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Am J Physiol Heart Circ Physiol 278: H1299-H1310, 2000;
0363-6135/00 $5.00
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Vol. 278, Issue 4, H1299-H1310, April 2000

Regulation of ET: pulmonary release of ET contributes to increased plasma ET levels and vasoconstriction in CHF

Harald Kjekshus1, Otto A. Smiseth1, Randi Klinge1, Erik Øie1,2, Marit E. Hystad1, and Håvard Attramadal1,2

1 Institute for Surgical Research and 2 Merck Sharp & Dohme Cardiovascular Research Center, National Hospital, University of Oslo, N-0027 Oslo, Norway


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
APPENDIX

Endothelin (ET) contributes to the increased systemic vascular resistance and elevated cardiac filling pressures seen in congestive heart failure (CHF). We investigated to what extent ET-mediated vasoconstriction in CHF occurs through an endocrine action of elevated plasma ET or by an autocrine/paracrine mechanism related to induction of vascular ET gene expression. Three weeks of pacing (225 beats/min) induced a marked release of ET-1 from the pulmonary circulation with a sixfold elevation of arterial plasma ET in CHF pigs compared with sham-operated pigs. Arterial plasma ET was the strongest and only independent predictor of systemic vascular resistance. In contrast, vascular preproET-1 and ET-receptor mRNA expression were unaltered or decreased in CHF pigs and did not correlate with indexes of vascular tone. However, myocardial preproET-1 mRNA expression increased twofold in CHF pigs. PreproET-2 and preproET-3 mRNAs were not detectable in cardiovascular tissues. In conclusion, plasma ET was markedly increased because of an augmented release from the pulmonary circulation during CHF, and arterial plasma ET correlated with systemic vascular resistance. The absence of ET induction in the peripheral vasculature suggests that ET increases vascular tone during CHF by an endocrine, not an autocrine/paracrine, mechanism.

heart failure; pacing; ribonucleic acid; swine; receptors


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
APPENDIX

THE ENDOTHELIN (ET) system consists of a family of three potent vasoconstrictor peptides, ET-1, ET-2, and ET-3 (16, 64), acting on two known receptor subtypes, ET-A and ET-B (1, 45). The ET system is activated in several cardiovascular diseases such as myocardial infarction and congestive heart failure (CHF), in which plasma endothelin (pET) levels have been shown to correlate strongly with morbidity and mortality (6, 25, 28, 39, 41, 50, 63). In various animal models of CHF and in patients with CHF, treatment with an ET-receptor antagonist has been shown to reduce systemic vascular resistance (SVR) and cardiac filling pressures and to improve cardiac output (CO) (18, 27, 37, 42, 51, 59). Thus the ET system may act in concert with the sympathetic nervous system and the renin-angiotensin-aldosterone system to elevate SVR and constrict the capacitance vessels, thereby elevating afterload and preload of the failing heart.

The prevailing notion has been that ET elicits its effect in an autocrine/paracrine fashion and that the elevated levels of pET observed during CHF are merely a spillover from an abluminal endothelial release. However, pathophysiological levels of circulating ET may elicit vasoconstriction and other biological effects (5, 7, 24, 58). Thus ET-mediated effects on arterial and venous tone could occur by a hormonal action of elevated circulating endothelin or by an autocrine/paracrine mechanism related to an induction of the ET system in peripheral vessels. Regulation of the ET system in the peripheral vasculature has not previously been studied in CHF. Furthermore, the source of circulating ET and its contribution to the increased ET-mediated vasoconstriction observed during CHF is as yet not clear. The aim of the present study was therefore to 1) analyze the regulation of ET-isopeptide and ET-receptor mRNA expression in the heart and vasculature during CHF, 2) quantify pET levels and localize the site of ET release to the circulation, and 3) determine to what extent increased SVR and elevated cardiac filling pressures in CHF could be attributed to a hormonal or an autocrine/paracrine action of endothelin.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
APPENDIX

Animal model. Norwegian farm pigs of either sex weighing 27-34 kg were premedicated with ketamine (20 mg/kg im), atropine sulfate (0.05 mg/kg im), and azaperone (3 mg/kg im); anesthetized with thiopental sodium (1 mg/kg iv); intubated; and ventilated (Servo 900B, Siemens-Elema, Solna, Sweden) with a mixture of air, oxygen (30%), and isoflurane (1.5-2%) anesthesia. Blood gases were monitored regularly and kept within normal limits by adjustment of ventilator settings. All pigs were subjected to a left thoracotomy at the fifth intercostal space and pericardiotomy. In the CHF group (n = 7 animals), a programmable pacemaker (Pasys 8329, Medtronic, Minneapolis, MN) was implanted with a unipolar shielded electrode sutured to the left atrium. The sham group (n = 7 animals) did not receive a pacemaker. To avoid contamination with systemic venous blood when sampling from the coronary sinus, we ligated the hemiazygous vein just before its opening into the coronary sinus in all pigs. The pericardium was then loosely approximated, the thoracotomy closed, and the pleural cavity evacuated for air. Ampicillin (1.5 mg/kg im) and buprenorphine (0.3 mg im bid) were given to all pigs the first three postoperative days. The pigs were allowed 1 wk to recover before entering the protocol.

