Spironolactone reduces fibrosis of dilated atria during heart failure in rats with myocardial infarction

Paul Milliez1,2, Noeleen DeAngelis3, Catherine Rucker-Martin4, Antoine Leenhardt1,2, Eric Vicaut5, Estelle Robidel1, Philippe Beaufils2, Claude Delcayre1 and Stéphane N. Hatem6,7,*

1Centre de Recherches Cardiovasculaires Inserm Lariboisière—Unité U689, Paris, France
2Service de Cardiologie, Hôpital Lariboisière, Paris, France
3Istituto di Ricerche Farmacologiche Mario Negri, Milano, Italy
4CNRS-UMR 8078, Hôpital Marie Lannelongue, Le Plessis Robinson, France
5Centre d'investigation clinique, Hôpital Lariboisière, Paris, France
6INSERM Unité 621, Faculté de Médecine Pitié Salpétrière, Université Pierre et Marie Curie, 91 Boulevard de l'Hôpital, 75634 Paris Cedex 13, France
7Centre d'Exploration Fonctionnelle Intégrée Faculté de Médecine Xavier Bichat, Paris, France

Received 12 February 2005; revised 22 July 2005; accepted 11 August 2005; online publish-ahead-of-print 1 September 2005.

* Corresponding author. Tel: +33 1 40 77 96 49; fax: +33 1 40 77 98 72. E-mail address: stephane.hatem{at}chups.jussieu.fr

See page 2079 for the editorial comment on this article (doi:10.1093/eurheartj/ehi477)


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgement
 References
 
Aims Congestive heart failure (CHF) is associated with severe structural changes of atria, contributing to impaired atrial function and the risk of arrhythmia. This study investigated the effects of CHF treatments on atrial remodelling.

Methods and results Three months after myocardial infarction (MI), rats were treated for 1 month with spironolactone, lisinopril, or atenolol alone or in combination. Echocardiography–Doppler tissue imaging, haemodynamic measurements, and 24-h Holter monitoring were used to characterize the cardiomyopathy. Atrial fibrosis was quantified with Picrosirius Red staining. Left atrial diameter was increased (5.8±0.6 mm in MI vs. 3.6±0.3 mm in sham; P<0.0001), as was atrial fibrosis (26.7±3.8% in MI vs. 10.5±2.2% in sham; P<0.0001), which correlated with left ventricular (LV) dysfunction after 3 months of MI. P-wave duration was also increased and premature atrial beats were frequent on the 24-h electrocardiogram. Similar improvements in LV dysfunction were observed after 1 month of spironolactone, ACE-inhibitor, or beta-blocker therapy alone or in combination. Atrial hyperexcitability was reduced by all the treatments, but only spironolactone attenuated atrial fibrosis and reduced P-wave duration.

Conclusion Atrial fibrosis caused by chronic CHF is reduced by spironolactone.

Key Words: Heart failure • Fibrosis • Atrial myocardium • Spironolactone • Atrial fibrillation


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgement
 References
 
Congestive heart failure (CHF) is often associated with severe structural and functional changes of the atria1,2. Increased fibrosis has been observed around trabeculae and in the interstitial spaces of the atrial myocardium during CHF in both animal models and human biopsies.1,35 This extracellular matrix remodelling is associated with extensive myolysis and with the presence of a number of enlarged atrial myocytes, which re-express a foetal phenotype.3,4 Moreover, there are profound changes in the atrial electrical properties including depressed excitability, increased refractoriness, and conduction slowing or block, which have been related to the degree of interstitial fibrosis.1,6,7 CHF is also associated with abnormalities of cell electrophysiology.6,8 This atrial remodelling is likely responsible for the composition of the substrate for atrial fibrillation (AF), the incidence of which is increased during CHF.912

These observations raise the important question about the prevention or reversion of the atrial remodelling associated with CHF. In the dog model of pacing-induced heart failure with AF, angiotensin-converting enzyme (ACE) inhibitor therapy started at the onset of cardiopathy protects against atrial fibrosis.13,14 Clinical studies also show that enalapril reduces the incidence of AF in patients with left ventricular (LV) dysfunction, most likely by preventing the constitution of the arrhythmic substrate.15 The efficacy of ACE-inhibitors on the atrial remodelling is consistent with the activation of the renin–angiotensin system in haemodynamic overloaded and dilated atria and with the effects of angiotensin on structural and electrical properties of the atrial myocardium.16

