© 2008 European Society of Cardiology
Comparison of the effects of intrapericardial and intravenous aldosterone infusions on left ventricular fibrosis in rats
Department of Pharmacology and Toxicology, Cardiovascular Research Institute, Maastricht University Netherlands
* Corresponding author. Department of Pharmacology and Toxicology, Cardiovascular Research Institute, Maastricht University, PO Box 616, 6200 MD Maastricht, The Netherlands. Tel.: +31 433881417; fax: +31 433884149. E-mail address: r.hermans{at}farmaco.unimaas.nl (J.J.R. Hermans).
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Abstract |
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 Top Abstract 1. Introduction2. Material and Methods3. Results4. DiscussionReferences |
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Background: Aldosterone plays a detrimental role in the pathology of chronic heart failure. An important mechanism resides in its ability to evoke extensive fibrosis in the heart. However, the locations of the aldosterone interaction sites responsible for triggering cardiac fibrosis are puzzling. Extra-cardiac aldosterone actions are known to contribute to cardiac fibrosis but whether local cardiac aldosterone actions are involved is unclear.
Aims: This study aimed to investigate whether local aldosterone actions contribute to cardiac fibrosis in vivo.
Methods: Saline treated male Wistar rats were intravenously (systemically) or intrapericardially (locally) infused for 8 weeks with 75 or 750 ng/h aldosterone to monitor end point left ventricular epicardial collagen levels (histology).
Results: Perivascular fibrosis was observed only at high dose aldosterone infusions and was not different for the administration routes. Regarding interstitial fibrosis however, clear differences between the administration routes were seen. Intrapericardial aldosterone infusions increased interstitial collagen levels 1.72x (P<0.05) at low dose, but –surprisingly– had no significant effect at high dose. In contrast, intravenous aldosterone had no significant effect at low dose but increased interstitial collagen 1.72x (P<0.05) at high dose.
Conclusion: Our data suggest that local cardiac aldosterone actions contribute to the development of left ventricular interstitial fibrosis.
Key Words: Aldosterone • Cardiac fibrosis • Heart failure • Intrapericardial application
Received April 25, 2008; Revised August 1, 2008; Accepted September 22, 2008
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1. Introduction |
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TopAbstract1. Introduction2. Material and Methods3. Results4. DiscussionReferences |
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Impressive clinical evidence exists, which shows that aldosterone plays a detrimental role in the pathogenesis of chronic heart failure and cardiovascular remodelling. Mortality of heart failure patients has been shown to correlate with plasma aldosterone levels [1] and furthermore to be greatly reduced by treatment with mineralocorticoid receptor antagonists [2,3]. The therapeutic successes of mineralocorticoid receptor antagonists in heart failure patients appear to be directly linked to the extent by which these drugs reduce cardiac collagen [4,5]. This suggests that cardiac fibrosis - which is considered a hallmark of heart failure related cardiac remodelling [6] - is a key mechanism via which aldosterone contributes to the pathogenesis of heart failure.
In the early nineties, Brilla and Weber [7] demonstrated that in saline treated rats, aldosterone infusion evoked extensive interstitial- and perivascular collagen deposition in the heart. Later, these observations were confirmed and extended by various groups (reviewed e.g. in [8-10]). In addition -although the mechanistic basis is unclear- it was shown that dietary NaCl intake is a crucial factor for the occurrence of cardiac fibrosis: aldosterone infusion was shown to cause cardiac fibrosis only if aldosterone levels are increased in an inappropriate fashion regarding NaCl intake [7-10]. Nevertheless, despite the well known intrinsic profibrotic properties of aldosterone and the recognized role of salt, it is still not completely understood where the interaction sites via which aldosterone promotes cardiac fibrosis are located.
