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Cardiovascular Research 1999 44(1):113-120; doi:10.1016/S0008-6363(99)00197-2
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Copyright © 1999, European Society of Cardiology

Transregulation of adenylyl-cyclase-coupled inhibitory receptors in heart failure enhances anti-adrenergic effects on adult rat cardiomyocytes

Mathias M Borst*, Palma Szalai, Nicole Herzog, Wolfgang Kübler and Ruth H Strasser

Department of Cardiology, Angiology, and Pulmonary Medicine, University of Heidelberg Medical Centre, Bergheimer Str. 58, D-69115 Heidelberg, Germany

* Corresponding author. Tel.: +49-6221-568-676; fax: +49-6221-568-554 mathias_borst{at}med.uni-heidelberg.de

Received 16 March 1999; accepted 19 May 1999


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: In congestive heart failure (CHF), a desensitisation of stimulatory β-receptors and of adenylyl cyclase in the heart is associated with an increase in inhibitory Gi proteins. To investigate whether the regulation of the Gi-mediated inhibitory side of the adenylyl cyclase system may be of functional importance in the failing myocardium, the contractile response of isolated adult cardiomyocytes to stimulation of inhibitory muscarinic M2 and A1 adenosine receptors was analysed. Methods: CHF was induced in rats by banding of the ascending aorta and was verified by doubling of lung wet weight. After four weeks, contraction amplitude ({Delta}L) and the velocity (dL/dtmax) of isolated ventricular cardiomyocytes during electrical field stimulation in the presence of 1 mM Ca2+ were measured using video micrometry. Results: Contractile responses of failing cardiomyocytes to 5 mM Ca2+ were unchanged. The response to increasing concentrations of the β-adrenergic agonist, isoproterenol (0.1–30 nM), and to forskolin (0.1 nM–1 µM) were significantly blunted. When A1 receptors were activated with N6-(R-phenyl-isopropyl)-adenosine (PIA; 0.01–1 µM) in the presence of 3 nM isoproterenol, contractility was unchanged in cells compared with those from sham-operated rats, but {Delta}L was reduced by up to 23% and dL/dtmax by 35% in failing cardiomyocytes (P<0.01), demonstrating an enhanced inhibitory effect of A1 receptors. The response to the M2 receptor agonist, carbachol (0.01–3 µM), was augmented to a comparable extent ({Delta}L, –22%, dL/dtmax, –39%; P<0.01). Conclusions: In CHF, the inotropic responses to β-receptor-stimulation and to direct stimulation of adenylyl cyclase, but not to Ca2+, are diminished due to desensitisation of the stimulatory side of the adenylyl cyclase signal transduction system. In parallel, the responses to inhibitory receptors are augmented, leading to a pronounced Gi-mediated negative inotropic effect on failing heart muscle cells. Those anti-adrenergic effects could contribute to the contractile dysfunction of the failing heart. Reversal of the sensitisation to inhibitory stimuli might be one of the desirable mechanisms of medical therapy in CHF.

KEYWORDS Adenosine; Adrenergic; Heart failure; Muscarinic agonists; Myocytes


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
The contractile function of the heart is regulated by the interplay between stimulatory receptors of the adenylyl-cyclase-coupled signal transduction system, such as the β-adrenergic receptor, and inhibitory receptors, such as the A1 adenosine or the muscarinic M2 receptors. In congestive heart failure (CHF), the balance between competing adrenergic and ‘anti-adrenergic’ stimuli may be chronically disturbed, as characterised by a down-regulation of myocardial β-adrenergic receptors and a reduction in receptor-independent cardiac adenylyl cyclase activity [1–11]. Functionally, a reduced inotropic response to direct adenylyl cyclase stimulation by the diterpene forskolin has been observed in experimental CHF [8,12,13] and in isolated cardiomyocytes from failing human ventricles [14].