The present study was designed to examine whether treatments currently used for CHF, such as spironolactone, ACE-inhibitors, and beta-blockers, can reverse atrial remodelling. We were particularly interested in determining whether spironolactone, which has marked antifibrotic properties in the ventricle,17 also has an effect on extracellular matrix remodelling in the left atria (LA). A rat model of myocardial infarction (MI) complicated by LV dysfunction, in which LA remodelling and severe fibrosis occur within 3 months after MI, was used in our study.5


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgement
 References
 
Experimental design
In accordance with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, Revised 1996), MI was produced in 180 anaesthetized, ventilated 10-week-old male Wistar rats by ligation of the left coronary artery (Charles River, France), as previously described.8 Fifty-four rats died during the 24-h post-operative period and 38 in the 3 months preceding randomization. No rats died during treatment. Hence, 88 rats were included in the study. Three months after MI, LV function was assessed by echocardiography and rats with LV dysfunction were randomly allocated to the various treatments. Eleven rats were sham operated, 11 were untreated MI, and the remaining were treated for 1 month with spironolactone (10 mg/kg per day, n=11), lisinopril (1 mg/kg per day, n=11), atenolol (1 mg/kg per day, n=11), spironolactone+lisinopril (n=11), lisinopril+atenolol (n=11), or spironolactone+lisinopril+atenolol (n=11). These are the therapeutic doses used in this model.17,18 Concomitantly, we used the same drugs to assess their effects on ventricular as well as vascular remodelling, and we found a similar decrease in fibrosis with all these drugs at the same doses. All rats were euthanized, 4 months after surgery.

Echocardiographic study
Transthoracic echocardiography was performed on anaesthetized rats using an echocardiography system (Toshiba Powervision-6000, SSA-370A) equipped with a 8–14-MHz linear transducer. Data were transferred online to a computer for analysis (Ultrasound Image Workstation-300-A, Toshiba). LV diameter and LA diameter (LAD) were measured in the parasternal long-axis view in M-mode. LV end-diastolic diameter was defined as the largest LV diameter, and LAD as the distance between posterior walls of the aorta and the LA. The ejection fraction (EF) was measured using a modified version of Simpson's monoplane analysis.19 Pulsed-wave Doppler spectra of mitral annular velocity measurements by Doppler tissue imaging in the apical four-chamber view were used to calculate left ventricular end-diastolic pressure (LVEDP), as described earlier.20 In a pilot study, the LVDEP calculated using the Doppler method correlated with that measured invasively, as previously reported in this model.21

Holter monitoring
One day before the end of treatment, a telemetry transmitter (DataScience International, USA) was implanted in the peritoneum of anaesthetized rats, with electrodes placed in a ‘lead 2-like’ position. Rats were housed for 24 h in a cage placed on a receiver, which captured the digitized one-channel ECG signal (1 kHz). Acquisition and analysis were performed using Chart v4.1.2 software (PowerLab, AdInstruments, USA). Automatic evaluation of atrial premature complexes (APCs) was performed using ECG-Auto software (EMKA technologies, France). An APC was defined as an unexpected premature narrow supraventricular QRS with a prematurity exceeding the normal variation of RR interval by 60% and often preceded by a P-wave. P-wave duration, defined as the time elapsed between P-wave onset and offset, was assessed by measuring the first derivative of the P-wave using dedicated software. These measurements were repeated randomly during several diurnal and nocturnal periods of the 24-h telemetry recording. No differences were observed between nocturnal and diurnal measurements. Furthermore, each sample was validated manually by two different observers, to avoid inappropriate spotting of the onset and offset of the P-wave. In the case of disagreement, the measurement was excluded.

LV pressure measurements
In preliminary validation studies, LVEDP was measured invasively in anaesthetized, ventilated rats, using a 3-Fr Mikro-Tip pressure transducer catheter (Model TC50, Millar Instruments, Inc.) introduced into the LV through the right carotid artery and connected through a data acquisition unit (PowerLab/4s, ADInstruments, Inc.) to a computer running MacLab software. LVEDP was read directly from the pressure curve as being the diastolic pressure immediately preceding the pressure rise associated with LV contraction.

