INTRODUCTION

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ENDOTHELIN-1 (ET-1) IS A CARDIOVASCULAR peptide whose circulating concentrations are increased in congestive heart failure (CHF) (10, 15, 35). This increase has been demonstrated to have functional importance as Kiowski et al. (8) elegantly demonstrated that acute administration of a dual ET_A- and ETA_B-receptor antagonist reduced systemic and pulmonary vascular resistance as well as cardiac filling pressures in human CHF (8). More recently, we and others reported that chronic ET receptor antagonism also has beneficial actions in rapid ventricular pacing-induced CHF and leads to sustained decreases in cardiac filling pressures, systemic, renal and pulmonary vascular resistance, increased cardiac output, and attenuated myocyte remodeling (1, 16, 18, 27).

Despite the emerging role of ET-1 for CHF, the activation of ET-1 in local tissues during CHF remains poorly defined. This is particularly relevant as ET-1 is thought to be predominantly an autocrine and paracrine rather than a circulating hormone. Indeed, circulating concentrations of ET-1 in CHF increase to a significantly lesser extent compared with other neurohormones (28). To better understand ET-1 in the pathophysiology of CHF, it is therefore essential to assess local activation in important target organs of the disease, such as the heart, kidney, and lung. Although previous studies focused primarily on left ventricular activation of the prepro-ET-1 gene in models of cardiac dysfunction or hypertrophy (7, 24, 31) and only two studies have addressed cardiac and pulmonary prepro-ET-1 gene expression (6, 32), a combined assessment of atrial, ventricular, pulmonary, and renal prepro-ET-1 gene expression, together with local accumulation of ET-1, has so far not been carried out in a well-documented model of overt experimental CHF.

It was therefore the objective of the present study to assess regional accumulation of ET-1 in atrial, ventricular, renal, and pulmonary tissue in a large-animal model of CHF. To further determine whether regional activation of ET-1 reflects local production or accumulation, we also defined regional expression of the prepro-ET-1 gene. The utilized model of progressive rapid ventricular pacing-induced left ventricular dysfunction shares many similarities with human CHF and results in overt CHF with severe cardiac dysfunction and atrial and ventricular hypertrophy as well as pulmonary hypertension, systemic hypotension, marked neurohormonal activation, and avid renal sodium retention (14, 28). On the basis of recent findings of functional improvement of renal and pulmonary vascular as well as cardiac function (1, 18) and selective pulmonary and atrial prepro-ET-1 gene activation in a vena caval constriction model of cardiac low output in the dog (34), we hypothesized that progressive rapid ventricular pacing-induced canine CHF would be characterized by cardiac, renal, and pulmonary activation of ET-1.

To address our hypothesis, we assessed ET-1 tissue concentrations and prepro-ET-1 gene expression in atrial and ventricular myocardium as well as in renal and pulmonary tissue in normal dogs and dogs with overt CHF after 38 days of progressive rapid ventricular pacing.

	METHODS

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Study Protocol

For the induction of CHF, dogs underwent pacing with a stepwise increase of stimulation frequencies over 38 days. As previously described, this pacing regimen results in overt CHF, as characterized by severe cardiac dysfunction, increased left ventricular mass, impaired systemic and regional hemodynamics, broad neurohumoral activation, and avid renal fluid and sodium retention (14, 28). All pacemakers were checked for proper pacing at the time of programming and then weekly and on completion of the study. At baseline (control) and at the end of the protocol (overt CHF), cardiac filling pressures and cardiac output by thermodilution (model 9510-A, American Edwards Laboratories) were measured in the conscious dog via a Swan-Ganz catheter, and mean arterial pressure was measured via the arterial port catheter. In addition, urine was collected over a 24-h period from animals in a metabolic cage for assessment of renal sodium excretion, and a two-dimensional guided M-mode echocardiogram was obtained. Arterial blood was collected in EDTA tubes, centrifuged at 2,500 rpm and 4°C, and the plasma was stored at 20°C until measurement of ET-1. After the dogs were euthanized (Sleepaway solution iv, Fort Dodge Laboratories, Fort Dodge, IA), tissue was rapidly harvested. Hearts were quickly trimmed, and atria and left ventricles were weighed for the calculation of mass indices. Left ventricular, left atrial, renal, and pulmonary tissues were snap-frozen in liquid nitrogen and stored at 80°C until further processing. A second group of normal dogs served as tissue donors for the control group. All studies were approved by the Institutional Animal Care and Use Committee of the Mayo Clinic and conducted in accordance with the Animal Welfare Act.