Heart failure was induced in the CHF group by rapid pacing (225 beats/min). An electrocardiogram was recorded immediately after the onset of pacing and on days 1, 7, 14, and 21 to confirm 1:1 atrioventricular conduction.

To evaluate the temporal pattern of pET, venous blood samples were drawn at baseline and on days 1, 7, 14, and 21 of pacing. After 21 days, pacing was terminated, and 24 h later the pigs were anesthetized again as described, except that thiopental was substituted with inhalation of isoflurane (2-3%) to avoid the cardiodepressant effects of thiopental.

A 7-F fluid-filled catheter was positioned in the abdominal aorta and a thermodilution catheter (Swan-Ganz 93A-131H-7F, connected to a 9520A Cardiac Output Computer, American Edwards, Santa Ana, CA) in the pulmonary artery for pressure and CO measurements. A 7-F fluid-filled catheter was positioned in the coronary sinus for blood sampling with its position verified by contrast injection (Omnipaque, Nycomed, Oslo, Norway) during fluoroscopy. The catheters were connected to AE840 transducers (SensoNor, Horten, Norway) and pressures recorded on a Gould ES-2000. After instrumentation, the pigs rested 15 min before hemodynamic measurements were performed. Blood samples were then drawn from the abdominal aorta, coronary sinus, and right atrium into prechilled vacutainers containing 1 mg/ml EDTA, kept on ice, and centrifuged, and the plasma was recovered and stored at -70°C until use. The pigs were then killed, and tissue samples for mRNA analysis were obtained from all four heart chambers (CHF, n = 6; sham, n = 6) and from the abdominal aorta, inferior vena cava, portal vein, hepatic artery and vein, renal artery and vein, femoral artery and vein, and a mesenteric vein (CHF, n = 6; sham, n = 7), rinsed in saline, snap-frozen in liquid N2, and stored at -70°C. Tissue samples for immunohistochemistry were taken from two sham-operated and two CHF animals, rinsed in saline, fixed in Bouins solution (2% paraformaldehyde and 0.2% picric acid in PBS), embedded in paraffin wax, and stored at 4°C. All tissue sampling was completed within 50 min after the circulation was stopped.

All animal handling, experiments, and proceedings were approved by the local laboratory animal science specialist under the surveillance and registration of the Norwegian Experimental Animal Board, conforming with "Guiding Principles for Research Involving Animals and Human Beings."

Neurohormonal assays. Plasma ET-like immunoreactivity (pET) was measured with a commercial RIA kit (Nichols Institute Diagnostics, Wichen, The Netherlands) after extraction on a Sep-Pak C18 column (Millipore, Bedford, MA; recovery rate 75%, not corrected for). The cross-reactivity using this assay system was 100% toward ET-1, 52% toward ET-2, 96% toward ET-3, and 7% toward Big ET-1. Within-assay and between-assay variability was 11.8% and 10.9%, respectively. For the analysis of ET gradients, we used a more ET-1-selective ELISA kit (BBE 5, R&D Systems, Minneapolis, MN) with cross-reactivity of 100% toward ET-1, 45% toward ET-2, 14% toward ET-3, and <1% toward Big ET-1. Within-assay and between-assay variability was 4.6% and 6.5%, respectively. Plasma ANG II and plasma atrial natriuretic peptide immunoreactivity were analyzed by RIA as previously described (14, 20). Within-assay and between-assay variability was 6.6% and 10.0%, respectively, for ANG II and 4.2% and 15.1%, respectively, for atrial natriuretic peptide. Plasma norepinephrine and plasma epinephrine were measured using HPLC with electrochemical detection (Chromsystems Instruments and Chemicals, Munich, Germany) after Sep-Pak Alumina (Millipore) extraction. Between-assay variability was 12% for norepinephrine and 13% for epinephrine.

cDNA cloning and plasmid vector constructs. A 520-bp fragment of porcine preproET-1 mRNA [corresponding to bp 7-526 (64)] was amplified by RT-PCR from porcine lung and subcloned into the pBluescript SK(+) vector (Stratagene, La Jolla, CA) and verified by DNA sequencing. The vector construct was cleaved in the vector and the insert with Xmn I and allowed for synthesis of an antisense RNA probe using T7 RNA polymerase, which protected a 265-bp fragment in the RNase protection assay (RPA).

A 159-bp fragment (APPENDIX) of porcine preproET-2 mRNA was amplified by RT-PCR from porcine small intestine and subcloned into the pBluescript SK(+) vector. DNA sequence analysis verified that the open reading frame of the DNA fragment encoded a deduced peptide sequence identical to that of mature human ET-2. The vector construct was linearized in the vector at the unique restriction site BamH I and allowed for synthesis of an antisense RNA probe using T7 RNA polymerase, which protected a 159-bp fragment in the RPA.