On the inhibitory side of the adenylyl cyclase signal transduction system, it has been shown repeatedly that the expression of inhibitory Gi proteins is enhanced on the protein and on the mRNA level [6,9,15–17]. Activation both of muscarinic M2 and of A1 adenosine receptors — coupling to adenylyl cyclase via Gi proteins — inhibits adenylyl cyclase activity [9,18,19]. This effect is called ‘anti-adrenergic’ because it is usually observed in the presence of β-adrenergic receptor stimulation. Cardiac muscarinic M2 receptors have been reported to be up-regulated in dogs with pacing-induced heart failure [17,20] and in humans with CHF [21]. Adenosine may exert a tonic inhibitory effect on adenylyl cyclase since the inotropic response to catecholamines of normoxic isolated hearts is augmented if endogenous adenosine is removed using adenosine deaminase [22]. However, there is little data about the anti-adrenergic effects of adenosine in CHF. In one study, no differences in the effects of A1 adenosine receptor stimulation on adenylyl cyclase activity was seen between failing and non-failing explanted human hearts [9]. A Gi protein-dependent, 90% reduction of isoproterenol-stimulated contractility by N6-(R-phenyl-isopropyl)-adenosine (PIA) was demonstrated in failing human cardiomyocytes, but, unfortunately, no cells from normal hearts were available as controls [23].

Since new therapeutic strategies in CHF could be targeted at a reversal of chronic negative inotropic stimulation of the failing heart, a better understanding of the functional role of the inhibitory side of the adenylyl-cyclase-coupled signal transduction system is warranted. Therefore, in the present investigation, isolated rat cardiomyocytes were chosen to compare the anti-adrenergic effects of the muscarinic agonist carbachol and of the specific A1 adenosine receptor agonist, PIA, in CHF, which was induced by aortic banding. As opposed to intact animals or isolated perfused hearts, this experimental model was selected for several reasons: it virtually eliminates the influence of endogenous adenosine release during adrenergic stimulation [24] and the interference with vagal nerve activity; the contractile responses of isolated cells can be investigated without confounding effects due to myocardial hypertrophy, fibrosis or perfusion inhomogeneity; and individual cells are studied at a resting tension of zero, therefore eliminating the need to correct for varying degrees of myocardial wall stress. The data indicate that induction of heart failure indeed alters the inotropic responses not only to stimulatory agents but also to inhibitory agonists.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Reagents
Thiopental–Na+ was purchased from BYK-Gulden (Konstanz, Germany), ketamine–HCl was from Parke-Davis (Berlin, Germany), and diazepam was from Hoffmann-La Roche (Grenzach-Wyhlen, Germany). Carbamylcholine chloride (carbachol), forskolin, (–)-isoproterenol-(+)-bitartrate, taurine, creatine and L-carnitine, and Krebs–Henseleit buffer with glucose were purchased from Sigma (Deisenhofen, Germany). HEPES and (–)-N6-(R-phenyl-isopropyl)-adenosine (PIA) were bought from Boehringer Mannheim (Mannheim, Germany). Fatty acid-free bovine serum albumin and medium M199 with Earle’s salts and L-glutamine were purchased from Serva (Heidelberg, Germany). Collagenase type CLS II was from Biochrom (Berlin, Germany). All other chemicals were of analytical grade and were obtained from Merck (Darmstadt, Germany).

2.2 Animal experiments
Male Wistar rats (90 g), obtained from Thomae, Biberach/Riss, Germany, were anaesthetised by intraperitoneal injection of ketamine (50 mg/kg body weight) and diazepam (5 mg/kg) and ventilated with oxygen. As described previously [25], the left hemithorax was opened, the ascending aorta was partially occluded using tantalum haemostatic clips with a defined internal diameter of 0.711 mm (Edward Weck, Research Triangle Park, NC, USA), and the wound was sutured (n=26). Sham-operated animals (n=16) underwent the same surgical procedure without insertion of the clip. The perioperative mortality ranged around 10%.