Aldosterone has a range of effects in e.g. the kidneys, central nervous system and blood vessels. These effects include sodium and fluid retention, potassium and magnesium loss, increased vascular tone and stiffness, hypertension and sympathetic/parasympathetic imbalance [11] that -alone or in combination- change the workload of the heart and may indirectly contribute to aldosterone induced cardiac fibrosis. Young et al have shown that aldosterone induced cardiac fibrosis can occur independently of potassium loss, cardiac hypertrophy and hypertension [12] and in recent work also demonstrated that the mineralocorticoid induced cardiac fibrosis is preceded by vascular inflammation, oxidative stress and macrophage infiltration [13]. Weber and co-workers [14,15] postulated that an important contributing factor in the profibrotic effect of aldosterone resides in the fact that aldosterone induces magnesium loss in peripheral blood mononuclear cells, leading to an activation of these cells and a subsequent cascade of events, resulting in cardiac fibrosis. More recently Lal et al hypothesized that part of the profibrotic action of aldosterone in the heart is the result of its effects in the central nervous system [16]. Thus, the extra-cardiac actions of aldosterone do seem to contribute to its profibrotic effects in the heart.
On the other hand, since the heart is known to possess mineralocorticoid receptors [17-19], whereas aldosterone has been shown to exert a variety of effects in the heart itself [20] it seems likely that at least a part of the aldosterone induced cardiac fibrosis results from direct local cardiac actions of aldosterone. Studies with isolated cardiac fibroblasts are however not conclusive in this respect, as aldosterone was shown to either stimulate or to inhibit collagen production or to have no effect [10]. Similarly, studies in mice in which the mineralocorticoid receptor was overexpressed or knocked down are puzzling. Cardiospecific [21] or general [22] human mineralocorticoid receptor overexpression did not result in cardiac fibrosis, whereas partial knock-down [23] of the native cardiac mineralocorticoid receptor gene expression evoked extensive cardiac fibrosis, thus leaving open the questions of how aldosterone can act as a profibrotic compound and why mineralocorticoid receptor antagonists can reduce fibrosis in the heart and prevent aldosterone from being profibrotic.
The present study aimed to evaluate whether local actions of aldosterone in the heart play a role in the cardiac fibrosis caused by aldosterone. For this reason, saline treated Wistar rats were provided with catheters in the pericardial space as well as with intravenous catheters, allowing prolonged local (intrapericardial) or systemic infusions of aldosterone. Two doses of aldosterone were infused for eight weeks either intrapericardially or intravenously: a high dose (750 ng/h) which is a standard dose used by others [7,12] to induce cardiac fibrosis in saline treated rats and a 10 times lower (low) dose (75 ng/h). Since intrapericardial drug application has been successfully used by our [24,25] and other groups to enhance the local cardiac effects of a variety of drugs (see e.g. [26]), this seems to be a suitable approach to reveal a possible contribution of local cardiac components of aldosterone action to the fibrosis in the heart.
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2. Material and Methods |
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TopAbstract1. Introduction2. Material and Methods3. Results4. DiscussionReferences |
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2.1. Animals
Male Wistar rats (280-320 g body weight) were obtained from Charles River The Netherlands. The animals were housed at the animal facilities of the University of Maastricht with a 12 h light/dark cycle and received regular rat chow and saline in tap water (0.9% NaCl) ad libitum. Experiments were performed according to institutional guidelines, that conform 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 have been approved by the local ethics committee for the use of experimental animals.
2.2. Surgical Procedure
All rats (including control rats) underwent the same surgical procedures. To enable intrapericardial or intravenous substance infusions, all rats (5 groups, 7-9 rats per group) were provided with intrapericardial and intravenous catheters, to which s.c. implanted osmotic minipumps were connected, as previously described [24]. Briefly, for the intrapericardial catheter insertion, rats were anaesthetized with sodium pentobarbitone (60 mg/kg intraperitoneal) and placed on a heating pad, kept at 37 °C. A high midline thoracotomy was performed and a retractor applied. Following careful cleavage of the sternohyoid muscle, thymus lobules were separated to expose the pericardium. The pericardial sac was opened by making a small incision (1-2 mm) with iris scissors in order to place the catheter in the pericardial space. Subsequently, the pericardial sac was closed by sealing it to the thymus with histoacryl tissue glue (Braun Melsungen Germany). After removing the retractor, the catheter was guided to the neck and externalized via a small incision in the skin. Osmotic minipumps (Alzet 2004, Durect, Cupertino, USA) were connected to the catheters and placed subcutaneously. These pumps provided a constant infusion rate (0.25 µl/h) for at least 4 weeks. After 4 weeks of treatment the minipumps were exchanged with new ones.