Histological analysis
Excised hearts were rinsed in ice-cold 0.9% saline buffer. Atria were fixed in 10% formalin, and the LV was frozen in OCT. Infarct size was measured by two independent investigators on 10-µm-thick cryostat sections. Infarct size was calculated as the total length of the scar as a percentage of the LV circumference and averaged over the endocardium and epicardium. Seven-micrometre-thick LA sections were stained with Picrosirius Red F3BA, and fibrosis was quantified blindly on five sections per atrium (8–10 fields per section). The interstitial collagen volume fraction expressed as a percentage of the total atrial surface area was determined as described elsewhere.17

Statistical analysis
The variance of the studied parameters was not known before the study. We chose to study 11 rats in each group in order to ensure a power >80% in order to detect a size effect (i.e. ratio of difference in mean on standard deviation) of at least 1.5 when comparing a group of treated MI rats against untreated MI rats.

First, we tested that echocardiographic measurements between MI and sham-operated rats before treatments were statistically different. Then, we checked that 1 month after randomization, clinical, electrical, and anatomical remodelling was different among untreated MI and sham-operated rats. Finally, we compared the treated MI groups with untreated MI groups. For these purposes, echocardiographic values between sham-operated rats and all MI rats before randomization were compared using Student's t-test. The same test was used to compare values of untreated MI rats 1 month after randomization with those of sham-operated rats. Then, treated MI rats were compared with untreated MI rats, using Dunnett's test—the most appropriate for the comparison of several groups with a control group, because we decided as a priori not to compare treated groups. In addition, when analyzing the effects of treatment on atrial fibrosis, we checked that similar results were obtained when LVEDP was included in the test as a covariate in a covariance analysis, thus eliminating any confounding effect of this parameter on the results. Linear-regression analysis was used to test relationships between quantitative parameters. All tests were two-sided, and the significance level was fixed at 5%. All results are shown as mean±SD.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgement
 References
 
Echocardiographic parameters of sham-operated and MI rats before randomization
Three months after surgery, sham-operated rats displayed normal echocardiographic parameters, whereas MI rats showed clear signs of LV dysfunction. Substantial LV dilation was observed, and LVEDD was increased when compared with sham-operated rats (Table 1). Systolic performance was worsened in MI rats, as indicated by the reduced EF (Table 1). LVEDP was increased, indicating LV haemodynamic overload (Table 1 and Figure 1). All MI rats had dilated LA, and atrial diameter was correlated with both LVEDP (r2=0.49; P<0.0001) and EF (r2=0.69; P<0.0001) (Figure 1).


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Table 1 Echocardiographic parameters of sham-operated and MI rats
 


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Figure 1 Time-motion echocardiographic images obtained in the parasternal long-axis view (A and C) and Doppler tissue imaging of the mitral annulus (B and D) in sham-operated (A and B) and MI (C and D) rats. Relationship of LAD with LVDEP (E) and EF (F). Ao, aorta.

 
Atrial electrical properties were also altered in MI rats. P-wave duration was increased (31.7±0.9 ms vs. 17.3±0.2 ms in sham-operated rats; P<0.0001) (Figure 2), and there was a high incidence of APCs (8652±2715 APC/24 h vs. 12±0.5 in sham-operated rats; P<0.01) (Figure 2). However, no episodes of AF, and only rare not sustained atrial tachyarrhythmia was found on the 24-h electrocardiogram.



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Figure 2 Characteristics of the P-wave in sham-operated (upper panel) and MI (middle panel) rats. (Lower panel) Representative examples of APC recorded in an MI rat.

 
Fibrosis in LA of untreated rats
Picrosirius Red staining of LA revealed narrow myocytes surrounded by a faint collagen network in sham-operated rats (Figure 3A) and extensive fibrosis in both interstitial spaces and between bundles of fibres in the dilated LA of MI rats (Figure 3B). This collagen accumulation was increased almost 2.5-fold in MI rats (26.7±3.8% vs. 10.5±2.2% in sham controls; P<0.0001); fibrosis was correlated with the increase in both LAD (r2=0.753, n=16; P<0.0001) and LA weight (r2=0.4, n=16; P<0.001).



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Figure 3 Picrosirius Red staining of atrial sections from untreated and treated MI rats: (A) sham-operated; (B) untreated MI; (C) MI+spironolactone; (D) MI+atenolol; (E) MI+lisinopril; (F) MI+spironolactone+lisinopril; (G) MI+lisinopril+atenolol; (H) MI+spironolactone+lisinopril+atenolol. Bar, 50 µM.

 
Regression of LA myocardial remodelling in treated rats
After 1 month of treatment, LVEDP was reduced in all treated MI groups when compared with untreated MI rats (Table 2 and Figure 4A). Weights of whole-heart, LV, and lung were not different among treated and untreated MI rats. The LA weights tended to decrease in MI-treated rats when compared with that of untreated MI rats, without reaching statistical significance (Table 2). Interestingly, the lowest value was observed with tri-therapy. Atrial fibrosis was variably reduced in treated MI rats with a marked effect of spironolactone on this parameter (Figures 3C–H and 4B). However, fibrosis remained higher than in sham-operated rats with all treatments.


