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Endocrinology Vol. 147, No. 5 2526-2534
Copyright © 2006 by The Endocrine Society

Spironolactone Preserves Cardiac Norepinephrine Reuptake in Salt-Sensitive Dahl Rats

Sebastian J. Buss1, Johannes Backs1, Michael M. Kreusser, Stefan E. Hardt, Christiane Maser-Gluth, Hugo A. Katus and Markus Haass

Departments of Cardiology (S.J.B., J.B., M.M.K., S.E.H., H.A.K.) and Pharmacology (C.M.-G.), University of Heidelberg, 69120 Heidelberg, Germany; and Department of Cardiology, Theresienkrankenhaus (M.H.), 68165 Mannheim, Germany

Address all correspondence and requests for reprints to: Dr. Johannes Backs, Department of Molecular Biology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390-9148. E-mail: johannes.backs{at}web.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
An impairment of cardiac norepinephrine (NE) reuptake via the neuronal NE transporter (NET) enhances the effects of increased cardiac NE release in heart failure patients. Increasing evidence suggests that aldosterone and endothelins promote sympathetic overstimulation of failing hearts. Salt-sensitive Dahl rats (DS) fed a high-salt diet developed arterial hypertension and diastolic heart failure as well as elevated plasma levels of endothelin-1 and NE. Cardiac NE reuptake and NET-binding sites, as assessed by clearance of bolus-injected [3H]NE in isolated perfused rat hearts and [3H]mazindol binding, were reduced. Treatment of DS with the mineralocorticoid receptor antagonist spironolactone preserved the plasma levels of endothelin-1 and NE, cardiac NE reuptake, and myocardial NET density. Moreover, the ventricular function and survival of spironolactone-treated DS were significantly improved compared with untreated DS. The {alpha}1-inhibitor prazosin decreased blood pressure in DS similar to spironolactone treatment, but did not normalize the plasma levels of endothelin-1 and NE, NE reuptake, or ventricular function. In a heart failure-independent model, Wistar rats that were infused with aldosterone and fed a high-salt diet developed impaired cardiac NE reuptake. Treatment of these rats with the endothelin A receptor antagonist darusentan attenuated the impairment of NE reuptake. In conclusion, spironolactone preserves NET-dependent cardiac NE reuptake in salt-dependent heart failure. Evidence is provided that aldosterone inhibits NET function through an interaction with the endothelin system. Selective antagonism of the mineralocorticoid and/or the endothelin A receptor might represent therapeutic principles to prevent cardiac sympathetic overactivity in salt-dependent heart failure.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE RENIN-ANGIOTENSIN-aldosterone system plays a central role in the pathophysiology of heart failure (1, 2, 3, 4, 5). Its activation is associated with a poor prognosis of heart failure patients (6, 7). Since the Randomized Aldactone Evaluation Study (3) revealed a reduced mortality of heart failure patients treated with the mineralocorticoid receptor (MR) antagonist spironolactone, it has become of interest to determine how MR antagonism improves survival. Aldosterone, the endogenous MR agonist, is known to not only regulate renal electrolyte and body fluid balance, but also promote cardiac and vascular fibrosis, baroreceptor dysfunction, parasympathetic inhibition, and sympathetic activation (5, 8). However, it remained unclear which action of MR antagonism contributes in particular to an improved survival rate of heart failure patients. Because in clinical studies the etiology of heart failure is heterogeneous, as are the type and dosages of background medications, it is difficult to conclude from clinical data the underlying mechanisms. Therefore, experimental heart failure models with a homogenous etiology and pathophysiology are helpful tools to dissect aldosterone actions leading to morbidity and mortality. Salt-sensitive Dahl rats (DS) are an established model for heart failure with predominant diastolic dysfunction and have been shown to develop an activation of the local cardiac aldosterone system (9, 10, 11, 12, 13).

Activation of the sympathetic nervous system is a major characteristic in heart failure patients (14). Elevated plasma levels of norepinephrine (NE) are associated with a poor prognosis of heart failure patients (15). An impairment of cardiac NE reuptake by NET down-regulation contributes to an increased cardiac net release of NE in heart failure, which is associated with a depletion of cardiac NE stores, down-regulation of cardiac ß-adrenoceptors, and profound alterations of postreceptor signal coupling (16, 17, 18, 19, 20, 21). Clinical studies have shown that impaired cardiac NE reuptake is also associated with a poor prognosis of heart failure patients (reflected by worsening of heart failure and increasing incidence of sudden death) (22, 23). Recently, we have shown that endothelin (ET)-1 is a potent negative regulator of NET and that inhibition of the ET-A receptor (ETA) attenuates NET impairment in aortic-banded rats (24). Interestingly, increasing evidence suggests an interaction between the aldosterone and ET systems (25).

In the present study we tested the hypothesis that MR antagonism improves cardiac sympathetic nerve function in DS (11). Determinants of cardiac NE homeostasis and left ventricular function were evaluated. Moreover, we asked whether aldosterone mediates its effects on cardiac sympathetic nerve function through an interaction with the endothelin system.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
This investigation conformed with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health and was approved by the authorities of the Regierungspräsidium Karlsruhe, Germany.