Analytic Methods

For analysis of prepro-ET-1 gene expression, mRNA was extracted from tissues by utilizing a commercially available kit (Fasttrack, Invitrogen). Briefly, tissue was homogenized (Polytron PT 1200) in a detergent-based buffer containing RNAse/protein degrader and incubated in a slowly shaking waterbath. DNA was precipitated and sheared, and oligo(dT) cellulose was added for adsorption of polyadenylated mRNA. DNA, proteins, cell debris, and nonpolyadenylated RNA were washed off, and mRNA was eluted off the oligo(dT) cellulose. The yield of mRNA was determined in a spectrophotometer by absorption of 260-nm ultraviolet light. mRNA was loaded on a 1.2% agarose-formaldehyde gel and electrophoresed for 2-3 h at 75 V. The gel was blotted downward overnight (Turbo-Blotter, Schleicher & Schuell) onto a nylon membrane (maximum strength nytran membrane, Schleicher & Schuell). A bovine endothelial prepro-ET-1 full-length cDNA (34) or a 283-base pair partial cDNA were random primed with P32-dCTP (Random Primed DNA Labeling Kit, Boehringer Mannheim Biochemical) and column purified. Membranes were prehybridized (QuickHyb Hybridization Solution, Stratagene, La Jolla, CA) for 10 min at 68°C and then hybridized with the labeled probe for 80 min at 68°C. Membranes were then washed stringent [2× standard sodium citrate (SSC)/0.1% SDS at 22°C for 5 min, then 0.2× SSC/0.1% SDS at 22°C for 5 min, then 0.2× SSC/0.1% SDS at 55°C for 20 min] and exposed to X-ray film for autoradiography. To control for loading conditions and mRNA transfer onto the membranes, blots were rehybridized with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe. The respective autoradiographic bands for prepro-ET-1 and GAPDH were quantified with a scanning spectrophotometer, and prepro-ET-1 mRNA was expressed in arbitrary units as the ratio of autoradiographic densities of the prepro-ET-1 band and the GAPDH band.

Urinary sodium was measured by using ion-selective electrodes (Beckman Instruments, Brea, LA).

Echocardiography

Statistical Analysis

	RESULTS

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Functional Characteristics and Cardiac Mass

Circulating and Tissue ET-1

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Endothelin-1 (ET-1) concentrations in plasma (pg/ml) and tissue (pg/mg protein). Values are means ± SE; n = 5 for control (open bars) and n = 6 for overt congestive heart failure (CHF) tissues (filled bars). LA, left atrium; LV, left ventricle.* P < 0.05 for CHF vs. control; ** P < 0.01 for CHF vs. control; a P < 0.0083 vs. lung (ANOVA); b P < 0.0083 vs. kidney (ANOVA).

A similar pattern was present when local ET-1 was expressed per gram tissue wet weight. Here, significant increases of ET-1 in overt CHF were present in lung (439.8 ± 73.5 vs. 226.0 ± 17.3 pg/g, P < 0.05) and kidney (259.4 ± 30.9 vs. 89.2 ± 43.0 pg/g, P < 0.05), whereas increases in left ventricle (166.8 ± 16.3 vs. 127.4 ± 20.8 pg/g) and left atrium (272.7 ± 46.5 vs. 224.0 ± 69.4 pg/g) did not reach statistical significance. The highest concentration of ET-1 per gram wet weight was observed in pulmonary tissue in overt CHF (P < 0.0083 vs. left ventricle, ANOVA).