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Table 2 Haemodynamic and macroscopic characteristics of euthanized
 


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Figure 4 Effects of treatment on LV function and atrial fibrosis. (A) LVEDP in sham-operated rats and in untreated and treated MI rats. (B) Atrial fibrosis in sham-operated, untreated, and treated MI rats. All values are mean±SD. *P<0.05 and **P<0.01 are significantly different from MI. S, spironolactone; L, lisinopril; A, atenolol.

 
In the LV, all treatments reduced fibrosis similarly (collagen volume fractions were (in %): 5.8±1.4 in MI group vs. 4.1±1.2, 4.3±0.9, and 4.4±0.9 in spironolactone, lisinopril, and atenolol groups, respectively; P<0.05 for all groups). Moreover, drug combinations further regressed ventricular fibrosis (3.8±1.1%, 3.8±0.4%, and 3.7±1.3% in spironolactone+lisinopril, lisinopril+atenolol, and spironolactone+lisinopril+atenolol groups, respectively; P<0.01 for all groups). Values for collagen volume fractions in all treated groups were not different from those of sham-operated group (3.6±0.2%).

There was a good correlation between LA weight and LA fibrosis (r2=0.45; P<0.0001). In an attempt to identify factors associated with the decrease in atrial remodelling, we did not find a significant correlation between the LVEDP and LA fibrosis of treated rats. When comparing the percentage of fibrosis between groups, using LVEDP as the covariate, spironolactone proved to be the most effective drug for the reversal of fibrosis (Figure 4B).

Only spironolactone treatments reduced P-wave duration, although the latter remained higher than in sham controls (Figure 5A). The number of APC was dramatically reduced in all treated rats. This reduction was more pronounced with spironolactone treatment, but the difference did not reach significance (Figure 5B).



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Figure 5 (A) P-wave duration in sham-operated, untreated, and treated MI rats. (B) Number of APC during 24-h Holter recording in sham-operated, untreated, and treated MI rats. *P<0.05 and **P<0.01 are significantly different from MI. S, spironolactone; L, lisinopril; A, atenolol.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgement
 References
 
Previous studies have shown that CHF is accompanied by severe structural alterations of the atrial myocardium, which may result in AF. We found that this atrial remodelling depended on the severity of LV dysfunction caused by MI in rats. Pharmacological improvement of cardiac function was associated with a significant attenuation of the structural alterations of the atrial myocardium, only when spironolactone was used.

Several mechanisms may underlie the atrial remodelling observed in this rat model. Dilated LA and fibrosis are probably a direct consequence of LV dysfunction and haemodynamic atrial overload. Indeed, it has been shown that both atrial pressure and volume are important factors in determining atrial fibrosis.1,5 Various neuromediators or hormones which are upregulated during CHF may also be involved in LA remodelling. The renin–angiotensin system is upregulated during CHF, and the density of angiotensin type-2 receptors is enhanced in fibrillating atria.22,23 Moreover, ERK1/2, a protein kinase, coupled to the angiotensin-II regulatory pathway is upregulated in atrial myocytes of dogs with CHF.13 Furthermore, in dogs with pacing-induced CHF, ACE-inhibitor therapy started at the onset of the cardiopathy reduces atrial fibrosis, even though this treatment is not fully protective.13,14

Surprisingly, attenuation of LV dysfunction in treated rats with MI was not always associated with the regression of atrial myocardium remodelling. Indeed, only spironolactone, used alone or in combination with other drugs, induced a significant decrease in atrial fibrosis. This is reminiscent of the spontaneously hypertensive rat model, in which captopril fails to reduce myocardial fibrosis when therapy is started after CHF has occurred.24 Once established, atrial remodelling might be inadequately sensitive to haemodynamic conditions and/or to the activity of the renin–angiotensin system.25 In addition, angiotensin-independent pathways could regulate the composition of the AF substrate during CHF.26 Spironolactone's efficacy may be due to its potent antifibrotic properties, as demonstrated in several experimental models,17,27 resulting in a more complete blockade of aldosterone's fibrogenic effect than that obtained with ACE-inhibitors. In the RALES trial patients randomized to spironolactone had a significant reduction of both pro-collagen I and III when compared with patients receiving placebo.28 Moreover, in patients with heart failure, the plasma levels of aldosterone escape after several months of treatment with ACE-inhibitor.29