A cDNA library from porcine lung (Porcine Lung 5'-STRETCH cDNA Library, Clontech, CA) was screened with a rat preproET-3 cDNA probe, and positive colonies were isolated and subcloned into pBluescript SK(+). DNA sequence analysis showed that the open reading frame of the cDNA encoded a peptide sequence identical to that of mature human ET-3 (APPENDIX). The vector construct was digested with Sac I to remove the 3'-end of the insert and then religated. The resulting vector construct was linearized at a unique Sty I site in the insert, allowing for RNA probe synthesis with T3 RNA polymerase that protected a 192-bp fragment in the RPA.

Porcine ET-A and ET-B receptor cDNA corresponding to the fragments between the third and sixth transmembrane loops were amplified from porcine lung by RT-PCR using degenerate oligonucleotide primers and then subcloned into PCRscript (Stratagene, La Jolla, CA). Southern blotting using rat cDNA from the same receptor regions was used to identify ET-A and ET-B receptor clones. DNA sequence analysis verified an ET-A receptor fragment corresponding to bp 593-1026 (34) and an ET-B receptor fragment corresponding to bp 578-1025 (12). To facilitate probe synthesis of an appropriate size, we digested the ET-A vector construct with Sph I and Not I, removed the fragment between the sites, and blunt ended, religated, and linearized the vector construct with BamH I, allowing for probe synthesis using T3 RNA polymerase that protected a 173-bp fragment in the RPA. The ET-B vector construct was digested with Sph I and EcoR V, the fragment between the sites was removed, and the vector construct was blunt ended, religated, and linearized with Sty I, allowing for probe synthesis using T7 RNA polymerase that protected a 211-bp fragment in the RPA.

A 417-bp fragment (GenBank accession no. U82261; corresponding to bp 1-399) of porcine glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified by RT-PCR from porcine myocardial mRNA, subcloned into pBluescript SK(+), and verified by DNA sequence analysis. The vector construct was linearized at a unique BspM I site, which allowed synthesis of an antisense RNA probe protecting a 99-bp fragment in the RPA.

All linearized DNA fragments were separated on agarose gels, purified by GeneClean, and phenol-chloroform extracted twice to eliminate contaminating RNase activities before in vitro transcription reactions were performed. DNA sequence analysis was performed using the dideoxy chain-termination technique.

Synthesis of radiolabeled RNA probes. Continuously labeled antisense RNA transcripts of preproET-1, ET-A receptor, ET-B receptor, and GAPDH were generated from ~1 µg of linearized template DNA by in vitro transcription using 30 units of T3 or T7 RNA polymerase (Promega, Madison, WI) in the presence of 3 µM [alpha -32P]CTP (specific activity 730 Ci/mM; DuPont NEN), 500 µM each of the unlabeled ribonucleotides ATP, GTP, and UTP, 40 mM Tris · HCl, pH 7.5, 6 mM MgCl2, 2 mM spermidine, 10 mM NaCl, 10 mM dithiothreitol, and 30 units of RNasin (Promega, Madison, WI) in a total volume of 30 µl. In vitro transcription of preproET-2 and preproET-3 were performed as described above, but with the concentration of radiolabeled CTP increased to 7 µM (specific activity 1,900 Ci/mM). After the transcription reaction was completed, the DNA template was removed by digestion with RQ1 DNase I (Promega). The radiolabeled RNA was subsequently extracted with phenol-chloroform and purified by PAGE and electroelution.

RNase protection assay. Total RNA was purified from the tissues by acid-phenol extraction in the presence of chaotropic salts (TRIzol, Life Technologies, Gaithersburg, MD) and subsequent isopropanol-ethanol precipitation. RNA concentrations were calculated from the absorbance at 260 nm.

Total RNA (20 µg) from each tissue sample was mixed with the antisense preproET-1, ET-A, ET-B, and GAPDH RNA probes and coprecipitated with ethanol. Antisense preproET-2, preproET-3, and GAPDH RNA probes were mixed with 50 or 100 µg of total RNA from sham and CHF pools from each type of tissue before coprecipitation with ethanol. The RPA conditions, gel separation, and autoradiography with phosphorimaging were performed as we have previously described (38).

All myocardial samples were analyzed together in one assay. The vessels were analyzed in three assays because of space limitations. Samples from both sham-operated and CHF animals from the aorta, vena cava, and portal vein were analyzed together in one assay. The remaining vascular samples were analyzed together, utilizing the same master probe mix and hybridization conditions, and separated on two identical gels running for the same length of time, and autoradiography was performed simultaneously on the same screen to ensure comparability between the samples. Myocardial tissues were probed for preproET-1 mRNA in two separate assays to ensure the reproducibility of the preproET-1 mRNA upregulation in the heart. Reported values are the means of the two assays. Differences in RNA loading were corrected for by normalizing the mRNA expression levels of preproET-1, ET-A, and ET-B to the GAPDH mRNA expression in each sample.