Dahl salt-resistant (DR) and -sensitive (DS) rats
Male DR and DS (9 wk of age; mean weight, 200 g; M&B, Ry, Denmark) were fed a high-salt diet (8% NaCl) for 40 d. DS were randomized to receive either spironolactone (50 mg/kg·d; DS + spironolactone) or prazosin (5 mg/kg·d; DS + prazosin) in the drinking water (was changed twice the day) beginning with onset of the diet. Water and drug intake was controlled daily.

Aldosterone infusion
Male Wistar rats (Charles River Laboratories, Sulzfeld, Germany; mean weight, 200 g) were fed on a high-salt diet (8% NaCl) and simultaneously received 0.75 µg/h D-aldosterone (Fischer Scientific Germany, Schwerte, Germany) via implanted osmotic minipumps (model 2004, Alzet Co., Charles River Laboratories, Sulzfeld, Germany) for 28 d as previously described (25). One group (aldosterone + darusentan) was treated with the specific ETA antagonist darusentan (Abbott Laboratories, Ludwigshafen, Germany; 30 mg/kg·d) in the drinking water. The control group received vehicle and standard chow (0.2% NaCl).

Hemodynamic studies
Rats were anesthetized with pentobarbital (50 mg/kg, ip), and a polyethylene catheter (Aorta 24; Medex Medical, Klein-Winternheim, Germany) was implanted into the abdominal aorta, sc tunneled to the suprascapular area, and brought out through a steel tether that allowed the animals free movement and access to water during the recovery period and the experiments. Using this catheter, arterial blood pressure was recorded with a transducer (Statham P 23 XL; Spectramed, Oxnard, CA) connected to a PowerLab personal computer system (ADInstruments Pty. Ltd., Colorado Springs, CO). Blood pressure was recorded after a 45-min rest of the awake animal in a dark, quiet room, and mean arterial blood pressure and heart rate (beats per minute) were calculated. Intraventricular pressure was recorded as previously described (26). The left ventricular end-diastolic pressure (LVEDP), left ventricular contractility [(+)dP/dtmax] and left ventricular relaxation [(–)dP/dtmax] were calculated using PowerLab Chart software (ADInstruments Pty. Ltd.).

Determination of plasma neurohormones
For measurement of plasma NE, the animals were put into individual cages in a quiet room between 1700–1900 h, and the implanted catheter was connected to a syringe. Forty-five minutes later, a 400-µl blood sample was taken from the awake animal and was replaced by an equal volume of saline. The plasma concentration of NE was determined by a radioenzymatic assay as previously described (27). Plasma concentrations of ET-1, the stable N-terminal part (amino acids 1–98) of the prohormone form of atrial natriuretic peptide (proANP), and aldosterone were determined in venous blood samples taken from the femoral vein. ET-1 and proANP were determined using enzyme immunoassays according to the manufacturer’s instructions (Biomedica, Vienna, Austria). Aldosterone was measured using a specific in-house RIA established at the Steroid Laboratory of University of Heidelberg, using tritiated aldosterone ([1,2,4,6-3H]aldosterone; Amersham Biosciences, Freiburg, Germany) and an antibody raised and characterized in the steroid laboratory as described previously (28, 29). Before RIA a recovery-corrected extraction and chromatographic purification were performed, thereby efficiently removing cross-reaction steroids. The standard curve ranged from 1–200 pg/tube, and the sensitivity was 2 pg/tube (1 ng/100 ml). The recovery of a known amount of aldosterone determined repeatedly in quality control samples was 103.8 ± 8.2% (n = 12; mean ± SD). The intraassay coefficient of variation was 3.5–8.5%, and the interassay coefficient of variation was 9.6–12.2%.

NE tissue levels
The left ventricle was rinsed in ice-cold 0.9% saline and frozen in liquid nitrogen until HPLC and electrochemical detection as previously described in detail (30, 31).

Isolated heart perfusion
Wistar rats were anesthetized with thiopental (100 mg/kg, ip). The hearts were rapidly cut out and rinsed in ice-cold buffer, and the aorta was cannulated for perfusion according to the method described by Langendorff (32). Within one experiment, eight to 12 spontaneously beating hearts were perfused simultaneously at a constant coronary flow and a constant temperature of 37.5 C. The perfusion medium was a modified Krebs-Henseleit solution (125 mmol/liter NaCl, 16.9 mmol/liter NaHCO3, 0.2 mmol/liter Na2HPO4, 4.0 mmol/liter KCl, 1.85 mmol/liter CaCl2, 1.0 mmol/liter MgCl2, 11 mmol/liter glucose, and 0.027 mmol/liter EDTA). The buffer was gassed with 95% O2 and 5% CO2, and the pH was adjusted to 7.4. Cardiac [3H]NE uptake was determined as described previously (16). Briefly, a bolus of [3H]NE (1 ml, 3 µCi, 100 pmol NE; Amersham-Buchler, Braunschweig, Germany) was injected into the perfusion system and proportionally distributed to the hearts and blank channels. Radioactivity was measured in the effluent. The amount of [3H]NE extracted by the hearts (uptake) was expressed as the percentage of radioactivity measured in the blank channels.

[3H]Mazindol binding
Plasma membranes of right and left ventricles were prepared as described previously (16). Radioligand binding assays were performed in a total volume of 250 µl containing 50 µg plasma membranes and increasing concentrations of [3H]mazindol (specific activity, 52,5 Ci/mmol; NEN Life Science Products, Dreieich, Germany) as a specific ligand. Nonspecific binding was determined by measuring the residual binding in the presence of desipramine (100 µmol/liter). The incubation was carried out at 30 C and was terminated by rapid vacuum filtration through a MultiScreenHTS-FB filter plate (Millipore Corp., Schwalbach, Germany). All experiments were performed in triplicate. The remaining filter radioactivity was determined, and the binding capacity was calculated using PRISM version 4.00 software (GraphPad, Inc., San Diego, CA).