2.3. Treatment
Total treatment time was 8 weeks for all animals. During this time, control rats were continuously infused with vehicle (40% polyethylene glycol in water; 6 µl/day) via both catheters. Experimental groups obtained continuous infusions (at 6 µl/day) with: —1) a low dose (0.075 µg/h) of aldosterone intravenously and vehicle intrapericardially or —2) a low dose (0.075 µg/h) of aldosterone intrapericardially and vehicle intravenously or —3) a high dose (0.75 µg/h) of aldosterone intravenously and vehicle intrapericardially or —4) a high dose (0.75 µg/h) of aldosterone intrapericardially and vehicle intravenously.
2.4. Echocardiography
At the end of the treatment, transthoracic echocardiography was performed under isoflurane anaesthesia in some of the rats (5-7 per group) using the SONOS 5500 echocardiographic system equipped with a 15-MHz linear-array transducer (Philips Medical Systems Corp., Netherlands). In brief, rats were anaesthetized with 2-3% isoflurane (Abbott Laboratories, Kent, UK) placed on a heating pad, kept at 37 °C with transducer placed on the left hemithorax. The 2-dimensional parasternal long- and short-axis view of the left ventricle (LV) and the parasternal short-axis M-mode tracings were recorded.
Measurements and calculations performed are as follows: percent LV fractional shortening (FS) was calculated as: FS=(LVIDd–LVIDs)/LVIDdx100%, where LVIDd and LVIDs are end-diastolic and end-systolic LV internal dimensions, respectively. End-diastolic (EDV) and end-systolic volumes (ESV) were calculated from LV systolic (LVAs) and diastolic (LVAd) areas via the method of discs. Ejection fraction (EF) was calculated from systolic and diastolic volumes with the formula: EF=(EDV–ESV)/EDVx100%. Other measurements include heart rate (HR-derived from the M-mode by determining the RR interval; M-mode R-R interval), stroke volume (SV=EDV–ESV) and cardiac output (CO=SVxHR).
2.5. Mean Systolic Blood Pressure Measurement
Mean arterial blood pressures and heart rates were measured at day 56 of treatment in conscious rats as previously described [27].
2.6. Measurement of LV Epicardial Fibrosis
Hearts were harvested for histological analysis in order to determine LV epicardial fibrillar interstitial and perivascular collagen by Picro Sirius Red staining and subsequent light microscopy.
For this purpose, the apical parts of the hearts were embedded in Tissue-tek and frozen in freezing 2-methyl-butane and subsequently stored at –80 °C until cutting histological sections. Cryostat cross-sections (6 µm) were prepared, air dried, fixed in 10% buffered formalin for 1 h and washed for 30 min in PBS, followed by 5 min in distilled water. After 5 min in 0.2% phosphomolybdic acid for prevention of background staining all sections were stained for 90 min with 0.1% Sirius Red (Polyscience, Warrington PA, USA) in saturated picric acid. Before covering with Entellan (Merck, Darmstadt Germany) all sections were rapidly dehydrated by subsequent sequential dipping in 70 v/v, 96 v/v ethanol/milliQ, and 100% ethanol, followed by xylene.
Interstitial fibrosis (fibrosis in the interstitial space) and perivascular fibrosis (fibrosis around the coronary arteries) were determined separately in the epicardial part of the LV. Collagen volume fraction of each section was determined using a computer image analysis system (Leica Q-Win). Interstitial collagen density of heart sections was evaluated based on the average of ten randomly selected fields (containing no coronary arteries) per heart (magnificationx20) and the stained area was calculated as percentage of the total tissue area within a field. Perivascular collagen was expressed as the ratio of the area of the collagen surrounding the coronary vessels (with diameters <150 µm) divided by the media area of the coronary arteries.
All analyses were performed in a blinded fashion.
2.7. Statistics
All data are expressed as mean±SEM. Statistical significance was assessed by one-way ANOVA followed by calculation of the LSD (least significant difference). Differences were considered significant at P<0.05.