Immunohistochemistry. Sections (7 µm) of paraffin-embedded tissues were made on a sliding microtome, dewaxed in xylene, and rehydrated in descending concentrations of ethanol. To block the presence of endogenous peroxidase activity, we preincubated the slides with 0.3% hydrogen peroxide in methanol for 30 min at room temperature. The sections were blocked with 3% normal goat serum, subsequently incubated with a rabbit polyclonal anti-preproET-1 antiserum (catalog no. IHC 6904, Peninsula, Belmont, CA) at a 1:500 dilution for 30 min at room temperature, and washed in PBS for 10 min. Immunostaining was done with an avidin-biotin-peroxidase system (Vectastain Elite kit, Vector Laboratories, Burlingame, CA) according to the manufacturer's instructions. Briefly, the sections were incubated with biotinylated goat anti-rabbit IgG for 30 min at room temperature, washed in PBS, and incubated with the avidin-biotin peroxidase complex for 30 min. After a final wash in PBS, the slides were incubated with diaminobenzidine as the chromagen in the commercial metal-enhanced system (Pierce Chemical). The sections were counterstained with hematoxylin. Nonimmune normal rabbit serum was used as negative control.

Calculations and statistical analysis. SVR was calculated as the difference between mean arterial pressure and central venous pressure (CVP) divided by CO. Pulmonary vascular resistance (PVR) was calculated as the difference between mean pulmonary arterial pressure (PAP) and pulmonary capillary wedge pressure (PCWP) divided by CO. Values are means ± SE unless otherwise stated. Shapiro-Wilk's test for normality of the data was performed, and Mann-Whitney rank sum tests were performed when the data did not conform with the normal distribution. Otherwise, Student's two-tailed independent-samples t-test was used for comparison between heart failure and sham groups. Overall cardiac, arterial, and venous mRNA values from CHF and sham-operated pigs were also compared with Student's two-tailed independent-samples t-test after normalization of the data. Normalization was done by dividing the individual sample values by the mean sham value for each tissue. The values from the individual heart chambers, arteries, and veins were subsequently averaged in each pig to obtain overall cardiac, arterial, and venous values. Associations between pET values, tissue mRNA levels, neurohormones, and hemodynamic variables were explored by calculating Spearman's correlation coefficients (rs) using a two-tailed bivariate model including both sham-operated and CHF animals in the analyses. The relative importance and independence of the variables were subsequently analyzed in stepwise multiple regression analyses (P < 0.05 to enter, P > 0.10 to remove), where sham or CHF status was adjusted for by including group status (sham = 0, CHF = 1) as an independent variable. P < 0.05 was considered statistically significant. Statistical analyses were performed using SPSS 7.5 for Windows (Chicago, IL).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
APPENDIX

Three weeks of rapid pacing induced severe heart failure in all CHF pigs as evidenced by clinical signs of CHF, including fatigue, shortness of breath, and loss of appetite, and significant alterations in hemodynamic and hormonal values as shown in Table 1. There were significant increases in both left and right filling pressures with PCWP ranging from 12 to 26 mmHg and CVP ranging from 3 to 18 mmHg in CHF pigs. Cardiac index was reduced by 31% (P < 0.01) and left ventricular stroke work by 41% (P < 0.02), whereas PVR and SVR increased 150% (P < 0.02) and 37% (P < 0.05), respectively. Cardiac hypertrophy was induced as evidenced by a 56% increase in heart-to-body weight ratio (P < 0.001).

                              
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  		Table 1.   Hemodynamic and hormonal values

Plasma endothelin. Venous pET levels during pacing were significantly higher in the CHF group than in the sham group already 1 day after the initiation of pacing and increased steadily throughout the pacing period (Fig. 1). At the time of hemodynamic measurements taken 24 h after cessation of pacing, arterial pET-levels were six times higher in CHF than in sham-operated pigs (P < 0.001; Table 1). The pET-1 gradient (as measured by ELISA) over the cardiopulmonary circulation (i.e., aortic minus right atrial values) increased in CHF pigs from -0.1 ± 0.3 to 4.5 ± 0.6 pg/ml (P < 0.001) and correlated strongly with arterial pET-1 levels [r2 = 0.95, P < 0.001 including all pigs in the analysis (Fig. 2) and r2 = 0.84, P = 0.004 including CHF pigs only in the analysis]. In contrast, the pET-1 gradient over the heart alone (i.e., coronary sinus minus aorta) decreased from 0.5 ± 0.2 to -0.6 ± 0.1 pg/ml (P < 0.001), changing from a positive gradient in sham-operated pigs to a negative gradient in CHF pigs. Thus in sham-operated-pigs there is a small net release of ET from the heart and a small net extraction of ET in the pulmonary circulation. These gradients were reversed in CHF animals, with a large net release of ET from the pulmonary circulation and a small net extraction of ET over the heart.


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  		Fig. 1.   Temporal pattern of venous plasma endothelin (pET) levels with 95% confidence intervals (CI). open circle , Sham-operated pigs; , congestive heart failure (CHF) pigs. *P < 0.05 vs. sham pigs.