Echocardiography
Transthoracic echocardiography was performed as previously described in detail (33). The investigator who conducted the echocardiography was blinded to the treatment status.

Survival analysis
DS were treated as described above (DS, DS + spironolactone, or DS + prazosin). Ten rats per group were monitored, and deaths were recorded every day. Survival was described by standard Kaplan-Meier analysis.

Statistics
The results are expressed as the mean ± SEM. Statistical analysis was performed using SPSS 12.0 software (SPSS, Inc., Chicago, IL). Differences between groups were tested by one-way ANOVA with post hoc comparisons by Fisher’s protected least significant difference test or unpaired Student’s t test where appropriate. Kaplan-Meier survival analysis was performed using the log-rank test. In all tests, P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of spironolactone on heart failure indices in DS
After 40 d of a high-salt diet, DS showed severe arterial hypertension, with a doubled mean arterial blood pressure compared with DR (Fig. 1AGo). Moreover, DS developed signs of heart failure, as indicated by biventricular myocardial hypertrophy combined with elevated lung wet weights (Table 1Go) and elevated LVEDPs (Fig. 1BGo). Treatment of these animals with spironolactone lowered the mean arterial blood pressure by only 15%, but markedly attenuated biventricular hypertrophy and pulmonary congestion (Table 1Go). In contrast, treatment with prazosin, which causes a similar effect on arterial blood pressure as spironolactone (Fig. 1AGo), did not result in a comparable attenuation of organ weights/body weight ratios (Table 1Go). By inserting a cannula connected to a pressure transducer via a short (<10 cm) water-filled polyethylene catheter, we measured the left ventricular pressure of these animals. The use of this method to assess LVEDP and especially (+)dP/dtmax has a limitation in that the distance between the LV and the pressure transducer may result in false low values. However, this method is suitable to compare the effects of different drugs, because this potential error would be a systemic error, affecting all groups equally. Treatment of DS with spironolactone, but not with prazosine, prevented the increase in LVEDP (Fig. 1BGo) but attenuated (+)dP/dtmax only partially (Fig. 1CGo). In accordance with previous findings by others (10, 11) that salt-induced heart failure of DS rats depends on a marked diastolic dysfunction, we observed a markedly impaired (-)dP/dtmax (Fig 1DGo). Treatment with spironolactone, but not with prazosine, prevented an impaired (-)dP/dtmax in DS (Fig. 1DGo). Likewise, transthoracic echocardiography revealed that treatment with spironolactone prevented an increase in anterior wall thickness in DS, but only affected marginally the decrease in ventricular fractional shortening (Table 2Go).


Figure 1
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  		FIG. 1. Hemodynamic characterization of DR and DS after 40 d of a 8% NaCl diet. Beginning with the onset of the high-salt diet, some DS rats were treated with either spironolactone (SP) or prazosine (P) as a blood pressure control. Mean arterial blood pressure (MAP; A), LVEDP (B), (+)dP/dtmax (C), and (–)dP/dtmax (D) are shown. n.s., Not significant.

 

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  		TABLE 1. Basic characterization of DR and DS after 40 d on an 8% NaCl diet

 

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  		TABLE 2. Echocardiography of DR (n = 5) and DS (n = 5) after 40 d on a 8% NaCl diet

 
Effects of spironolactone on survival of DS
To determine whether spironolactone-mediated beneficial effects on cardiac function also lead to an improved outcome for DS, a Kaplan-Meier survival (10 animals/group) analysis was performed. Treatment with spironolactone prolonged the median survival of DS to 72 d after onset of the diet compared with 42 d in nontreated and prazosin-treated DS (Fig. 2Go). However, spironolactone treatment of DS did not entirely prevent, but delayed, mortality of DS in response to a high-salt diet. No spironolactone-treated DS rats lived longer than 102 d after onset of the diet. The postmortem analysis of both spironolactone- and non/prazosine-treated animals revealed signs of heart failure (ascites, pleural effusion, and enlargement of LV and RV).


Figure 2
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  		FIG. 2. Kaplan-Meier survival curves of DS (n = 10), spironolactone-treated DS (DS + SP; n = 10), and prazosin-treated DS (DS + P; n = 10) during a 8% NaCl diet. The arrow above the diagram indicates the duration of the 8% NaCl diet. *, P < 0.05 vs. DS and DS + P.