Expression of Prepro-ET-1 mRNA

View larger version (30K): [in this window] [in a new window]   Fig. 2.   Representative autoradiography for prepro-ET-1 mRNA from pulmonary tissue. For quantitative analysis, prepro-ET-1 mRNA from control (open bar, n = 7) and overt CHF (filled bar, n = 9) tissues was quantified as ratio of autoradiographic densities of prepro-ET-1 mRNA over glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA and is expressed in arbitrary units (AU). Values are means ± SE. * P < 0.03 vs. control.

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Representative autoradiography for prepro-ET-1 mRNA from left atrial tissue. For quantitative analysis, prepro-ET-1 mRNA from control (open bar, n = 5) and overt CHF (filled bar, n = 9) tissues was quantified as ratio of autoradiographic densities of prepro-ET-1 mRNA over GAPDH mRNA and is expressed in AU. Values are means ± SE. * P < 0.02 vs. control.

	DISCUSSION

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The present study was designed to assess the regional accumulation of ET-1 together with prepro-ET-1 gene expression in cardiac, renal, and pulmonary tissue in a large-animal model of overt CHF. Together with an activation of circulating ET-1, we observed increased concentrations of ET-1 in left ventricular, renal, and pulmonary tissue. This activation was associated with a threefold increase in pulmonary and a twofold increase in left atrial prepro-ET-1 gene expression in overt CHF, whereas a moderate increase in left ventricular gene expression did not reach statistical significance and renal gene expression remained unaltered. During overt CHF, the strongest prepro-ET-1 gene expression was observed in pulmonary tissue and the highest concentrations of ET-1 were observed in pulmonary and renal tissue. Thus the present study demonstrates cardiorenal and pulmonary activation of tissue ET-1 in association with a differential activation of prepro-ET-1 gene expression.

The present study demonstrates increased pulmonary tissue ET-1 in rapid ventricular pacing-induced heart failure in the dog in association with increased gene expression of prepro-ET-1. Indeed, pulmonary tissue showed the strongest expression of prepro-ET-1 of all tissues and a particularly strong upregulation during overt CHF, an observation that may have important implications for our understanding of the lung in CHF. We and others have recently demonstrated a key role of ET-1 for pulmonary vasoconstriction associated with CHF on the basis of a reduction in pulmonary vascular resistance in the presence of acute and chronic ET-receptor antagonists (1, 8). The present finding extends previous reports and suggests that pulmonary tissue is not only a target for ET-1 in CHF but also a major source of ET-1 production. As a result, locally generated and bound ET-1 may importantly contribute to pulmonary vasoconstriction in CHF, and this adaptation may largely represent an autocrine phenomenon. Such an association would also be consistent with studies that have linked the pulmonary ET-1 system to pulmonary hypertension (11). Furthermore, as the lung is characterized by the strongest prepro-ET-1 gene expression and the highest ET-1 concentrations of all tissues studied, pulmonary tissue may as well contribute significantly to increased circulating concentrations of ET-1 in CHF. And although ET-1 gradients were not performed in the present study to prove pulmonary secretion, this concept is also consistent with findings by Tsumamoto et al. (33), who have reported an increase in plasma ET-1 between the main pulmonary artery and the pulmonary capillary wedge region in patients with CHF.