After four weeks, the animals were anaesthetised with thiopental (50 mg/kg i.p., dissolved in isotonic saline). Hearts were rapidly excised and ventricular cardiomyocytes were isolated according to the method of Piper et al. [26]. In brief, hearts were perfused using a modified Langendorff technique with a recirculating, oxygenated HEPES buffer. After ~2 min of nominally Ca2+-free perfusion, collagenase and CaCl2 were added, and the hearts were perfused for a total of ~20 min. The ventricles were dissected using a pair of scissors and a tissue chopper (McIlwain, Bachofer, Reutlingen, Germany). Bovine serum albumin (free fatty acid-free, 2 g) was added to 40 ml of the recirculating buffer and the tissue fragments were suspended in it for 15 min, while bubbling with oxygen. Finally, the cells were filtered through nylon gauze, centrifuged (300 g for 2 min), resuspended in 20 ml of M199 medium and stored at room temperature for subsequent use. The fraction of morphologically intact, rod-shaped cardiomyocytes ranged at ~40% of the cells isolated from animals with aortic clips and at ~ 65% in controls. The lungs were also excised, rinsed in isotonic saline, blotted, and weighed. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) and was performed with approval by the authorities of the Regierungspräsidium Karlsruhe.

2.3 Cell experiments
Cell contractions were measured according to the technique developed by Harding et al. [27]. In brief, cardiomyocytes were placed in a custom-designed Lucite chamber mounted on an inverted polarisation microscope (Olympus IMT-2, Hamburg, Germany) and were allowed to adhere to its glass floor. The cells were superfused with modified Krebs–Henseleit buffer (25°C, pH 7.40, Ca2+ 1.0 mM equilibrated with oxygen) at 2 ml/min and electrically stimulated with strictly bipolar impulses (1 Hz, 2 ms, 5 to 7 V) using platinum field electrodes and a custom-designed stimulator (type 04, Montgomery, London, UK). For contraction studies, cells were selected according to the criteria established by Harding et al. [27], and cell length at rest was measured. The contractions of single cardiomyocytes were monitored at a detection rate of 50 Hz using a CCD video camera connected to a cell length monitor (SP 144, HVS Image, Hampton, UK). The analogue signal was digitised and 20 consecutive contraction cycles recorded during steady state were averaged for each experimental condition. The cell length signal was differentiated to obtain maximal contraction velocity (MacLab 2e, ADInstruments, Castle Hill, Australia). Pharmacological substances, such as CaCl2, isoproterenol, forskolin, carbachol and PIA, were added to the superfusate, as indicated by the experimental protocol.

2.4 Data analysis
Data are expressed as means±SEM. Statistical comparison of group means was performed using a two-tailed Student’s t-test. Concentration response curves were obtained from multiple regression analysis with variable Hill slopes, based on the least squares method. Only one cell from each heart was used in a particular contraction experiment. Statistical comparison of the curves was performed by analysis of variance (two-way ANOVA). Differences between groups or curves were considered significant at a P value<0.01 [28].


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Induction of heart failure
Four weeks after banding of the ascending aorta, considerable myocardial hypertrophy and congestive heart failure were present (Fig. 1). In rats with CHF, heart weight (after removal of the atria) was increased by >50% and the lung wet weight was doubled, indicating pulmonary congestion due to left heart failure. To allow better comparison of contraction data, cells of uniform size were selected for videomicrometric analysis. The mean cell length of cells from rats with CHF was 114±4 µm, compared to 108±4 µm in controls (P=0.28).


Figure 1
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  		Fig. 1 Myocardial hypertrophy, with a significant increase in heart weight, and pulmonary congestion due to heart failure, indicated by a doubling in lung wet weight, were present four weeks after aortic banding (filled bars, n=23) compared to that found in sham-operated animals (open bars, n=13). Means±SEM, *P<0.01 by t-test.

 
3.2 Desensitisation to β-adrenergic and direct adenylyl cyclase stimulation
All contraction experiments were performed at a basal Ca2+ concentration in the superfusate of 1 mM. Elevation of cytosolic Ca2+ levels by increasing the extracellular CaCl2 concentration to 5 mM approximately doubled the contraction amplitude of isolated cells (Fig. 2). No differences between cells from failing and control hearts were observed. As opposed to the effects of Ca2+, cardiomyocytes from failing hearts were desensitised to stimulation of β-adrenergic receptors and of the adenylyl cyclase. Basal contraction amplitude was 19±3 µm in controls (n=16) and 24±2 µm in failing cardiomyocytes (n=20; not significant). The β-adrenergic agonist isoproterenol (3 nM) induced an increase in contraction amplitude of 14±2 µm in the controls and of only 6±2 µm in the failing cells (P<0.01). Dose response curves to isoproterenol were obtained in six hearts from each group. A significant rightward shift of the inotropic response was observed (Fig. 2). To stimulate the adenylyl cyclase independent of the activation of adrenergic receptors, the diterpene, forskolin, was used. The inotropic response to forskolin was also significantly shifted to the right (Fig. 3), indicating a decreased sensitivity of the adenylyl cyclase not only to receptor-mediated, but also to direct, stimulation.