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3. Results |
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TopAbstract1. Introduction2. Material and Methods3. Results4. DiscussionReferences |
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As shown in Table 1, bodyweights or lung/body weight ratios did not differ between the treatment groups. Cardiac hypertrophy (i.e. a higher heart/bodyweight ratio) was observed in the animals that received the high dose of aldosterone, both when the aldosterone was infused intravenously or intrapericardially. Low dose aldosterone infusions also resulted in higher heart/bodyweight ratios, but only if the aldosterone was applied intrapericardially. Kidney/body weight ratios and drinking water intake were similarly elevated in the animals that were infused with the high dose of aldosterone intravenously or intrapericardially, but were unchanged in the low dose aldosterone infused rats.
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Haemodynamic parameters were monitored on day 56 of treatment. In comparison to vehicle treated animals, mean arterial blood pressures (Fig. 1A) were significantly higher in animals that received intravenous and intrapericardial high dose aldosterone infusions, and were also slightly higher in animals that received an intrapericardial low dose aldosterone infusion. Low dose intravenously infused animals did not show significantly changed mean arterial blood pressures. Heart rates (Fig. 1B) were lower in all aldosterone infused rats and were similar for the intrapericardially or intravenously infused rats.
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Histological assessment of LV epicardial collagen levels showed that the treatments affected the perivascular (Fig. 2A) and the interstitial fibrosis (Fig. 2B) in a different manner. Substantial perivascular fibrosis was only observed in the animals that had received high dose aldosterone infusions either intrapericardially or intravenously. For the interstitial fibrosis, the observed pattern was more complex. For intravenously infused aldosterone, only the high dose resulted in significantly higher interstitial collagen levels. For intrapericardially infused aldosterone however, the low dose resulted in significantly higher interstitial collagen levels, whereas high dose treated animals did not display significantly elevated interstitial collagen levels.
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Echocardiography (Table 2), showed that the animals that received intravenous or intrapericardial high dose aldosterone infusions, displayed significantly larger inter ventricular septum wall thicknesses and lower heart rates under isoflurane anaesthesia, the latter being more pronounced (P<0.05) for the intrapericardially infused rats. Elevated LV end-diastolic volumes were observed in rats that were given the high dose of aldosterone intrapericardially, but not intravenously. No differences between the treatment groups were observed for the end-systolic inter ventricular septum wall thickness and volume, stroke volume, cardiac output, fractional shortening and ejection fraction.
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4. Discussion |
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TopAbstract1. Introduction2. Material and Methods3. Results4. DiscussionReferences |
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Although it is well established that aldosterone evokes extensive heart failure related cardiac remodelling and fibrosis, it is currently still unclear whether this is solely due to aldosterone acting at various extra-cardiac sites and thereby indirectly affecting cardiac architecture, or whether direct local cardiac aldosterone actions play a role as well.
Here we show that aldosterone induced LV epicardial interstitial fibrosis in a dose and administration route dependent manner. Intrapericardial aldosterone infusion increased LV epicardial interstitial collagen levels (by 72%, P<0.05) at a dose of 75 ng/h, which was ineffective when infused into the bloodstream. Similar observations were seen for heart/bodyweight ratios that were increased by the low dose of aldosterone only if it was applied intrapericardially. Thus, at low dose, locally but not systemically applied aldosterone induced cardiac hypertrophy and interstitial cardiac fibrosis, suggesting that aldosterone interacts with target sites within the heart itself and thereby promotes cardiac remodelling. Together with the observed changes in interstitial cardiac fibrosis, a moderate increase in blood pressure was observed in rats that received the intrapericardial low dose aldosterone infusions, but it is unclear how these two phenomena are related. Previously, others have shown that the profibrotic effects of aldosterone or the anti-fibrotic effects of mineralocorticoid receptor antagonists are not related to changes in blood pressure [7,12] and overall inspection of Figs. 1A and 2B fails to reveal a clear-cut parallel between blood pressure and the extent of interstitial fibrosis.
Interestingly, if the aldosterone dose was increased by a factor of ten, intrapericardial aldosterone infusion no longer resulted in significant increases in epicardial LV interstitial collagen levels. In contrast, intravenous infusion of this aldosterone dose elevated LV epicardial interstitial collagen by 72% (P<0.05), corroborating the findings of others who applied this dose systemically [7,12]. The contrasting results of the high versus the low dose of intrapericardially infused aldosterone on LV epicardial interstitial collagen deposition may either indicate that additional local mechanisms or receptors are activated at the high local dose of aldosterone, counteracting the intrinsic profibrotic effect of aldosterone, or that the high dose of intrapericardially infused aldosterone may have led to enhanced activity of cardiac or pericardial collagen degrading enzymes.