 
Effects of spironolactone on neurohormonal indices in DS
Elevated NE plasma levels in DS compared with DR indicated activation of the sympathetic nervous system (Fig. 3AGo). We observed higher plasma levels of ET-1 in DS compared with DR, indicating activation of the endogenous ET system (Fig. 3BGo). In accordance, a similar increase in plasma ET-1 was reported by others (10, 11). Although plasma levels of aldosterone were comparable in DR and DS that were fed a standard diet (~13 ng/dl in both groups; data not shown), high salt intake induced a reduction of plasma aldosterone in DR (<5 ng/dl), but not in DS (11 ng/dl; Fig. 3CGo), suggesting that the endogenous aldosterone system of DS is unresponsive to salt. Consistently, Nishikimi et al. (9) reported doubled plasma aldosterone levels in DS compared with DR. As a marker for the hemodynamic overload of the heart, proANP plasma levels were 10-fold increased. Treatment with spironolactone, but not prazosine, prevented increased plasma levels of both NE and ET-1 (Fig. 3Go, A and B). ProANP plasma levels were attenuated by spironolactone and, although less pronounced, by prazosine (Fig. 3DGo). As expected, treatment with spironolactone elevated plasma aldosterone levels in DS, indicating efficient MR antagonism by spironolactone (Fig. 3CGo). In accordance, a similar increase in plasma aldosterone due to spironolactone was observed by others (34).


Figure 3
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  		FIG. 3. Plasma levels of NE (A), ET-1 (B), aldosterone (AL; C), and proANP (D) in DR, DS, DS + SP, and DS + P rats after 40 d of an 8% NaCl diet. n.s., Not significant.

 
Effects of spironolactone on cardiac NE homeostasis in DS
To test the hypothesis that an endogenously activated aldosterone system in DS impairs NE reuptake in vivo, the uptake of exogenous [3H]NE in a Langendorff preparation was determined. Cardiac [3H]NE uptake was reduced by approximately 30% in DS compared with DR, and in fact, treatment with spironolactone, but not prazosin, preserved cardiac NE reuptake in DS (Fig. 4Go). There was no difference between DR, DS, DS + spironolactone, and DS + prazosine rats in the residual uptake of [3H]NE after specific blockade of NET with desipramine, indicating that the diminished cardiac elimination of [3H]NE was entirely due to reduced uptake via the NET and not, for example, to the extraneuronal NE transporter (uptake2 carrier; Fig. 4Go). Impaired cardiac NE reuptake might induce depletion of cardiac NE stores (16), reflecting an overactivity of the cardiac sympathetic nervous system. Tissue NE stores in the right and left ventricles (Fig. 5Go, A and B) were markedly depleted in DS compared with DR. Treatment with spironolactone, but not prazosin, attenuated NE depletion in the left ventricle only partially, but prevented it in the right ventricle (Fig. 5Go, A and B). This discrepancy led us to ask whether aldosterone impairs NE reuptake only in the right ventricle. We reported previously that in aortic banded rats, impaired NE reuptake of the whole isolated perfused heart is caused by reduced NET density in the right and left ventricles (16, 24). Consistently, NET density, as assessed by [3H]mazindol binding, was also reduced in both ventricles of DS compared with DR rats. However, in contrast to the NE stores, spironolactone normalized NET density in the right and left ventricles (Fig. 5Go, C and D).


Figure 4
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  		FIG. 4. Cardiac [3H]NE uptake with (+) and without (–) blockade of NET with desipramine (DMI) in DR, DS, DS + SP, and DS + P rats after 40 d of an 8% NaCl diet. n.s., Not significant.

 

Figure 5
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  		FIG. 5. Left (A) and right (B) ventricular tissue concentrations of NE in DR, DS, DS + SP, and DS + P rats after 40 d of an 8% NaCl diet. Cardiac [3H]mazindol binding in left (C) and right (D) ventricles of DR, DS, DS + SP, and DS + P rats after 40 d of an 8% NaCl diet is shown. n.s., Not significant.

 
Effects of ETA antagonism on NE reuptake in aldosterone-infused Wistar rats
To determine whether impaired NE reuptake is directly associated with the aldosterone system, we used an experimental animal model that mimics activation of the aldosterone system. Male Wistar rats received aldosterone via osmotic minipumps and simultaneously a high-salt diet for 28 d. Because these animals did not develop heart failure, it is unlikely that potential effects on NE reuptake were mediated by activation of other heart failure-associated neurohormonal systems. Sufficient application of aldosterone via osmotic minipumps was confirmed by the almost tripled plasma aldosterone levels compared with control rats (vehicle infusion and normal salt diet; Fig. 6AGo). Consistently, comparable levels were reported by others (34). Compared with control animals, in aldosterone-infused rats we observed impaired cardiac [3H]NE uptake (Fig. 5BGo). This result confirmed that activation of the aldosterone system is sufficient to inhibit NET function. Treatment with the specific ETA receptor antagonist darusentan did not attenuate increased plasma aldosterone levels (Fig. 6AGo), indicating that in this model ETA antagonism does not prevent activation of the aldosterone system, e.g. by inducing its degradation. Despite high aldosterone levels, treatment of aldosterone-infused rats with darusentan attenuated a decreased elimination of NE via NET (Fig. 6BGo), suggesting that activation of ETA is required for aldosterone-mediated impairment of NET.


Figure 6
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  		FIG. 6. Plasma aldosterone (AL) levels (A) and cardiac [3H]NE uptake (B) in rats after 28 d of infusion with aldosterone via osmotic minipumps (0.75 µg aldosterone/h) and a simultaneous diet of 8% NaCl (AL) compared with control animals that received the vehicle and standard chow (0.2% NaCl) for 28 d. Beginning with the onset of aldosterone infusion and high-salt diet, some rats were treated with darusentan (DA). n.s., Not significant.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have previously shown that an impairment of NE reuptake in experimental heart failure is mediated by posttranscriptional down-regulation of neuronal NET within the failing heart (16). More recently, we provided evidence that ETA activation via ET-1 negatively regulates NE reuptake, and that activation of the endogenous ET system mediates NET down-regulation in heart failure (24). However, other groups demonstrated that aldosterone might also be involved in the regulation of NET function (1, 35). In the present study we confirm that MR antagonism attenuates cardiac sympathetic dysfunction and improves cardiac contractile function in an experimental model of salt-induced heart failure. In addition, we show for the first time that ETA activation contributes to aldosterone-induced NET impairment.

