The sympathetic nervous system and the renin-angiotensin system are two neurohormonal compensatory mechanisms that are stimulated in chronic heart failure. Their initial beneficial hemodynamic effects are, however, associated progressively with adverse effects on cardiac remodeling and in particular fibrosis, which results from the imbalance between the synthesis and the degradation of type I and III collagen.1
The activity of fibroblasts, which produce collagen, may be regulated by numerous interacting factors, including stimulation by aldosterone and angiotensin II.2 Synthesis of aldosterone occurs in the heart itself, but the relative role of such cardiac aldosterone compared with that of plasma aldosterone coming from the adrenal glands remains to be determined.3
Blockade of β-adrenergic receptors4,5 or of aldosterone receptors6 or inhibition of angiotensin II synthesis7 provides long-term benefit in chronic congestive heart failure in humans. However, because β1-adrenergic stimulation induces renin release,8 and because stimulation of presynaptic angiotensin II receptors increases norepinephrine release,9 part of the benefit of β-blocker treatment could involve reduction of fibrosis formation linked to angiotensin II and aldosterone. We have demonstrated previously10 that acute administration of a high dose of isoproterenol induces an important cardiac fibrosis, which is partially prevented by the administration of potassium canrenoate, an antagonist of aldosterone receptors.
The present experiments were designed to evaluate the impact of angiotensin II and aldosterone receptor blockade on cardiac fibrosis induced by chronic β-adrenergic stimulation in an experimental model of heart failure.
MATERIALS AND METHODS
Normotensive male Wistar rats (body weight 250-300 g, age 7 weeks) were purchased from CERJ (Saint Berthevin, France). The rats were nursed and housed in a 12-hour dark/light room under controlled environmental conditions: dark cycle at 21°C of ambient temperature. Food and water were given ad libitum. All the procedures and protocols involving animals and their care were conducted in conformity with institutional guidelines that are in compliance with national and international laws and policies (Council directive # 87.848, October 19, 1987, Ministère de l'Agriculture et de la Forêt, Service vétérinaire de la Protection Animale, permission #0299 to Ministry of Health) and were approved by the institutional animal care commitee.
Heart failure was induced in Wistar rats after ligation of the left anterior coronary artery under pentobarbital anesthesia (50 mg/kg IP).11 Animals were connected to a respirator (Harvard, Rodent Ventilator, model 683, frequency of 42 ventilations per minute, and a volume of 1 mL/100 g). The chest was opened by a left lateral thoracotomy, and the left coronary anterior artery was ligated approximately 2 to 3 mm from its origin with 5-0 silk suture (Ethicon). Control rats were submitted to a sham operation by using a similar procedure without coronary ligation. The perioperative mortality was around 50% in rats submitted to coronary artery ligation.
Ten weeks after surgery, the hemodynamic state of heart failure is present after coronary ligation with neurohormonal stimulation. We have shown previously that isoproterenol administration for a 15-day period with osmotic minipumps greatly enhances collagen deposition in the myocardium.12 Sham and infarcted surviving animals were then submitted to such a chronic stimulation of β-adrenergic receptors with isoproterenol. The possible prevention of isoproterenol-induced remodeling with antagonists of aldosterone and angiotensin II receptors was studied.
Administration of Isoproterenol
Chronic stimulation of β-adrenergic receptors was performed by means of subcutaneously implanted micropumps (Alzet, Palo Alto, CA, model 2002) filled with isoproterenol and delivering 30 μg/kg/h for a 2-week period. Isoproterenol was dissolved in sterile water.
Administration of Antagonists of Aldosterone and of Angiotensin II
Blockade of aldosterone receptors was obtained by administration of potassium canrenoate at a dose of 20 mg/kg, adjusted each day, in drinking water.10 Blockade of AT1, angiotensin II receptors was obtained with oral administration of losartan 10 mg/kg/d by oral gavage.13
Sham or infarcted animals were then randomized into 4 groups: control, isoproterenol, isoproterenol with administration of potassium canrenoate, isoproterenol with potassium canrenoate and coadministration of losartan. Potassium canrenoate and losartan were administrated during the 2-week period of infusion of isoproterenol.
After 2 weeks of such treatment with isoproterenol (12 weeks after coronary ligation), the animals were anesthetized (pentobarbital 50 mg/kg, IP), and invasive measurements were performed. The heart was then removed after thoracotomy for morphometry study and quantification of the fibrosis. Extent of myocardial infarction was calculated as described previously by Pfeffer et al.11
A microtip pressure transducer catheter (Millar Instruments, 2-French) was inserted into the right carotid artery and connected to a Gould recorder (Model SR 3200, Gould Instruments Co). The following pressures were obtained: aortic blood pressure, left ventricular pressure and maximal positive and minimal negative left ventricle dP/dt. The heart rate was determined from the ventricular pressure tracing.
After completion of hemodynamic measurements, the heart was removed rapidly, auricles and ventricles were weighed, and the heart to body weight ratio was calculated. They were weighed again. All ventricles were immersed in 10% buffered formalin and dehydrated in 95° ethanol and then in acetone. They were impregnated with methyl salicylate and embedded in paraffin. Three cross sections (short axis) of 6 μm, separated by 1.2 mm, were obtained for each heart.