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  		Fig. 2.   Scatter plot of pET-1 arteriovenous gradient (ET-1AV; i.e., aortic minus right atrial pET-1 values) vs. arterial pET with linear regression line (solid line) and 95% CI (dashed lines). open circle , Sham-operated pigs; , CHF pigs.

Stepwise multiple regression analysis with adjustment for sham or CHF group status indicated PAP and ANG II as the strongest predictors of arterial pET (Table 2).

                              
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  		Table 2.   Prediction of pET and SVR

Arterial pET and the other neurohormones were explored as possible predictors of SVR, PCWP, and CVP (Table 3). Arterial pET correlated strongly with SVR, and it remained the only significant positive predictor when sham or CHF group status was adjusted for in a stepwise multiple regression analysis (Fig. 3, Table 2). In the analysis, the vasodilator atrial natriuretic peptide showed an inverse correlation to SVR (Table 2). Arterial pET correlated more strongly than the catecholamines and ANG II to PCWP and CVP, but it did not remain an independent predictor when sham or CHF group status was adjusted for.

                              
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  		Table 3.   Associations between neurohormones and vascular tone



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  		Fig. 3.   Scatter plot of arterial pET vs. systemic vascular resistance (SVR) with linear regression line (solid line) and 95% CI (dashed lines) for unadjusted raw data. open circle , Sham-operated pigs; , CHF pigs.

Vascular endothelin expression. PreproET-1 mRNA and ET-A and ET-B receptor mRNA were all clearly expressed in the vasculature (Fig. 4, A-C). There was no significant regulation of preproET-1 mRNA between sham-operated and CHF animals in any of the vessels examined. There was a small nonsignificant trend for downregulation of preproET-1 mRNA in both arteries (-16%) and veins (-9%) in CHF compared with sham-operated pigs. Immunohistochemical analysis with an anti-preproET-1 antibody showed an intense immunostaining in the endothelial layer and weaker, diffuse staining in the media and adventitia of all the vessels (see Fig. 5, F and G). There were no apparent differences in staining intensity or distribution in the vessel wall between sham-operated or CHF animals (data not shown).





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  		Fig. 4.   Autoradiographs from RNase protection assays of preproET-1, ET-A receptor, ET-B receptor, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. Data in A and B and in C and D, respectively, are derived from same RNase protection assays, separated on identical gels and autoradiographed on same phosphorimaging screen. Histograms show results from densitometric analyses of target mRNA levels normalized to GAPDH expression. Data on histograms are means + SE. Values are arbitrary units but are comparable within each assay.

There was no upregulation of vascular ET-receptor mRNA levels during CHF. If anything, a trend toward downregulation of venous ET-A receptor mRNA levels [-21%, not significant (NS)] was observed. ET-A-to-ET-B receptor mRNA ratios in both CHF and sham-operated animals were higher in the arteries than in the veins (2.8 ± 0.3 and 1.4 ± 0.1, respectively, P < 0.001). With the exception of the hepatic circulation, CHF did not alter the vascular ET-A-to-ET-B receptor mRNA ratio.

The expression of ET-A receptor mRNA in the hepatic vein was reduced by 52% (P = 0.03) in CHF compared with sham-operated pigs. There was a strong trend for a downregulation of the ET-A-to-ET-B receptor ratio in the hepatic circulation with a 56% reduction (P = 0.02) in the hepatic vein, a 42% reduction in the hepatic artery (NS), and a 29% reduction in the portal vein (NS). Hepatic vein ET-A-to-ET-B receptor mRNA ratio showed a negative association with CVP (rs = -0.71) and pET (rs = -0.69) but did not remain an independent predictor when sham or CHF group status was adjusted for. Renal vein ET-B receptor mRNA expression was also downregulated, but to a lesser degree (38%, P = 0.02) than in the hepatic vein. Neither renal vein ET-B receptor mRNA nor renal vein ET-A-to-ET-B receptor mRNA ratio was associated with any hemodynamic or hormonal variables.

Myocardial endothelin expression. CHF increased myocardial preproET-1 mRNA levels (Fig. 4D) with a 2.7-fold upregulation in the left ventricle (P = 0.007), a 2.6-fold upregulation in the left atrium (P < 0.001), a 2.4-fold upregulation in the right ventricle (P = 0.002), and a 1.5-fold upregulation in the right atrium (NS). The levels of preproET-1 mRNA differed among the cardiac chambers in both CHF and sham-operated animals. The highest levels were seen in the right atrium, followed by the left atrium and right ventricle, which were, respectively, 2.8 [95% confidence interval (CI): 2.2-3.5], 1.9 (95% CI: 1.6-2.3), and 1.2 (95% CI: 1.1-1.4) times the observed levels in the left ventricle.

Immunohistochemical analyses with an anti-preproET-1 antibody revealed marked staining of the cardiomyocytes that appeared more intense in CHF animals (Fig. 5, A-E). The endothelium of small and medium-sized intramyocardial vessels stained heavily, contrasting with the media, which stained weakly compared with the endothelium and surrounding cardiomyocytes in both sham-operated and CHF pigs.