With respect to cardiac ET-1 in CHF, our results demonstrate differential activation in atrial and ventricular myocardium. Although ET-1 was significantly increased in left ventricular tissue in the absence of significant alterations in prepro-ET-1 gene expression, left atrial tissue ET-1 was unchanged, despite significantly increased gene expression. The observation of increased left ventricular ET-1 in the absence of significant activation of prepro-ET-1 gene expression confirms a previous report in a rabbit model of CHF (12) and supports the concept that local gene activation is not a necessary prerequisite for increased accumulation of ET-1 in the left ventricle. Indeed, this finding challenges the concept of a predominantly autocrine-paracrine function of left ventricular ET-1 in CHF, which has been advanced by studies in rodents with myocardial infarction and pressure-overload hypertrophy (7, 24, 31), as it suggests that increased circulating ET-1 contributes to increased local ET-1 in the left ventricle during CHF.

Recent studies in vitro have suggested that the positive inotropic actions of ET-1 on the myocyte may be attenuated (21, 27) or even reversed in CHF (30), and it has been suggested that ET-1-induced coronary vasoconstriction and increased afterload may offset or even reverse residual positive inotropic effects of ET-1 on the myocyte in CHF. Indeed, negative effects of endogenous ET-1 in CHF on total left ventricular contractile performance and relaxation have been reported (20), and studies utilizing chronic ET receptor antagonists have demonstrated an improvement in myocardial structure and function in CHF (1, 27). The present study clearly does not provide functional data. It does, however, support activation of left ventricular ET-1 at an important site of action in CHF and suggests that local mechanisms participate in the detrimental effects of ET-1 on the left ventricle in CHF.

The observed significant gene activation in atrial but not ventricular myocardium during CHF underscores the role of atrial myocytes in cardiac endocrine function. Such a role is also supported by the attenuated increase in local ET-1 in left atrial tissue despite the twofold increase in gene transcription, which is consistent with secretion of ET-1 into the circulation. In addition to an endocrine role of left atrial tissue to generate and release ET-1, local activation of ET-1 may also have an important regulatory function for the secretion of atrial natriuretic peptide (ANP) in CHF. Indeed, a permissive effect of ET-1 for ANP secretion has previously been suggested by studies in vitro and in vivo (2-5, 26, 29). Furthermore, a blunted increase in circulating ANP during experimental heart failure has been reported in the presence of ET receptor antagonism (1).

To our knowledge, the present studies are the first to address alterations of local ET-1 in the kidney during overt CHF with avid renal sodium retention. We observed a significant increase in renal ET-1 concentrations that was even more pronounced compared with those in pulmonary or cardiac tissue and almost achieved a concentration equivalent to pulmonary ET-1 during overt CHF. This finding further underscores an important role of ET-1 in the pathogenesis of renal dysfunction associated with CHF, which has been suggested by studies that reported significantly increased renal vascular resistance, decreased glomerular filtration, and increased tubular reabsorption of sodium in response to the infusion of exogenous ET-1 (9, 17) and which were then partially reversed by ET_A receptor blockade (1, 19). In addition to renal ET-1 concentrations, the present study also assessed whether renal prepro-ET-1 gene expression is regulated in overt CHF. The potential for regulation of renal prepro-ET-1 gene expression has previously been demonstrated by studies that reported activated gene expression in association with renal artery stenosis and hypoxia (23) as well as cytokine stimulation (25). Our present observations extend and complement these studies as they demonstrate that renal prepro-ET-1 gene expression is not activated in a model of overt CHF and sodium retention and that increased renal ET-1 during overt CHF, similar to left ventricular ET-1, must therefore represent local accumulation rather than synthesis.

In summary, the present studies report ET-1 and prepro-ET-1 gene expression in cardiorenal and pulmonary tissues in CHF produced by progressive rapid ventricular pacing. Significantly increased ET-1 concentrations in the left ventricle, lung, and kidney were associated with enhanced gene expression of prepro ET-1 only in the left atrium and the lung. The relatively strongest increases in tissue ET-1 were observed in lung and kidney, and the relatively strongest increase in prepro-ET-1 gene expression was observed in the lung. These studies therefore suggest differential mechanisms by which ET-1 is activated in experimental CHF.

Perspectives