study was sufficiently high to almost completely block the potent pressor effect of exogenously applied ET-1.

Statistical Analysis

Data are expressed as mean±SEM. All statistical comparisons were performed with a commercially available statistical package for the Macintosh personal computer (stat view, version 4.0, Abacus Concepts). Differences in data between the CHF rats and the sham-operated rats were assessed by unpaired t test. ANOVA for repeated measures followed by Bonferroni’s multiple-comparison tests was used for statistical comparison of changes in heart rate and arterial blood pressure during BQ-123 infusion. Paired two-tailed t test comparisons were used to analyze the changes in LVSP, LVEDP, LV+dP/dt_max, and LV[+dP/dt/Pi]_max induced by BQ-123 infusion. Differences were considered significant at the level of P<.05.

Results

First Series of Experiments

Hemodynamic Parameters and Tissue Weights

MAP and LVSP were significantly lower in the CHF rats than in the sham-operated rats (Table 1). LV+dP/dt_max was also significantly lower in the CHF rats than in the sham-operated rats (Table 1). LVEDP was markedly elevated in the CHF rats (Table 1). RVSP and CVP were significantly higher in the CHF rats than in the sham-operated rats (Table 1). These data indicated that the CHF rats had developed CHF.

The CHF rats weighed less than the sham-operated rats (Table 2). The lung weight/mass index for body weight was markedly higher in the CHF rats than in the sham-operated rats, suggesting the presence of pulmonary congestion in the CHF rats (Table 2). The right ventricular mass index for body weight was significantly higher in the CHF rats in accordance with the elevation of RVSP. The left ventricular wet weight was significantly lower in the CHF rats than in the sham-operated rats (Table 2), although the left ventricular mass index for body weight did not differ between the two groups (Table 2). These results indicated that there was substantial left ventricular thinning in the CHF rats. The infarcted area of the left ventricular free wall was replaced by a thin fibrous tissue. The mean myocardial infarct size was 48±4%.

ET-1 Levels in Plasma and Tissues (Peptide, mRNA) and Levels of ET Receptors

The plasma ET-1 concentration of the CHF rats was ≈3.3-fold higher than that of the sham-operated rats (Fig 1). Plasma ET-1 level was significantly correlated with LVEDP (r=.71, P<.01) and LV+dP/dt_max (r=−.69, P<.01) in the CHF rats. However, in the sham-operated rats, plasma ET-1 level was significantly correlated with neither LVEDP (r=.49, P=NS) nor LV+dP/dt_max (r=−.33, P=NS). Plasma ET-1 level was significantly correlated with the level of the percent infarct size (r=.65, P<.05).

The expression of prepro–ET-1 mRNA in the left ventricle of both CHF rats and sham-operated rats was determined with Northern blot analysis. Typical examples of the left ventricle 3 weeks after surgery are shown in Fig 2A. Fig 2B depicts densitometric analysis of these blots corrected for the levels of GAPDH mRNA, which was used as normalization for a constitutively expressed message in the left ventricle. The expression of prepro–ET-1 mRNA in the left ventricle was markedly higher in the CHF rats than in the sham-operated rats (Fig 2A and 2B). The expression of prepro–ET-1 mRNA in the hypertrophied right ventricle in the CHF rats was markedly enhanced (Fig 2A). The densitometric analysis of these blots also showed that the ratio of the levels of prepro–ET-1 mRNA to GAPDH mRNA in the right ventricle was significantly higher in the CHF rats than in the sham-operated rats (1.5±0.3 versus 0.2±0.1, both n=5, P<.05). However, in the kidney, the expression of prepro–ET-1 mRNA did not differ between the CHF rats and the sham-operated rats (data not shown).

The peptide level of ET-1 in the left ventricle was approximately fivefold higher in the CHF rats than in the sham-operated rats (Fig 3).