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  		Fig. 5.   Photomicrographs of sections of porcine cardiovascular tissues immunostained with an anti-preproET-1 antiserum. A: section from normal left ventricular myocardium after sham operation with nonimmune normal goat serum as negative control. B: same section as in A, but with anti-preproET-1 antiserum showing moderate preproET-1-like immunoreactivity primarily in cardiomyocytes. C: section from left ventricular myocardium after pacing-induced CHF showing enhanced preproET-1-like immunoreactivity in cardiomyocytes compared with normal myocardium. Note moderate preproET-1-like immunoreactivity in endothelial cells of vessels, but with almost no immunostaining in media. D and E: high-power magnification of B and C, respectively. F and G: sections through aorta and caval vein, respectively, after pacing-induced CHF. Note intense preproET-1-like immunoreactivity in endothelial cells with moderate immunostaining in media of both vessels.

The neurohormones, and hemodynamic indexes of preload and afterload were explored as predictors of myocardial preproET-1 mRNA. Although all were highly associated with myocardial preproET-1 mRNA levels, none remained as independent predictors when sham or CHF group status was adjusted for in a stepwise multiple regression analysis. However, ANG II came close with a partial correlation coefficient of 0.59 (P < 0.06).

Both ET-receptor mRNAs were clearly expressed in the heart. ET-A receptor mRNA dominated, with an ET-A-to-ET-B receptor mRNA ratio of 4.6 ± 0.3 in the ventricles and 3.0 ± 0.3 in the atria (P < 0.001 compared with ventricles). There was a minor upregulation of left ventricular ET-A receptor mRNA levels (+32%, P = 0.04) in CHF compared with sham-operated animals. However, overall cardiac ET-receptor mRNA expression was not altered by the induction of CHF.

ET-2 and ET-3 mRNA. Neither preproET-2 nor preproET-3 mRNAs were detectable in vascular or myocardial tissues, but both were detected when total RNA was probed from normal small intestine, and preproET-3 mRNA was also detected in normal lung tissue (Fig. 6).


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  		Fig. 6.   Autoradiographs from RNase protection assays of preproET-2, preproET-3, and GAPDH mRNA. Total RNA was pooled from different segments of cardiovascular system from sham-operated pigs and from pigs with pacing-induced CHF to probe 50 or 100 µg of total RNA. PreproET-2 and preproET-3 mRNA were not detectable in heart or vessels despite increased amount of total RNA probed and 5.3-fold increase in [alpha -32P]CTP concentration used in synthesis of antisense RNA probes for preproET-2 and preproET-3 compared with that used for preproET-1.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
APPENDIX

The present study demonstrated a sixfold increase of pET in CHF compared with sham-operated animals. This large increase of pET could primarily be ascribed to a marked release of ET from the cardiopulmonary circulation with an arteriovenous difference of >70% of the venous value. Selective catheterization of the coronary sinus revealed a small extraction of pET in the heart during CHF. Thus the pulmonary circulation appears to be the primary source of ET release in this porcine model of severe pacing-induced CHF. This is consistent with previous reports of increased levels of ET-1 in the lungs of CHF animals (15, 46). Furthermore, Tsutamoto and co-workers (56) found a marked increase of pET levels from the pulmonary capillary wedge region compared with the pulmonary artery in patients with CHF, consistent with our findings. However, not all studies report such a large magnitude of pulmonary ET release (8, 10, 23). Whether this relates to species differences, degree of heart failure, or methodological aspects such as varying cross-reactivity toward Big ET, for example, remains to be studied. Finally, the pulmonary circulation is a putative site not only for increased de novo production of ET but also for ET clearance (9, 11, 47). Pulmonary clearance of pET appears to be inversely related to the degree of pulmonary hypertension and may represent an additional mechanism whereby the lungs indirectly contribute to the increased pET levels observed in CHF (8, 10, 21).