Figure 2
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  		Fig. 2 Contraction amplitude of isolated cardiomyocytes from failing hearts (filled bars/symbols) and sham-operated controls (open bars/symbols). One cell from each animal was included in the analysis (means±SEM). The contractile response to β-receptor-mediated stimulation with isoproterenol is shifted to the right in CHF (P<0.01 by ANOVA), whereas maximal contraction amplitude remains unaltered. Contraction amplitude is shown as % of the maximal response obtained with 5 mM Ca2+. Concentration response curves were fitted using the least squares method. Insert: Receptor-independent maximal stimulation of cells by elevating the extracellular Ca2+ concentration from 1 to 5 mM results in identical changes in contraction amplitude (shown as % of baseline) in both groups (sham: n=12; CHF: n=14; Student’s t-test; means±SEM).

 

Figure 3
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  		Fig. 3 Contraction amplitude of isolated cardiomyocytes from failing hearts (filled symbols) and sham-operated controls (open symbols). The contractile response to direct stimulation of the adenylyl cyclase using forskolin is shifted to the right in CHF (P<0.01 by ANOVA). Contraction amplitude is shown as % of the maximal response. One cell from each animal was included in the analysis (means±SEM). Concentration response curves were fitted using the least squares method.

 
3.3 Sensitisation to anti-adrenergic effects of inhibitory receptor stimulation
Carbachol (10 nM to 3 µM) was used to elicit muscarinic M2 receptor-mediated anti-adrenergic responses in isolated cardiomyocytes. These experiments were performed in the presence of 3 nM isoproterenol. As shown in Fig. 4, contraction amplitude and maximal contraction velocity were decreased by carbachol in a dose-dependent fashion (n=9). This response was significantly augmented in cells derived from failing hearts (n=9; P<0.01 by ANOVA). At a concentration of 1 µM, carbachol reduced the contraction amplitude from 29±3 to 25±3 µm in control cells and from 28±4 to 22±4 µm in failing cells, i.e. by 15.5 and 22.5%, respectively (Fig. 4A). The changes in contractility were more evident when regarding the first derivative of the contraction amplitude, i.e. the maximal contraction velocity (Fig. 4B). The anti-adrenergic effect of carbachol was more pronounced in cells derived from rats with heart failure although these cells were less responsive to isoproterenol, as delineated above (Fig. 2). These findings indicate that heart failure resulted in a significant sensitisation of heart muscle cells towards negative inotropic stimulation via muscarinic receptors. A similar result was obtained when PIA (10 nM to 1 µM) was used to analyse A1 adenosine receptor-mediated responses. There was only a slight anti-adrenergic effect in cells from sham-operated animals (n=7; Fig. 5A). However, a significant PIA-induced reduction in contraction amplitude and in contraction velocity became evident in failing cardiomyocytes (n=11; P<0.01 by ANOVA; Fig. 5B). These findings indicate that the anti-adrenergic effect of A1 adenosine receptor stimulation on myocardial contractility is less pronounced than that of muscarinic activation in normal heart cells. The sensitivity to stimulation of both pathways is significantly up-regulated in the failing heart. The extent of this newly characterised functional up-regulation is comparable for both inhibitory receptor systems.


Figure 4
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  		Fig. 4 Contraction amplitude (A) and contraction velocity (B) of isolated cardiomyocytes from failing hearts (filled symbols) and sham-operated controls (open symbols) in response to stimulation of muscarinic M2 receptors using carbachol. The negative inotropic response is augmented in CHF (P<0.01 by ANOVA). Experiments were performed in the presence of 3 nM isoproterenol. One cell from each animal was included in the analysis (means±SEM). Concentration response curves were fitted using the least squares method.