Despite a similar effect on arterial blood pressure, only treatment with spironolactone, but not the {alpha}1-inhibitor prazosin, significantly improves the cardiac function and survival of DS. In addition, compared with DR, the arterial blood pressure of spironolactone-treated DS was still markedly enhanced by approximately 60 mm Hg. This indicates that the beneficial effects of spironolactone are blood pressure independent. These findings are consistent with previous experimental (36, 37) and clinical (2, 3) studies and reveal that the aldosterone system plays a key role in the progression of heart failure.

Our results demonstrate that MR antagonism normalizes plasma NE levels and attenuates depletion of cardiac NE stores in salt-dependent heart failure. Both could be explained by a preservation of NE reuptake via the neuronal NET. In this study we show that spironolactone prevents an impairment of NE reuptake and a reduction of NET density, suggesting that aldosterone inhibits NET function by down-regulation of NET per sympathetic nerve ending. One could argue that the spironolactone-induced preservation of cardiac NE reuptake in salt-sensitive Dahl rats was secondary due to an improvement of cardiac function as a consequence of other beneficial effects of spironolactone (e.g. antifibrotic effect) (36). In this regard, the observation that activation of the aldosterone system via aldosterone infusion and a simultaneous high-salt diet also caused NET impairment supported the hypothesis that aldosterone, and not other independently activated neurohormonal systems, inhibit the NET.

In accordance, using iodine-123-metaiodobenzylguanidine (MIBG) uptake, clinical studies (1, 35) provided indirect evidence that spironolactone treatment caused an improvement of NE reuptake. Iodine-123-MIBG is thought to use the same transport mechanisms as NE (38). However, this method can distinguish neither between neuronal or extraneuronal uptake nor between reduced NE uptake or enhanced NE release. Because the present study performed [3H]NE uptake measurements in the absence and presence of the specific NET antagonist desipramine, we provide direct evidence that spironolactone preserves NE reuptake via NET.

Our recent findings that ET-1 inhibits NET in aortic banded rats (24) and DS (our unpublished observations) together with the present observation that aldosterone does the same demonstrate that the possibility exists that ET-1 mediates its effect via aldosterone or vice versa. Because ET-1 exerts its effect on NET function in a rapid manner (24), whereas the MR-mediated effect occurs in a chronic manner and is only observed in the presence of a high-salt diet, it seems more likely that aldosterone affects NET function through salt-dependent activation of the ET system. Interestingly, it was demonstrated that aldosterone infusion to salt-loaded rats induced vascular expression of ET-1, and that MR antagonism normalized vascular ET-1 levels that were increased in liquorice-induced hypertension (25, 39). Likewise, we observed that spironolactone treatment of DS normalizes plasma ET-1 levels. We have previously shown that endogenous ET-1 inhibits NET function by selective activation of ETA in experimental heart failure (24). In this study we demonstrate that ETA antagonism attenuates salt-dependent and aldosterone-induced impairment of NET in a heart failure-independent model. One could argue that the impairment of NET function in this model is a consequence of high blood pressure, and the improvement after ETA antagonism is the result of lowered blood pressure. However, high blood pressure alone is unlikely to cause NET impairment independent of salt loading and aldosterone infusion, because in a salt-independent model of arterial hypertension, we did not observe any NET impairment (40). Moreover, our observation that DS rats still have a high mean arterial blood pressure, by far higher than those of aldosterone-infused and salt-loaded rats (25), after treatment with spironolactone, but show normalized NE reuptake, strongly suggests that high blood pressure per se is not a negative stimulus for NET function. In fact, NET function seems to depend, rather, on the activation status of the aldosterone system. We postulate that aldosterone inhibits NET function in a salt-dependent manner via activation of the ET system. Our findings that both MR and ETA antagonism preserve NET function in heart failure models suggest that activated aldosterone and ET systems enhance cardiac sympathetic activity by inhibiting NET-mediated NE reuptake.

In the present study the effect of spironolactone on left ventricular NET density was more dramatic than the effect on left ventricular cardiac NE stores. A potential explanation for this discrepancy is that other aldosterone-independent presynaptic mechanisms also contribute to the depletion of cardiac NE stores. Likewise, we and others (19, 40, 41) reported that enhanced exocytotic NE release (e.g. by activation of angiotensin receptors or inhibition of {alpha}2-receptors) also causes sympathetic overdrive resulting in depleted cardiac NE stores. Another possibility is that MR antagonism by itself exerts dual effects on NE reuptake and exocytotic NE release. Interestingly, it has been reported that MR antagonism induced a decrease in cardiac NE stores in the viable myocardium of myocardial infarcted rats (42, 43). Although the significance and underlying mechanism of this finding remain elusive, these reports suggest that aldosterone might also inhibit exocytotic NE release. In this regard, the interaction between the aldosterone and ET systems is of great interest, because it was demonstrated that ET-1 not only inhibits cardiac NE reuptake via ETA, but also attenuates cardiac exocytotic NE release via ETB (24). Therefore, MR antagonism could exert opposite effects on cardiac NE stores via reduced production of ET-1 and, consequently, attenuated activation of ETA and ETB, explaining the relatively mild restoration of left ventricular NE in DS in response to spironolactone. The finding that right ventricular NE stores were completely normalized could be explained by the possibility that the right ventricle benefits more than the left ventricle from a reduced volume overload in response to the slight diuretic effect of spironolactone. As a consequence, the right ventricle might be more relieved from the neurohormonal stress that induces exocytotic NE release.