The marked increase in circulating ET contrasts with the findings in the vasculature. No induction of vascular preproET-1 mRNA or any increase in vascular ET-receptor mRNA expression could be demonstrated in the present study. These findings raise interesting implications as to the mode of action of ET-1. Vascular tone is increased during CHF as a compensatory mechanism operating to maintain adequate perfusion pressures to vital organs. However, the increased vascular resistance and the redistribution of blood from the capacitance to the central circulation impose an added burden on the failing myocardium. The significant reductions in cardiac filling pressures and SVR observed during endothelin antagonism in both experimental and human CHF (13, 18, 30, 37, 54) suggest that endothelin is causally involved in the regulation of vascular tone by mediating clinically significant vasoconstriction during CHF. With regard to a putative ET-mediated regulation of SVR, we found no evidence for increased vascular levels of preproET-1 mRNA or altered ET-receptor mRNA expression that might lead to an increased sensitivity to ET-1 in the arteries during CHF. Thus our data do not support an autocrine/paracrine mode of action of ET-1 in the arteries that would account for the increased SVR. Exploring the correlation between SVR and possible regulators of arterial tone such as pET, norepinephrine, ANG II, atrial natriuretic peptide, and epinephrine, we found that pET was the strongest and only independent positive predictor of SVR. We therefore propose that ET-dependent arterial vasoconstriction during CHF is caused by an endocrine effect of the increased levels of circulating ET rather than an autocrine/paracrine effect of ET-1 produced in the vasculature. An endocrine role for ET is consistent with studies of exogenous ET-1 infusions demonstrating that elevating pET-1 to levels observed during CHF leads to increased SVR (5, 24, 58). Although the arterial ET system does not appear to be upregulated in CHF, increased production of ET-1 in the microvasculature of the systemic circulation cannot be excluded and could contribute to the increased SVR in CHF. However, if CHF were to cause an extensive upregulation of microvascular preproET-1 mRNA, elevations of preproET-1 mRNA levels in peripheral tissues would be expected. Thus inferences about the regulation of microvascular preproET-1 might be drawn from analysis of total RNA from entire organs. We have previously performed Northern blot analysis of total RNA isolated from hepatic tissues from rats with and without CHF and did not demonstrate any alterations in preproET-1 mRNA levels (H. Kjekshus, unpublished data). Sakai et al. (44) found no change in preproET-1 mRNA expression in rat kidneys during CHF. Furthermore, in the present study immunohistochemical analysis of myocardial tissues revealed anti-preproET-1 immunostaining of both microvessels and cardiomyocytes. However, only cardiomyocyte immunostaining appeared to be enhanced in the CHF group compared with that in sham-operated animals, consistent with the hypothesis that vascular ET-1 production is unaltered in CHF. Finally, if increased ET-production in the microvasculature were the operating mechanism of ET-mediated vasoconstriction during CHF, one would expect an increased spillover from the vessels. Contrary to this hypothesis, Tsutamoto and co-workers (55) found a decrease in ET-1 spillover in the femoral circulation (i.e., femoral vein minus femoral artery pET-1) in patients with increasing severity of CHF, with a net release of ET-1 observed in New York Heart Association (NYHA) class II patients, no arteriovenous difference in NYHA class III patients, and a net extraction of ET-1 in NYHA class IV patients. Thus the available evidence from the present study and previously published reports does not support an increased production of ET in the microvasculature of the systemic circulation during CHF.

Regulation of venous tone determines the distribution of the circulating blood volume between the capacitance and the systemic circulation. During CHF, capacitance vascular tone has been shown to increase, causing a redistribution of blood from the capacitance vasculature to the central circulation contributing to the increased cardiac filling pressures (35). ET-1 appears to be involved in the regulation of capacitance vascular volume. Studies in healthy humans have shown that decreased venous pressure, hypovolemia, and postural changes elicit increased ET-1 secretion (32, 33, 50). Furthermore, infusion of ET-1 in dogs and rats has been shown to increase mean circulatory filling pressure, a measure of capacitance vessel tone (52, 61). Kiowski et al. (18) found that ET-receptor antagonist treatment in CHF patients significantly reduced right atrial pressure and PCWP, which are indirect measures of capacitance vascular tone (36). In the present study we found no evidence of increased gene expression of the ET system in the capacitance vessels that could account for an increased capacitance vascular tone during CHF. We therefore suggest that the contribution from the ET system to a CHF-induced increase in capacitance vascular tone must occur through an endocrine action of the elevated pET levels.

Vasoconstriction is consistently reported as the dominating effect of vascular ET-A receptor activation, whereas stimulation of vascular ET-B receptors may lead to both vasodilatation and vasoconstriction (18, 26, 53, 57). The 56% reduction of hepatic venous ET-A-to-ET-B receptor mRNA ratio observed in CHF compared with sham-operated animals may potentially attenuate a vasoconstrictive response to ET in the liver. The splanchnic circulation contains the largest capacitance blood volume reservoir in the body and contributes substantially to blood volume homeostasis (19, 40, 60). Thus a selective compensatory regulation of hepatic ET receptors in CHF would be expected to influence the overall effect of ET on total vascular capacitance volume.

The increased levels of myocardial preproET-1 mRNA observed during pacing-induced CHF are consistent with previous reports from several animal models of CHF (2, 15, 38, 43) and testify to the validity and sensitivity of RNase protection assays utilized in the study. In contrast to the pulmonary circulation, the myocardial circulation extracted pET during CHF, consistent with the findings in human cardiomyopathy (31).

ANG II has been shown to increase ET-1 production in cardiomyocytes in vitro (17) and in normal vessels in vivo (29). The correlation between ANG II and both pET and myocardial preproET-1 mRNA expression found in the present study is in keeping with these studies and suggests a link between ANG II and ET-1 production in CHF.