A similar difference between high and low doses of aldosterone has also been described by Sato et al for the glucose stimulated hypertrophic effect of aldosterone in isolated cardiomyocytes, which proved to be due to aldosterone activating glucocorticoid receptors next to mineralocorticoid receptors, with opposing effects [28]. Also regarding interstitial cardiac fibrosis, glucocorticoid receptor activation seems to have effects opposite to mineralocorticoid receptor activation in vivo, as we (manuscript in preparation) and others [29] have found that glucocorticoid agonists reduce interstitial fibrosis in rat hearts. Thus the ligand-mineralocorticoid receptor-ligand-glucocorticoid receptor balance seems to play an important role in determining interstitial collagen levels in the heart.
Comparison of the perivascular and interstitial LV epicardial collagen deposition (Fig. 2) reveals diverging patterns in response to the aldosterone administrations and shows that aldosterone can evoke interstitial fibrosis independently of perivascular fibrosis. Combined with the non-parallel changes in cardiac perivascular and interstitial fibrosis in response to corticosteroids reported by others [7], this indicates that both types of fibrosis are differentially regulated by mineralocorticoids. Unlike for the interstitial fibrosis, we did not find clear indications that local cardiac aldosterone effects might play a role in perivascular fibrosis.
Apart from effects on LV interstitial fibrosis, a difference between the administration routes for the high dose aldosterone infusions was also observed for LV end-diastolic volume, which was increased only by intrapericardially infused aldosterone, once more illustrating that local aldosterone actions are of importance for cardiac remodelling. However, none of the functional parameters that were assessed by echocardiography (fractional shortening, ejection fraction or cardiac output) were changed by any treatment.
A potential limitation of the current study seems to reside in the fact that intrapericardially applied substances will leak into the circulation, making it impossible to exclude that peripheral actions of the locally infused aldosterone contribute to its cardiac effects. Indeed, this leaking of the aldosterone into the circulation is reflected in the water intake and kidney weights that were similarly elevated by the intrapericardial and intravenous high dose aldosterone infusions, indicating that both application routes result in similar extra-cardiac aldosterone actions. In this respect (next to the different animal species), our experimental model differs fundamentally from the mouse models that were designed to clarify the local cardiac actions of aldosterone by cardiospecific overexpression of the mineralocorticoid receptor [21] or aldosterone synthase [30] and in which no fibrosis was detected in the heart. A number of studies have shown that aldosterone per se cannot induce cardiac fibrosis, but needs to be combined with an increased salt intake [7-10]. Furthermore, activation of peripheral blood mononuclear cells [14,15] and central mineralocorticoid receptors [16] were reported to be important determinants in the profibrotic effects of aldosterone in the heart. Hence, extra-cardiac factors are of importance for the aldosterone induced cardiac fibrosis. Clearly, intrapericardial aldosterone will exert peripheral actions that may contribute to the cardiac fibrosis but the differences that we observed in the effects of intrapericardially or intravenously administered aldosterone on LV interstitial fibrosis demonstrate that local factors do play a role in the profibrotic effect of aldosterone in the interstitial space of the heart.
Another potential limitation of the study is that it does not provide mechanistic information on the sequence of molecular events that are triggered by aldosterone, but it should be stressed that this was not our initial study-aim. Mineralocorticoid receptor activation induces early tissue remodelling, vascular inflammation and oxidative stress and triggers a variety of cellular signalling pathways in the heart, while cardiac fibrosis is a rather late event [13]. How far these processes are modulated by direct actions of aldosterone in the heart or by indirect extra-cardiac aldosterone actions still awaits further investigation.
To conclude, this study indicates that interaction of aldosterone with sites located in the heart itself contributes to aldosterone induced interstitial cardiac fibrosis. Local cardiac aldosterone actions probably act in concert with extra-cardiac aldosterone actions in causing interstitial fibrosis in the heart.
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Acknowledgement |
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This work was supported by a grant from the Dutch Heart Foundation (Nederlandse Hartstichting; grant number 2001B054).
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