Clinical implications
Myocardial NE reuptake is known to be a strong prognostic marker for overall mortality in heart failure (44). During the preparation of this manuscript, it was demonstrated that adenoviral gene transfer of NET into failing hearts of rabbits is sufficient to improve cardiac function (45). The present study identified aldosterone as a potent negative regulator of NE reuptake. Consequently, the beneficial effects of spironolactone on the survival of heart failure patients might be at least in part mediated by this effect. Likewise, improved NE reuptake would decrease the NE concentration in the synaptic cleft and, therefore, would explain the finding of the Randomized Aldactone Evaluation Study that spironolactone reduces the rate of sudden cardiac death by elevating the threshold for ventricular fibrillation (3). Thus, imaging techniques such as iodine-123-MIBG scintigraphy, which allow an assessment of NE reuptake, could identify a patient population that benefits in particular from MR antagonism. Moreover, our finding that aldosterone inhibits NE reuptake via activation of the ET system implies that such a patient population could also benefit from ETA antagonism. The identification of heart failure subpopulations that benefit from ETA antagonism is of particular interest, because, to date, clinical trials using ET receptor antagonists could not demonstrate an improvement in mortality in a broad heart failure population (46).

Conclusions
In conclusion, aldosterone inhibits NET-mediated cardiac NE reuptake. This action of aldosterone is at least partially mediated via an interaction with the ET system resulting in ETA activation. In salt-dependent heart failure, MR antagonism normalizes plasma ET-1 levels and consequently preserves cardiac NE reuptake and plasma NE levels. These findings provide insight into the regulatory mechanisms by which aldosterone enhances cardiac sympathetic activity in heart failure.


   Acknowledgments
 
The expert technical assistance of Silvia Harrack, Jutta Krebs, and Michaela Oestringer is gratefully acknowledged. We thank Dr. Klaus Muenter (Knoll AG, Ludwigshafen, Germany) for his generous gift of darusentan.


   Footnotes
 
All authors have nothing to declare.

First Published Online January 26, 2006

1 S.J.B. and J.B. contributed equally to this work and should both be considered as first authors. Back

Abbreviations: (+)dP/dtmax, Left ventricular contractility; (–)dP/dtmax, left ventricular relaxation; DS, Dahl rat; ET, endothelin; ETA, ET-A receptor; LVEDP, left ventricular end-diastolic pressure; MIBG, metaiodobenzylguanidine; MR, mineralocorticoid receptor; NE, norepinephrine; NET, norepinephrine transporter; proANP, prohormone form of atrial natriuretic peptide.

Received September 12, 2005.