Quantification of Fibrosis
Sirius red staining was used to characterize collagen fibers. Sirius red-stained slides of left ventricle were placed under a 3CCD color camera (KY-F55B, JVC, Japan), which was connected to a quantimeter Qwin (Leica, Rueil Malmaison, France) with the acquisition software Leica Win (version 2.2, Leica Microsystems, France). Area of fibrosis on each ventricle was then calculated, excluding fibrosis related to infarct scar.
Isoproterenol (dl-isoproterenol hydrochloride) was purchased from Sigma (Saint Quentin Fallavier, France). Soludactone® (potassium canrenoate) was purchased from Searle (Boulogne-Billancourt, France). Losartan was provided by Merck Pharmaceuticals. Sodium pentobarbital (Sanofi Santé Animal, France) was used in injectable solution. Isoproterenol was prepared under sterile conditions with NaCl 0.9%.
The statistical analysis for each studied parameter was carried out with analysis of variance (ANOVA). When the F test was significant, between-group comparisons were performed using a Newman-Keuls test. P values less than 0.05 were considered to be statistically significant. All results were expressed as means ± SEM. For the statistical analysis, Statistica software (Statsoft) was used.
Effects of Isoproterenol and Infarction
Both infarction and administration of isoproterenol increased the atrial and ventricular weights (Figs. 1 and 2). In particular, atrial weight was on average two-fold more important in all infarcted groups compared with respective sham groups.
Effects of Potassium Canrenoate and Losartan
The significant (P < 0.05) isoproterenol-induced ventricular mass increase in infarcted animals was significantly reduced by the simultaneous administration of potassium canrenoate and losartan but not completely by potassium canrenoate alone. The ventricular weights were as follows: control MI, 1.26 ± 0.02 g; MI + iso, 1.76 ± 0.05 g; MI + iso + pc, 1.67 ± 0.05 g; and MI + iso + pc + los, 1.53 ± 0.05 g (Fig. 2). Potassium canrenoate alone only partly inhibited the effect of isoproterenol on the ventricles but abolished its effect on the atria.
Atrial weights were as follows: control MI, 0.16 ± 0.01 g; MI + iso, 0.45 ± 0.06 g; MI + iso + pc, 0.30 ± 0.03 g; and MI + iso + pc + los, 0.27 ± 0.02 g. (Statistical comparisons are provided in the legend of Figure 2.)
Effect of Isoproterenol and Myocardial Infarction
The administration of isoproterenol increased heart rate (Table 1). Myocardial infarction did not change heart rate. The aortic blood pressures were not modified statistically by infarction or isoproterenol. However, in infarcted animals, the left ventricular systolic pressure (LVSP) was significantly decreased, and the left ventricular end-diastolic pressure was increased.
Peak +dP/dt and −2dP/dt were reduced significantly by infarction and increased by isoproterenol.
Effects of Potassium Canrenoate and Losartan
The administration of the two drugs had no significant effects on infarcted rats or isoproterenol-induced hemodynamic modifications.
Morphometry and Fibrosis
Effect of Potassium Canrenoate and Losartan in Sham Animals
The chronic perfusion of isoproterenol increased significantly the fibrosis of the left and right ventricles (P < 0.001) (Figs. 3 and 4). In the left ventricle, potassium canrenoate blunted such fibrosis compared with the isoproterenol group without any additional effect of losartan. The extents of left ventricular fibrosis were as follows: control sham, 0.16 ± 0.03%; sham + iso, 3.01 ± 0.37%; sham + iso + pc, 1.67 ± 0.45%; and sham + iso + pc + los, 2.06 ± 0.58%.
However, in the right ventricle, compared with sham + iso group, potassium canrenoate only partly reduced the fibrosis, and the addition of losartan inhibited nearly completely the isoproterenol-induced fibrosis. In right ventricle, the extents of fibrosis were as follows: control sham, 0.14 ± 0.03%; sham + iso, 1.05 ± 0.13%; sham + iso + pc, 0.83 ± 0.13%; and sham + iso + pc + los, 0.37 ± 0.07%.
Effect of Potassium Canrenoate and Losartan in Infarcted Rats
Myocardial infarction increased interstitial fibrosis in noninfarcted areas of ventricle (P = 0.01). Such fibrosis was completely inhibited in both ventricles by potassium canrenoate without an additional effect of losartan. The decrease of fibrosis in the left ventricle was significant in MI + iso + pc and in MI + iso + pc + los versus MI + iso. The values of the cardiac extents of fibrosis in the left ventricle were as follows: control MI, 1.30 ± 0.34%; MI + iso, 2.50 ± 0.27%; MI + iso + pc, 0.82 ± 0.11%; and MI + iso + pc + los, 1.47 ± 0.31%.
In the right ventricle, the reduction of the extent of fibrosis was statistically different in MI + iso + pc and in MI + iso + pc + los compared with MI + iso. The extents of cardiac fibrosis in right ventricle were as follows: control MI, 0.97 ± 0.25%; MI + iso, 1.41 ± 0.20%; MI + iso + pc, 0.32 ± 0.06%; and MI + iso + pc + los, 0.45 ± 0.10%.