In the left ventricle, [¹²⁵I]ET-1 binding density (B_max) was 57% higher in the CHF rats than in the sham-operated rats (Fig 4). However, the dissociation constant (K_d) in the CHF rats did not differ from that in the sham-operated rats (28.7±7.0 versus 29.8±1.9 pmol/L, both n=4). The displacement experiments with BQ-123 showed that the cardiac membranes of the sham-operated rats contained ET_A and ET_B receptors in a ratio of 91:9 (n=4).

Plasma Renin Activity

The plasma renin activity was significantly higher in the CHF rats than in the sham-operated rats (33.8±1.2 versus 25.6±2.1 ng·mL⁻¹·h⁻¹, both n=7, P<.01). There was no significant correlation between the plasma renin activity and plasma ET-1 level in the CHF rats (r=.52, P=NS).

Second Series of Experiments

Effects of Intravenous BQ-123 Infusion on Hemodynamics

Intravenous infusion by syringe pump of BQ-123, an ET_A receptor antagonist, significantly reduced heart rate in the CHF rats by 20% (Fig 5, right) but not MAP (Fig 6, right) or LVSP (Table 3). Intravenous BQ-123 infusion also decreased LV+dP/dt_max, a parameter of cardiac contractility, by 26% in the CHF rats (Fig 7, right). Intravenous BQ-123 infusion increased LVEDP in the CHF rats (Table 3). In the sham-operated rats, BQ-123 infusion did not alter these hemodynamic parameters (Figs 5 through 7, left; and Table 3). In the CHF rats, saline infusion did not affect the studied hemodynamic parameters (ie, heart rate, MAP, LV+dP/dt_max, LV[+dP/dt/Pi]_max, LVSP, and LVEDP; Figs 5 and 6, right; Fig 7, middle; and Table 3).

Because LV+dP/dt_max is reported to be heart rate dependent and because BQ-123 infusion significantly reduced heart rate in the CHF rats, we added the following experiments. The rat hearts were paced at their prior rates of BQ-123 application, and this pacing was continued for 2 hours during BQ-123 infusion. Ventricular pacing of the rat in vivo was performed according to the method of Bittl et al.²⁸ In brief, the CHF rats were artificially ventilated, and the chest was opened. Bipolar pacing electrodes were attached to the posterior wall of the left ventricle and ventricular pacing was performed with square-wave pulses of 5-millisecond duration. The pacing was continued during BQ-123 infusion (2 hours) at prior rates of BQ-123 application (323±8 beats per minutes, n=7). Under this condition, intravenous BQ-123 infusion for 2 hours also significantly decreased LV+dP/dt_max (by 16%; from 5601±357 to 4676±193 mm Hg/s, n=7, P<.05) in the CHF rats.

Because our preliminary study revealed that a decrease in heart rate by continuous BQ-123 infusion reached a plateau level at ≈2 hours in the CHF rats, we infused BQ-123 for 120 minutes in the present study.

Discussion

Cardiac myocytes¹ as well as vascular endothelial cells² produce ET-1. The present study revealed for the first time that the production of ET-1 in the heart is markedly increased in CHF rats as demonstrated by both mRNA and peptide levels and that the density of myocardial ET receptors is also significantly increased in CHF rats, suggesting that the ET receptor–mediated signal transduction system in the heart may be markedly stimulated in these rats. We also demonstrated that intravenous infusion of BQ-123, an ET_A receptor antagonist,¹⁷ significantly decreased both heart rate and LV+dP/dt_max without altering MAP in CHF rats, whereas intravenous BQ-123 infusion did not affect these hemodynamic parameters in sham-operated rats. Furthermore, the present study showed that under constant heart rate condition by ventricular pacing, intravenous BQ-123 infusion also significantly decreased LV+dP/dt_max in CHF rats. These data suggest that endogenous ET-1 is involved in the maintenance of both heart rate and myocardial contractility in CHF rats.