 

Figure 5
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  		Fig. 5 Contraction amplitude (A) and contraction velocity (B) of isolated cardiomyocytes from failing hearts (filled symbols) and sham-operated controls (open symbols) during stimulation of A1 adenosine receptors using PIA. No significant reduction in inotropy was present in control cells, whereas contractility was depressed by PIA in CHF (P<0.01 by ANOVA). Experiments were performed in the presence of 3 nM isoproterenol. One cell from each animal was included in the analysis (means±SEM). Concentration response curves were fitted using the least squares method.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
The novel finding of the present investigation is a functionally effective transregulation of the inhibitory side of the adenylyl-cyclase-coupled signal transduction system in congestive heart failure (CHF), including a dual sensitisation towards anti-adrenergic stimulation via A1 adenosine receptors and via muscarinic M2 receptors.

4.1 Desensitisation to stimulation of the adenylyl cyclase system
In the experimental model of severe CHF used in this study, which includes doubling of plasma norepinephrine levels [29], a desensitisation of isolated cardiomyocytes towards β-adrenergic receptor stimulation is observed. This is a well-characterised regulation mechanism in CHF, which has been shown to be due to a reduction in cardiac β-adrenergic receptor density and mRNA levels both in experimental and clinical investigations [1–3,30,31]. At the post-receptor level, adenylyl cyclase is also desensitised during CHF, a finding known to be associated with changes in the catalytic activity of the enzyme [5–8,11] as well as in its isoform composition [7,11]. It is in accordance with functional data obtained in the intact heart in several models of CHF [12] and in isolated cardiomyocytes from failing human hearts [14].

The reduced inotropic response to activation of the adenylyl cyclase system is not due to a reduction in Ca2+ sensitivity of the failing cardiomyocyte or to a defect in the contractile response to Ca2+, since elevation of extracellular Ca2+ elicited similar responses in failing and normal cells. Although a reduced contractile response to Ca2+ was observed in one study of cardiomyocytes derived from dogs with pacing-induced heart failure [32], both in failing right ventricular rat cardiomyocytes [33] and in ventricular cardiomyocytes from patients with congestive heart failure [14], the contractile response to Ca2+ was unchanged from controls whereas the response to isoproterenol was diminished by ~30%, similar to the present data. Taken together, isolated cardiomyocytes from failing rat hearts are desensitised to inotropic stimulation on the receptor and post-receptor level, but not beyond, indicating that the contractile apparatus per se is intact.

4.2 Sensitisation to inhibitory receptor activation
On the inhibitory side of the cardiac adenylyl-cyclase-coupled signal transduction system, the sensitisation of the failing heart to anti-adrenergic stimuli found in the present study may be mediated by an up-regulation of Gi proteins [6,9,15–17] or a transregulation of inhibitory receptors.

Muscarinic receptor regulation and its functional importance in heart failure have been studied in the past, but the results were inconclusive. In the pacing-induced heart failure model in the dog, an augmentation of negative inotropic responses of the heart in vivo and an increase in the inhibitory effects on adenylyl cyclase activity were associated with an increase in muscarinic receptor density [17,20]. In contrast, muscarinic receptor density was found to be unchanged in failing rat hearts, although the inhibitory effect of carbachol on forskolin-stimulated adenylyl cyclase activities was augmented [10]; moreover, no changes in Gi proteins were observed in that particular study [10], as opposed to most data obtained in CHF [6,15]. Desensitisation of β-adrenergic receptors in the rat using isoproterenol increased the negative inotropic response of isolated atria [34] and papillary muscles to a cholinergic agonist, but, again, did not change cardiac muscarinic receptor density [34]. In patients with CHF, a reduction of myocardial muscarinic agonist binding sites was detected using positron emission tomography [21]. In healthy humans, muscarinic receptor stimulation has been found to elicit anti-adrenergic responses on left ventricular function [35,36]. However, at present, there are no data to show that endogenous acetylcholine stimulation of muscarinic receptors may exert significant negative inotropic effects on the myocardium, that it might be enhanced in heart failure or that it may contribute to the impaired myocardial function in CHF.