All statistical comparisons are displayed on Figures 3 and 4.
In the present study, the isoproterenol-induced cardiac fibrosis was reduced significantly by administration of potassium canrenoate, an antagonist at the aldosterone receptor. The addition of losartan, an angiotensin II receptor antagonist, did not further reduce the extent of fibrosis in the left ventricle. The isoproterenol-induced cardiac fibrosis observed in this study therefore partly involves stimulation of cardiac aldosterone receptors.
The myocardial infarction model in rats is commonly used as an experimental model of heart failure and well characterized by a significant elevation of LVEDP, an alteration of contractility, and significant cardiac remodeling with development of fibrosis in left as well as right ventricles.11 The left ventricular remodeling after myocardial infarction is influenced by an excessive activation of the sympathetic nervous system and renin-angiotensin system (RAS)14 and can be attenuated by compounds blocking neurohormonal receptors of these 2 systems.4-7
The administration of isoproterenol, a β-adrenergic receptor agonist, induces myocardial remodeling with left ventricular hypertrophy and fibrosis.10,15,16 These effects are related to the stimulation of β-adrenergic receptors and involve several cellular mechanisms such as intracellular cAMP increase, intracellular calcium overload, ischemia, and oxidative stress.17,18 In the present experiments, cardiac hypertrophy and fibrosis were indeed present after isoproterenol administration at both levels of atria and ventricles.
Chronic administration of aldosterone in experiments increased cardiac collagen content in both left and right atria and ventricles both in rats19 and in guinea pigs.20 Robert et al have shown also that aldosterone increases the mRNA coding for type I and III collagen.19
According to these experimental results, the interest in blockade of aldosterone effects has been explored in human heart failure in the RALES study with spironolactone. The RALES study demonstrated that this aldosterone receptor blocker reduces mortality by 30%. A new aldosterone receptor antagonist, eplerenone, has also been used with success in the EPHESUS trial including patients with past myocardial infarction.21 Eplerenone has fewer side effects and has a greater selectivity for mineralocorticoid receptor (MR) than spironolactone.22
The present experiments suggest interactions between β-adrenergic receptors and angiotensin II and aldosterone receptors. Indeed, blockade of aldosterone receptors partly inhibited the effects of β-adrenergic stimulation on the induction of cardiac hypertrophy and fibrosis. Such interactions may occur at different levels, either at the receptor and cell membrane level or at the transcription-transduction level.
An interrelationship has been recently described between AT1 and β-adrenergic receptors. Indeed, the selective β1-adrenergic receptor blocker metoprolol inhibits response to AT1 receptor stimulation, whereas selective AT1 receptor blockade with valsartan inhibits the β-adrenergic inotropic effect on isolated myocyte preparations.23 Although the pathway of signal transduction is different in AT1 and β-adrenoceptors-the AT1 receptor is coupled to a Gq protein, and β-adrenoceptors to a Gs protein-these results suggested that blockade of either AT1 or β-adrenoceptors can induce a conformational change of the other receptor that is not longer favorable to support the interaction with its G protein.23 Other studies have shown that angiotensin II receptor blockade can prevent the negative cardiac effects of aldosterone,24 whereas eplerenone prevents angiotensin II-induced vascular inflammation in the heart. These results suggest that the cardiac effects of aldosterone on the heart are mediated, at least in part, via intervention of AT1 receptors, whereas those of angiotensin II are mediated partially by mineralocorticoid receptors (MR). The present study demonstrates that the isoproterenol-induced cardiac fibrosis is mediated, at least in part, through aldosterone action. Indeed, on one hand, the stimulation of the β-receptors by isoproterenol leads to the synthesis of aldosterone,10 and on the other hand, interactions at the transcription level may occur. The mineralocorticoid receptor that binds aldosterone has the ability to bind after formation of homodimer or heterodimer complexes to the glucocorticoid response element (GRE) localized in the DNA.25,26 GREs are present in target genes responsible for the formation of fibrosis such as transforming growth factor β127,28 and pro-α 1 and 2 collagen type I28,29 and in other genes such as angiotensin receptors30,31 and the β-adrenoceptors.32 Binding of spironolactone to the MR inhibits the effects of aldosterone by competing at its binding site on the MR. The spironolactone/MR complex could therefore interact with DNA, as has been demonstrated by Lombès et al in vitro,33 and inhibit the expression of genes coding for protein of fibrotic tissue. GREs regulate the transcription of target genes in two different ways, either by stimulation or suppression. In addition, binding of aldosterone to MR could induce β-adrenergic receptor transcription through intervention of GRE. This could then enhance response to β-adrenergic agonists through an increase in the number of β-adrenergic receptors. Blockade of the MR would induce the opposite effect, which we have observed in our experiments.
In conclusion, results of our study strongly suggest complex interactions between aldosterone and β-adrenergic receptor pathways with prevention of cardiac remodeling induced by β-adrenergic stimulation by blockade of aldosterone receptors. This interaction probably occurs at the transcriptional level.
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