There are three isopeptides of ET (ET-1, ET-2, and ET-3²⁹³⁰ ) and two subtypes of ET receptors (ET_A receptor³¹ ) and ET_B receptor³² ). The affinity rank order of ETs for the ET_A receptor is ET-1≥ET-2 >> ET-3, and that for the ET_B receptor is ET-1=ET-2=ET-3.³³ Both subtypes of ET receptor have been shown to exist on myocytes.³⁴ In the rat heart, binding experiments showed that the cardiac membranes of normal rats contained ET_A and ET_B receptors in a ratio of 91:9. Therefore, it appears that ET_A receptors are dominant in the rat heart. It has also been reported that ET_A receptors are dominant in the human heart.³⁵ Our previous study indicated that ET-1 has potent positive inotropic and chronotropic effects on isolated heart muscle.³⁴ Kasai et al³⁶ showed that the positive inotropic effect of ET-1 on isolated rabbit heart was partially antagonized by BQ-123, indicating the presence of positive inotropic responses elicited by the stimulation of ET_A receptors. Therefore, the inhibitory effect of intravenous BQ-123 infusion on cardiac contractility in CHF rats in the present study may have been due to the blocking of myocardial ET_A receptors. The amount of intravenously infused BQ-123 (0.1 mg·kg⁻¹·min⁻¹) used in the present study was sufficiently high to almost completely block the potent pressor effect of exogenously applied ET-1 (1 nmol/kg). Because intravenous BQ-123 infusion did not affect the cardiac function of the sham-operated rats, it is conceivable that the potency of the stimulation of myocardial ET_A receptors by endogenous ET-1 may not be sufficient to elicit inotropic effects in normal rats.

To our knowledge, there are no reports indicating which subtype of ET receptor is involved in the positive chronotropic effect of ET-1 in the normal heart in vitro. Because ET-1 has a potent positive chronotropic action, the reduction in heart rate induced by intravenous BQ-123 infusion in CHF rats may be due to the blocking effect of BQ-123 on ET_A receptors in the pacemaker cells of CHF rats. Therefore, it appears that the ET_A receptor–mediated signal transduction system was at least partially involved in mediating the positive chronotropic effects of ET-1 in CHF rats.

We recently demonstrated that prepro–ET-1 mRNA was increased in the heart under some pathological conditions in vivo: pressure overload to the left ventricle due to aortic banding¹⁵ or pressure overload to the right ventricle due to pulmonary hypertension.¹⁶ However, the studied rats did not have CHF, and the production of ET-1 in the failing heart was not known. The present study revealed for the first time that the production of ET-1 in the heart is markedly increased in CHF rats. The question arises of how the production of ET-1 is increased in the heart of CHF rats. The expression of c-jun proto-oncogene has been shown to be induced in the failing heart of rats with myocardial infarction.³⁷ It has been demonstrated that the 5′ flanking region of the prepro–ET-1 gene has three octanucleotide sequences that conform with a consensus of AP-1/Jun-binding elements and that phorbol ester, an activator of protein kinase C, which is a necessary upstream prerequisite for the regulation of the AP-1/Jun-binding elements, actually activates prepro–ET-1 mRNA expression in cultured endothelial cells.³⁸³⁹ Therefore, it is likely that failing of the heart may induce prepro–ET-1 mRNA expression via the expression of trans-acting transcription factors such as activator protein-1 in CHF rats. Because the expression of prepro–ET-1 mRNA in the kidney did not differ between the two groups, tissue-specific enhancement of the expression of prepro−ET-1 mRNA may occur in the heart of CHF rats. The present study revealed that the heart was one of the origins of elevated plasma ET-1 in CHF rats. In the present study, plasma ET-1 level was positively correlated with LVEDP and negatively correlated with LV+dP/dt_max. Furthermore, the present study showed that plasma ET-1 level was significantly correlated with the level of the percent infarct size. In patients with chronic heart failure, plasma ET-1 concentration is well correlated with the clinical class of heart failure, New York Heart Association functional class.⁶ Taken together, it is considered that plasma ET-1 concentration may reflect the severity of cardiac damage in CHF.