The mechanisms by which spironolactone reduces fibrosis are not fully understood. Mineralocorticoid receptors are expressed in both cardiac myocytes and fibroblasts, and aldosterone is produced locally in the failing heart.27 In the rat ventricle, the induction of pericoronary inflammation and increase in oxidative stress by aldosterone are prevented by spironolactone.30 Aldosterone stimulates rat cardiac fibroblasts to produce extracellular matrix and metalloproteinases. This effect is blocked by spironolactone.31 It is conceivable that the effect of spironolactone on atrial fibrosis may be partly due to mechanisms other than its antifibrotic properties. For instance, as a diuretic, spironolactone may also contribute to the attenuation of the volume overload to the LA. However, the fact that LVEDP is reduced by all treatments used does not support a marked effect of spironolactone on cardiac haemodynamic conditions. Note also that in experimental aldosteronism, 20 mg/kg per day spironolactone prevents cardiac fibrosis without affecting hypertension and LV hypertrophy.32

LA remodelling is associated with changes in atrial electrical properties in MI rats, with P-wave lengthening and a high incidence of APC. P-wave prolongation indicates that conduction of the electrical impulse is depressed in the dilated atria, probably because of marked interstitial fibrosis.3335 This is supported by our observation that the antifibrotic efficacy of spironolactone was associated with a decrease in P-wave duration. A relationship between the severity of atrial fibrosis and the duration of the P-wave has also been established in humans,36 and the risk of AF correlates with P-wave prolongation.37 However, the slight decrease in P-wave duration in spironolactone-treated rats suggests that other factors apart from fibrosis contribute to P-wave lengthening including alterations of cellular electrophysiology.6,8 For example, it has been recently observed that overexpression of MR in mice heart is associated with ion channel remodelling and increased occurrence of severe ventricular arrhythmias.38 All the CHF treatments used in the present study reduced the number of APC, suggesting a weak relationship between atrial hyperexcitability and structural alterations of the atrial myocardium. Changes in the activity of neuromediators or hormones may contribute to atrial hyperexcitability in the failing heart. The accumulation of cGMP in atrial myocytes of CHF rats may contribute to the downregulation of the calcium current and its abnormal sensitivity to beta-adrenergics. This may contribute to abnormal cell excitability.8 An increase in the sodium–calcium exchange current observed in the atria of failing heart may promote arrhythmogenic afterdepolarizations.6

Limitations of the study
The beta-blocker and ACE-inhibitor were used at clinically relevant doses.17,18 They improved haemodynamic working conditions of the failing LV and reduced fibrosis in ventricles of MI rats. These results point to a distinct pharmacological profile of ECM remodelling in the atria, which may involve distinct regulatory pathways.25,26 However, it is possible that beta-blockers and ACE-inhibitors may reduce atrial myocardial remodelling if used at higher doses or for longer periods. Note that the present study was designed to evaluate the effects of treatment and not to test differences between treatments. Another limitation is that treatments were started some time after establishment of the cardiopathy and thus the capacity of beta-blockers, ACE-inhibitors, and spironolactone to prevent LA remodelling was not studied. Finally, the lack of spontaneous episodes of AF in this model, probably due to the small size of the rat atria for the constitution of micro-re-entry circuits, rules out any conclusions on the efficacy of treatments tested in this study on the prevention of AF.

Potential clinical significance
Recently, we have shown that excess plasma aldosterone correlates with increased AF in patients with primary aldosteronism.39 Several other studies have demonstrated the beneficial effects of ACE-inhibitors on both atrial and ventricular arrhythmias in patients with severe heart failure.15,40 The decrease in arrhythmic events is mainly attributed to the improvement in LV function. In the RALES study,41 there was a significant reduction in sudden cardiac deaths in spironolactone-treated patients with severe heart failure, suggesting that the decrease in fibrosis can also have a beneficial effect on arrhythmias. Unfortunately, the incidence of AF was not examined in this trial. Our finding that spironolactone has a marked efficacy on the structural remodelling of the atrial myocardium suggests that clinical studies may be warranted to determine whether antifibrotic treatment might open new therapeutic perspectives for both the prevention and treatment of AF.


    Acknowledgement
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgement
 References
 
This study was supported by the INSERM, Association Française contre les Myopathies, Fondation de France, Pharmacia France and the Société Française de Cardiologie.

Conflict of interest: none declared.


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