Accepted for publication January 17, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Barr CS, Lang CC, Hanson J, Arnott M, Kennedy N, Struthers AD 1995 Effects of adding spironolactone to an angiotensin-converting enzyme inhibitor in chronic congestive heart failure secondary to coronary artery disease. Am J Cardiol 76:1259–1265[CrossRef][Medline]
  2. Pitt B, Remme W, Zannad F, Neaton J, Martinez F, Roniker B, Bittman R, Hurley S, Kleiman J, Gatlin M 2003 Eplerenone, a selective aldosterone blocker, in patients with left ventricular dysfunction after myocardial infarction. N Engl J Med 348:1309–1321[Abstract/Free Full Text]
  3. Pitt B, Zannad F, Remme WJ, Cody R, Castaigne A, Perez A, Palensky J, Wittes J 1999 The effect of spironolactone on morbidity and mortality in patients with severe heart failure. Randomized Aldactone Evaluation Study Investigators. N Engl J Med 341:709–717[Abstract/Free Full Text]
  4. Weber KT, Villarreal D 1993 Aldosterone and antialdosterone therapy in congestive heart failure. Am J Cardiol 71:3A–11A
  5. Struthers AD, MacDonald TM 2004 Review of aldosterone- and angiotensin II-induced target organ damage and prevention. Cardiovasc Res 61:663–670[Abstract/Free Full Text]
  6. Swedberg K, Eneroth P, Kjekshus J, Wilhelmsen L 1990 Hormones regulating cardiovascular function in patients with severe congestive heart failure and their relation to mortality. CONSENSUS Trial Study Group. Circulation 82:1730–1736[Abstract/Free Full Text]
  7. Rouleau JL, Packer M, Moye L, de Champlain J, Bichet D, Klein M, Rouleau JR, Sussex B, Arnold JM, Sestier F 1994 Prognostic value of neurohumoral activation in patients with an acute myocardial infarction: effect of captopril. J Am Coll Cardiol 24:583–591[Abstract]
  8. Weber KT 2001 Aldosterone in congestive heart failure. N Engl J Med 345:1689–1697[Free Full Text]
  9. Nishikimi T, Mori Y, Kobayashi N, Tadokoro K, Wang X, Akimoto K, Yoshihara F, Kangawa K, Matsuoka H 2002 Renoprotective effect of chronic adrenomedullin infusion in Dahl salt-sensitive rats. Hypertension 39:1077–1082[Abstract/Free Full Text]
  10. Yamamoto K, Masuyama T, Sakata Y, Mano T, Nishikawa N, Kondo H, Akehi N, Kuzuya T, Miwa T, Hori M 2000 Roles of renin-angiotensin and endothelin systems in development of diastolic heart failure in hypertensive hearts. Cardiovasc Res 47:274–283[Abstract/Free Full Text]
  11. Yamamoto K, Masuyama T, Sakata Y, Doi R, Ono K, Mano T, Kondo H, Kuzuya T, Miwa T, Hori M 2000 Local neurohumoral regulation in the transition to isolated diastolic heart failure in hypertensive heart disease: absence of AT1 receptor downregulation and ‘overdrive’ of the endothelin system. Cardiovasc Res 46:421–432[Abstract/Free Full Text]
  12. Takeda Y 2005 Role of cardiovascular aldosterone in hypertension. Curr Med Chem Cardiovasc Hematol Agents 3:261–266[CrossRef][Medline]
  13. Bayorh MA, Ganafa AA, Emmett N, Socci RR, Eatman D, Fridie IL 2005 Alterations in aldosterone and angiotensin II levels in salt-induced hypertension. Clin Exp Hypertens 27:355–367[CrossRef][Medline]
  14. Leimbach Jr WN, Wallin BG, Victor RG, Aylward PE, Sundlof G, Mark AL 1986 Direct evidence from intraneural recordings for increased central sympathetic outflow in patients with heart failure. Circulation 73:913–919[Abstract/Free Full Text]
  15. Cohn JN, Levine TB, Olivari MT, Garberg V, Lura D, Francis GS, Simon AB, Rector T 1984 Plasma norepinephrine as a guide to prognosis in patients with chronic congestive heart failure. N Engl J Med 311:819–823[Abstract]
  16. Backs J, Haunstetter A, Gerber SH, Metz J, Borst MM, Strasser RH, Kubler W, Haass M 2001 The neuronal norepinephrine transporter in experimental heart failure: evidence for a posttranscriptional downregulation. J Mol Cell Cardiol 33:461–472[CrossRef][Medline]
  17. Bohm M, La Rosee K, Schwinger RH, Erdmann E 1995 Evidence for reduction of norepinephrine uptake sites in the failing human heart. J Am Coll Cardiol 25:146–153[Abstract]
  18. Eisenhofer G 2001 The role of neuronal and extraneuronal plasma membrane transporters in the inactivation of peripheral catecholamines. Pharmacol Ther 91:35–62[CrossRef][Medline]
  19. Eisenhofer G, Friberg P, Rundqvist B, Quyyumi AA, Lambert G, Kaye DM, Kopin IJ, Goldstein DS, Esler MD 1996 Cardiac sympathetic nerve function in congestive heart failure. Circulation 93:1667–1676[Abstract/Free Full Text]
  20. Liang CS, Fan TH, Sullebarger JT, Sakamoto S 1989 Decreased adrenergic neuronal uptake activity in experimental right heart failure. A chamber-specific contributor to ß-adrenoceptor downregulation. J Clin Invest 84:1267–1275[Medline]
  21. Petch MC, Nayler WG 1979 Uptake of catecholamines by human cardiac muscle in vitro. Br Heart J 41:336–339[Abstract/Free Full Text]
  22. Nakata T, Miyamoto K, Doi A, Sasao H, Wakabayashi T, Kobayashi H, Tsuchihashi K, Shimamoto K 1998 Cardiac death prediction and impaired cardiac sympathetic innervation assessed by MIBG in patients with failing and nonfailing hearts. J Nucl Cardiol 5:579–590[CrossRef][Medline]
  23. Ogita H, Shimonagata T, Fukunami M, Kumagai K, Yamada T, Asano Y, Hirata A, Asai M, Kusuoka H, Hori M, Hoki N 2001 Prognostic significance of cardiac 123I metaiodobenzylguanidine imaging for mortality and morbidity in patients with chronic heart failure: a prospective study. Heart 86:656–660[Abstract/Free Full Text]