It has been reported that the production of ET-1 in vascular endothelial cells is increased by some humoral factors such as angiotensin II,¹¹ arginine vasopressin,¹¹ and norepinephrine.² Plasma concentrations of angiotensin II, arginine vasopressin, and norepinephrine have been demonstrated to be increased in CHF.⁴⁰⁴¹ Therefore, some researchers⁶⁴² have speculated that the increased plasma level of ET-1 in CHF may be due to increased production of ET-1 in systemic endothelial cells that were stimulated by increased circulating angiotensin II, arginine vasopressin, and norepinephrine.⁴² However, in the present study, the expression of prepro–ET-1 mRNA in the kidney, an endothelial cell–rich tissue, was almost the same in CHF rats as in sham-operated rats. Therefore, it was thought that circulating humoral factors did not cause the observed increase in the production of ET-1 in the endothelial cells of the vessels in the systemic circulation of the CHF rats. However, further study is needed to determine whether the production of ET-1 in the endothelial cells of the systemic vessels is higher in the more severe stages of CHF than in the present stage. On the other hand, it has been reported that angiotensin II induces prepro–ET-1 mRNA in cultured myocardium¹⁴ and that the activity of the intracardiac renin-angiotensin system is increased in CHF.⁴³⁴⁴ Therefore, it seems possible that the activation of the intracardiac renin-angiotensin system contributed to the enhanced production of ET-1 in the hearts of the CHF rats in an autocrine/paracrine fashion. This consideration is in accord with the present results that the renin-angiotensin system was stimulated in the CHF rats. Alternatively, because it has been demonstrated that mechanical factors such as shear stress¹² and endothelial stretching¹³ increase the production of ET-1, hemodynamic overload to the cardiac myocytes appears to be capable of directly increasing the production of ET-1 in the heart of CHF. Our previous report indicated that pressure overload to the left ventricle caused an increase in the production of ET-1 in the left, but not in the right, ventricle of rats.¹⁵

It has been reported that ET-1 binding sites on cultured cardiocytes are downregulated by pretreatment with ET-1.⁴⁵ Furthermore, we have demonstrated that the level of ET_B receptor mRNA is downregulated by ET-1 via a decrease in the intracellular stability of mRNA molecules in rat osteosarcoma cells.⁴⁶ Therefore, it can be anticipated that agonist-induced receptor downregulation exists in the ET system. Although ET-1 levels in plasma and in the heart were markedly increased in CHF rats, unexpectedly, ET-1 binding sites were also significantly increased in the hearts of CHF rats. The precise mechanism of the increase of ET receptors in the hearts of CHF rats is unclear at present; however, the results of this study strongly suggest that the ET receptor–mediated signal transduction system in the heart is markedly stimulated in CHF rats.

It has been reported that the acute administration of bosentan, an ET_A/_B combined receptor antagonist,⁴⁷ lowers blood pressure in rats with CHF due to myocardial infarction.⁴⁸ However, in the present study, BQ-123 at a dosage (0.1 mg·kg⁻¹·min⁻¹) that was sufficiently high to block the potent pressor effect of exogenously applied ET-1 (1 nmol/kg) almost completely unaltered MAP in CHF rats. Furthermore, Love et al⁴⁹ reported that intra-arterial BQ-123 infusion did not affect mean blood pressure in patients with heart failure. One possible explanation for the discrepancy in effects between bosentan and BQ-123 is that the blockade of both ET_A and ET_B receptors by bosentan may contribute to its blood pressure–lowering effect in CHF rats, because both ET_A- and ET_B-mediated mechanisms for vascular contraction exist in various blood vessels.⁴²⁵⁰ This argument is in good agreement with the fact that the blood pressure of spontaneously hypertensive rats is lowered by the acute administration of Ro 47-0203 (bosentan)⁵¹ and by SB 209670 (an ET_A/_B combined receptor antagonist⁵² ) but not by BQ-123.⁵³ ) The acute administration of bosentan did not reduce heart rate in rats with CHF.⁴⁸ Because bosentan lowered blood pressure in rats with CHF,⁴⁸ we considered the activation of the sympathetic nervous system by the reflex to hypotension to have occurred in these rats and that this activation may compensate for the decrease in heart rate due to the blockade of cardiac ET receptors, thereby maintaining the heart rate in these rats. This may be a reason for the discrepancy in the effects of BQ-123 (the present study) and bosentan⁴⁸ on the heart rate of rats with CHF.