The widespread distribution of ET-1 in the cardiovascular system is well established, but the distribution of ET-2 and ET-3 is not clear. Plumpton et al. (39a) detected ET-1 and ET-2, but not ET-3 in human myocardium. In rats, preproET-2 mRNA expression has been demonstrated in myocardial tissues (38) and preproET-3 mRNA in vascular tissues (62), but to a lesser degree than preproET-1 mRNA. Our failure to detect preproET-2 and preproET-3 mRNA in cardiovascular tissues under conditions up to 10 times more sensitive than those for the analysis of preproET-1 mRNA confirms the predominant role of ET-1 in the cardiovascular system.

Limitations of the study. The hemodynamic and neurohormonal features of pacing-induced heart failure resemble those in human CHF (3, 48, 49). Therefore, the model should be appropriate for studies of the peripheral vasculature during CHF. However, evidence of ultrastructural damage to the myocardium (48) indicates that extrapolation of myocardial findings from pacing-induced CHF to more common forms, i.e., ischemic CHF in humans, most be done with caution.

In the present study quantitative analysis of mature ET peptides or receptor proteins was not performed; rather, inferences as to the levels of these peptides in different segments of the cardiovascular system were made from analyses of mRNA expression. Normally, peptide levels will relate to the mRNA expression in a stable chronic situation. However, processing of preproET-1 to the mature ET-1 peptide by endothelin converting enzyme (ECE) may be altered in CHF. Kobayashi et al. (22) found no regulation of myocardial ECE mRNA expression during CHF, lending less credence to a regulatory role of ECE activity in CHF, but to what extent this is the case in the vasculature remains to be investigated.

In conclusion, in the present porcine model of severe pacing-induced CHF, preproET-1 mRNA was the dominant ET isopeptide expressed in the cardiovascular system. The pulmonary circulation contributed significantly to the markedly increased pET observed in CHF animals, and arterial pET was the strongest predictor of SVR. Vascular preproET-1 and ET-receptor mRNAs were unaltered or decreased in CHF. These findings suggest that ET increases vascular tone during CHF by an endocrine, and not an autocrine/paracrine, mechanism. CHF increased myocardial preproET-1 mRNA levels with enhanced anti-preproET-1 cardiomyocyte immunoreactivity and induced a net extraction of ET from the myocardial circulation. Judging by the mRNA levels, ET-A receptors dominated in the heart and arteries, whereas ET-B receptors were equally or more abundantly expressed in the veins.


    APPENDIX
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
APPENDIX

The sequence of porcine ET-2 mRNA fragment with protein translation follows. The mature ET-2 peptide sequence is highlighted in bold type.
The sequence of porcine ET-3 mRNA fragment with protein translation follows. The mature ET-3 peptide sequence is highlighted in bold type.


    ACKNOWLEDGEMENTS

We gratefully acknowledge the following for skillful analysis of plasma mediators: Dr. Asbjørn Aakvaag, Department of Clinical Biochemistry, Endocrine Section, Haukeland University Hospital, Bergen, Norway (pET RIA); Dr. Ole Djøseland, Medical Department B, The National Hospital, Oslo, Norway (epinephrine and norepinephrine); and Dr. Bengt Erik Karlberg, Department of Internal Medicine, University Hospital, Linköping, Sweden (ANG II). Heidi Kongshaug is warmly recognized for excellent technical assistance.


    FOOTNOTES

This study was supported by grants from The Norwegian Council for Cardiovascular Diseases, Oslo, Norway; the Novo Nordic Foundation; Johan Egeberg's Medical Fund; and The Blix Family Fund for the Promotion of Science, Oslo, Norway. H. Kjekshus, R. Klinge, and E. Øie were supported by fellowships from The Norwegian Council on Cardiovascular Diseases. M. E. Hystad was supported by a fellowship from The National Research Council, Oslo, Norway.

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

Address for reprint requests and other correspondence: H. Attramadal, Institute for Surgical Research, National Hospital, N-0027 Oslo, Norway (E-mail: havard.attramadal{at}klinmed.uio.no).

Received 17 March 1999; accepted in final form 14 October 1999.


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ABSTRACT
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Contribution of endothelin to coronary vasomotor tone is abolished after myocardial infarction
Am J Physiol Heart Circ Physiol, February 1, 2005; 288(2): H871 - H880.
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 Cardiovasc ResHome page
T. G von Lueder, H. Kjekshus, T. Edvardsen, E. Oie, S. Urheim, L. E. Vinge, M. S. Ahmed, O. A Smiseth, and H. Attramadal
Mechanisms of elevated plasma endothelin-1 in CHF: congestion increases pulmonary synthesis and secretion of endothelin-1
Cardiovasc Res, July 1, 2004; 63(1): 41 - 50.
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 Physiol. Rev.Home page
D. L. Brutsaert
Cardiac Endothelial-Myocardial Signaling: Its Role in Cardiac Growth, Contractile Performance, and Rhythmicity
Physiol Rev, January 1, 2003; 83(1): 59 - 115.
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 ChestHome page
A. A. El-Solh, E. Bozkanat, J. Mador, and B. J. B. Grant
Association Between Plasma Endothelin-1 Levels and Cheyne-Stokes Respiration in Patients With Congestive Heart Failure*
Chest, June 1, 2002; 121(6): 1928 - 1934.
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