  24. Backs J, Bresch E, Lutz M, Kristen AV, Haass M 2005 Endothelin-1 inhibits the neuronal norepinephrine transporter in hearts of male rats. Cardiovasc Res 67:283–290[Abstract/Free Full Text]
  25. Park JB, Schiffrin EL 2001 ETA receptor antagonist prevents blood pressure elevation and vascular remodeling in aldosterone-infused rats. Hypertension 37:1444–1449[Abstract/Free Full Text]
  26. Borst MM, Beuthien W, Schwencke C, LaRosee P, Marquetant R, Haass M, Kubler W, Strasser RH 1999 Desensitization of the pulmonary adenylyl cyclase system: a cause of airway hyperresponsiveness in congestive heart failure? J Am Coll Cardiol 34:848–856[Abstract/Free Full Text]
  27. Da Prada M, Zurcher 1976 Simultaneous radioenzymatic determination of plasma and tissue adrenaline, noradrenaline and dopamine within the femtomole range. Life Sci 19:1161–1174[CrossRef][Medline]
  28. Sander M, Bader M, Djavidani B, Maser-Gluth C, Vecsei P, Mullins J, Ganten D, Peters J 1992 The role of the adrenal gland in hypertensive transgenic rat TGR(mREN2)27. Endocrinology 131:807–814[Abstract]
  29. Vecsei P, Abdelhamid S, Mittelstadt GV, Lichtwald K, Haack D, Lewicka S 1983 Aldosterone metabolites and possible aldosterone precursors in hypertension. J Steroid Biochem 19:345–351[Medline]
  30. Haass M, Cheng B, Richardt G, Lang RE, Schomig A 1989 Characterization and presynaptic modulation of stimulation-evoked exocytotic co-release of noradrenaline and neuropeptide Y in guinea pig heart. Naunyn Schmiedebergs Arch Pharmacol 339:71–78[CrossRef][Medline]
  31. Kranzhofer R, Haass M, Kurz T, Richardt G, Schomig A 1991 Effect of digitalis glycosides on norepinephrine release in the heart. Dual mechanism of action. Circ Res 68:1628–1637[Abstract/Free Full Text]
  32. Langendorff O 1895 Untersuchungen am ueberlebenden Saeugetierherzen. Arch Ges Physiol 61:291–332[CrossRef]
  33. Hardt SE, Geng YJ, Montagne O, Asai K, Hong C, Yang GP, Bishop SP, Kim SJ, Vatner DE, Seidman CE, Seidman JG, Homcy CJ, Vatner SF 2002 Accelerated cardiomyopathy in mice with overexpression of cardiac Gs{alpha} and a missense mutation in the {alpha}-myosin heavy chain. Circulation 105:614–620[Abstract/Free Full Text]
  34. Virdis A, Neves MF, Amiri F, Viel E, Touyz RM, Schiffrin EL 2002 Spironolactone improves angiotensin-induced vascular changes and oxidative stress. Hypertension 40:504–510[Abstract/Free Full Text]
  35. Kasama S, Toyama T, Kumakura H, Takayama Y, Ichikawa S, Suzuki T, Kurabayashi M 2003 Effect of spironolactone on cardiac sympathetic nerve activity and left ventricular remodeling in patients with dilated cardiomyopathy. J Am Coll Cardiol 41:574–581[Abstract/Free Full Text]
  36. Brilla CG, Matsubara LS, Weber KT 1993 Antifibrotic effects of spironolactone in preventing myocardial fibrosis in systemic arterial hypertension. Am J Cardiol 71:12A–16A
  37. Stier Jr CT, Chander PN, Rocha R 2002 Aldosterone as a mediator in cardiovascular injury. Cardiol Rev 10:97–107[CrossRef][Medline]
  38. Wieland DM, Wu J, Brown LE, Mangner TJ, Swanson DP, Beierwaltes WH 1980 Radiolabeled adrenergic neuron-blocking agents: adrenomedullary imaging with [131I]iodobenzylguanidine. J Nucl Med 21:349–353[Abstract/Free Full Text]
  39. Quaschning T, Ruschitzka F, Shaw S, Luscher TF 2001 Aldosterone receptor antagonism normalizes vascular function in liquorice-induced hypertension. Hypertension 37:801–805[Abstract/Free Full Text]
  40. Zugck C, Lossnitzer D, Backs J, Kristen A, Kinscherf R, Haass M 2003 Increased cardiac norepinephrine release in spontaneously hypertensive rats: role of presynaptic {alpha}-2A adrenoceptors. J Hypertens 21:1363–1369[CrossRef][Medline]
  41. Akers WS, Cassis LA 2004 Presynaptic modulation of evoked NE release contributes to sympathetic activation after pressure overload. Am J Physiol 286:H2151–H2158
  42. Cittadini A, Monti MG, Isgaard J, Casaburi C, Stromer H, Di Gianni A, Serpico R, Saldamarco L, Vanasia M, Sacca L 2003 Aldosterone receptor blockade improves left ventricular remodeling and increases ventricular fibrillation threshold in experimental heart failure. Cardiovasc Res 58:555–564[Abstract/Free Full Text]
  43. Silvestre JS, Heymes C, Oubenaissa A, Robert V, Aupetit-Faisant B, Carayon A, Swynghedauw B, Delcayre C 1999 Activation of cardiac aldosterone production in rat myocardial infarction: effect of angiotensin II receptor blockade and role in cardiac fibrosis. Circulation 99:2694–2701[Abstract/Free Full Text]
  44. Merlet P, Valette H, Dubois-Rande JL, Moyse D, Duboc D, Dove P, Bourguignon MH, Benvenuti C, Duval AM, Agostini D 1992 Prognostic value of cardiac metaiodobenzylguanidine imaging in patients with heart failure. J Nucl Med 33:471–477[Abstract/Free Full Text]
  45. Munch G, Rosport K, Bultmann A, Baumgartner C, Li Z, Laacke L, Ungerer M 2005 Cardiac overexpression of the norepinephrine transporter uptake-1 results in marked improvement of heart failure. Circ Res 97:928–936[Abstract/Free Full Text]
  46. Anand I, McMurray J, Cohn JN, Konstam MA, Notter T, Quitzau K, Ruschitzka F, Luscher TF 2004 Long-term effects of darusentan on left-ventricular remodelling and clinical outcomes in the Endothelin A Receptor Antagonist Trial in Heart Failure (EARTH): randomised, double-blind, placebo-controlled trial. Lancet 364:347–354[CrossRef][Medline]



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