The role of adenosine for myocardial function in vivo has also not been clarified yet, although a negative inotropic, anti-adrenergic action on human atrial and ventricular myocardium [23,35,37] was observed in vitro. The failure to demonstrate such effects in patients [35] may have been due to insufficient tissue concentrations achieved after intravenous administration, or to confounding chronotropic, dromotropic or vascular effects. Moreover, those patients were not in cardiac failure. However, the finding that adenosine concentrations are elevated in the pericardial transudate of dogs with CHF [38] and in the plasma of patients with various forms of CHF [39] indicates that adenosine receptors might be chronically stimulated in heart failure. As shown previously, free myocardial adenosine levels may reach concentrations in the micromolar range, both in myocardial ischemia and during adrenergic stimulation [40,41]. These concentrations had significant anti-adrenergic effects on the contractility of cardiomyocytes, both in previous studies [23] and in the present study. Those findings suggest that endogenous adenosine might indeed exert a tonic inhibitory effect [22] on the adenylyl-cyclase-coupled signal transduction system in the failing myocardium, which might even be augmented during conditions such as myocardial underperfusion, increased wall stress or exercise [39,40]. Although the present data do not allow extrapolation to clinical conditions, it is suggested that anti-adrenergic effects on the failing cardiomyocyte mediated by A1 adenosine receptors could be of greater importance in vivo than those mediated by muscarinic M2 receptors.

4.3 Possible implications for the treatment of CHF
As suggested by the present data, the sensitisation of the inhibitory side of the adenylyl cyclase-coupled signal transduction system in CHF, which parallels the agonist-induced desensitisation of the stimulatory side, eventually results in augmented anti-adrenergic responses and possibly to some degree of tonic inhibition of the adenylyl cyclase. This complex regulation might have implications for the treatment of CHF. Böhm et al. [42] recently found a restoration of the blunted myocardial inotropic response to the phosphodiesterase inhibitor milrinone in patients with CHF after pre-treatment with the β-blocker metoprolol. They suggested that β-blocker therapy in CHF acted at the post-receptor level, including inhibitory Gi proteins and adenylyl cyclase. This concept is supported by data from our laboratory showing that chronic β1-subtype-selective receptor blockade leads to a reduction of cardiac Gi proteins below control values in rats [43]. Although this finding could not be confirmed by Mende et al. [44], who used propranolol, the same group found a reduction of Gi proteins in myocardial biopsies from patients with heart failure after metoprolol treatment [45]. Moreover, a desensitisation of Gi protein-coupled inhibitory receptors, such as the muscarinic M2 receptor or the A1 adenosine receptor, might also be involved in the beneficial effects of β-blocker therapy. On the receptor level, chronic β-blockade induced a down-regulation of muscarinic M2 as well as A1 adenosine receptors in rat heart, brain and lung [46] and a decrease in muscarinic M2 receptor density in the human right atrial appendage [47]. This transregulation of inhibitory receptors resulted in significant blunting of the negative inotropic response of isolated rat hearts to carbachol and adenosine [43]. Therefore, the reversal of the chronic sensitisation to anti-adrenergic stimuli in the failing heart could be an important target of modern therapy of CHF with β-receptor blockers.

In summary, the present data support the notion that the sensitisation to anti-adrenergic receptor stimulation has important functional significance in CHF. In addition to a functionally relevant desensitisation of both cardiac β-receptors and adenylyl cyclase, the anti-adrenergic effects of A1 adenosine and muscarinic M2 receptor stimulation on the contractile function of the cardiomyocyte are significantly increased. This regulation process, probably mediated both by elevated Gi proteins and changes in inhibitory receptor density, may contribute to the functional impairment of the failing myocardium. It may thus be the target for future therapeutic strategies reducing the response to anti-adrenergic receptor systems in heart failure.

Time for primary review 21 days.


   Acknowledgements
 
The authors are greatly indebted to Sian E. Harding, PhD, Imperial College of Medicine, National Heart and Lung Institute, London, UK, for her invaluable help. This work was supported by grants from Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 320.


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

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V. Vallon, C. Miracle, and S. Thomson
Adenosine and kidney function: Potential implications in patients with heart failure
Eur J Heart Fail, February 1, 2008; 10(2): 176 - 187.
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