In conclusion, the results of the present study indicate that the production of ET-1 in the heart is markedly increased and that the density of myocardial ET receptors is significantly elevated in CHF rats. These findings suggest that the ET receptor–mediated signal transduction system in the heart is markedly stimulated in CHF rats. Because intravenous BQ-123 infusion significantly reduced both heart rate and myocardial contractility in CHF rats but not in sham-operated rats, endogenous ET-1 may be involved in the maintenance of cardiac function in CHF rats.

Study Limitations

In general, heart rate increased in patients with CHF. However, in the present study, the heart rate of the CHF rats under anesthesia did not differ from that of the sham-operated rats in both the first and the second series. We consider the reason for this discrepancy to be the following. We used pentobarbital in both groups at the same dose (50 mg/kg IP) in the first series. In the second series, we used urethane in both groups at the same dose (750 mg/kg IP). Both anesthetic agents have a cardiodepressant effect. In the CHF rats, the metabolic rate of anesthetic agents might be decreased. Therefore, it was thought that a cardiodepressant action was augmented in the CHF rats, thereby resulting in no difference in heart rate between in these rats. Other researchers have also reported that there was no difference in heart rate between rats with CHF and control rats under pentobarbital anesthesia.⁵⁴ Because we have previously confirmed that plasma ET-1 levels were not affected by pentobarbital anesthesia in rats,¹⁶ we used this drug for anesthesia in the first series (measurement of ET levels). Because pentobarbital is short acting and the second series of experiments (effects of BQ-123 infusion [120 minutes] on the hemodynamics) required a long-lasting anesthesia, we switched anesthetic agent from pentobarbital to urethane. We considered urethane to be a more suitable anesthetic agent for the second series. However, it is not known whether urethane anesthesia affects plasma ET-1 levels.

The plasma renin activity in the sham-operated rats (25.6±2.1 ng·mL⁻¹·h⁻¹) appears to be higher than the level in the other report.⁵⁵ Because anesthesia with pentobarbital causes an increase in the plasma renin activity and we used this agent for anesthesia in the first series of experiments, this may be a major reason for a relatively high level in the plasma renin activity in the sham-operated rats.

LV+dP/dt_max is reported to be heart rate dependent. In the present study, BQ-123 infusion significantly reduced heart rate in CHF rats. The present study showed that the degree of the percent decrease in LV+dP/dt_max due to BQ-123 infusion in CHF rats with ventricular pacing is less than that in CHF rats without ventricular pacing (16% versus 26%). Therefore, it is possible that a decrease in LV+dP/dt_max due to BQ-123 infusion in CHF rats without ventricular pacing may be in part attributed to the decrease in heart rate due to BQ-123, in addition to the direct blocking effect of BQ-123 for the positive inotropic action induced by endogenous ET-1.

In the present study, the rats that had myocardial infarction 3 weeks earlier were used as CHF model animals. The right ventricle of the CHF rats was markedly hypertrophied by pressure overload, whereas there was substantial left ventricular thinning in the CHF rats. We consider this decline in left ventricular weight to possibly be due to a lack of opportunity for full compensation in the CHF rats. Therefore, it remains to be elucidated whether the production of ET-1 is altered in the left ventricle of rats with CHF that are fully compensated.

Selected Abbreviations and Acronyms

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Footnotes