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Endocrinology Vol. 145, No. 7 3135-3142
Copyright © 2004 by The Endocrine Society

Direct and Indirect Effects of Aldosterone on Cyclooxygenase-2 and Interleukin-6 Expression in Rat Cardiac Cells in Culture and after Myocardial Infarction

Michela C. Rebsamen, Emeline Perrier, Christine Gerber-Wicht, Jean-Pierre Benitah and Ursula Lang

Division of Endocrinology and Diabetology (M.C.R., C.G.-W., U.L.), University Hospital, CH-1211 Geneva, Switzerland; and Institut National de la Santé et de la Recherche Médicale U390-EA 3759 (E.P., J.-P.B.), CHU Arnaud de Villeneuve, 34295 Montpellier, France

Address all correspondence and requests for reprints to: Michela Rebsamen, Ph.D., Division of Endocrinology and Diabetology, University Hospital, 24 rue Micheli-du-Crest, CH-1211 Geneva 14, Switzerland. E-mail: rebsamenmichela{at}hotmail.com.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Aldosterone contributes to cardiac failure, which is associated with induction of inflammatory mediators. Moreover, aldosterone was shown to induce a vascular inflammatory phenotype in the rat heart. Using Western blotting and/or real-time RT-PCR, we examined the effect of aldosterone on the expression of the proinflammatory molecules, cyclooxygenase-2 (COX-2), and IL-6 in neonatal rat ventricular cardiac myocytes and fibroblasts as well as in adult cardiomyocytes after myocardial infarction. In cardiomyocytes, aldosterone induced COX-2 but not IL-6 expression. After 4–18 h of stimulation with 1 µM aldosterone, a significant increase in COX-2 protein expression was observed, preceded by an increase of COX-2 mRNA levels. After 18 h treatment, 100 nM and 1 µM aldosterone increased COX-2 protein amount by 2- and 4-fold, respectively. Consistently, aldosterone increased by 2.5-fold prostaglandin E2 secretion in cardiomyocytes. In cardiac fibroblasts, aldosterone increased neither COX-2 nor IL-6 mRNA expression. Interestingly, prostaglandin E2 (100 nM) strongly induced both proinflammatory molecules in fibroblasts and cardiomyocytes. Our results indicate that aldosterone directly induces COX-2 expression in cardiomyocytes and suggest that the subsequent increase in prostaglandin secretion may act in an autocrine and/or paracrine manner inducing in turn COX-2 and IL-6 expression. In vivo, myocardial infarction strongly increased both COX-2 and IL-6 expression in ventricular cardiomyocytes. Administration of the aldosterone antagonist RU28318 completely prevented COX-2 induction by infarction and partially inhibited the increase in IL-6 mRNA. These data suggest that after myocardial infarction, mineralocorticoid receptor activity is responsible for COX-2 induction and indirectly participates in IL-6 expression in cardiomyocytes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
BESIDES ITS WELL-ESTABLISHED role in cardiovascular homeostasis via its effects on sodium retention and blood volume, aldosterone exerts direct adverse effects on the heart that are independent of blood pressure (1). Mineralocorticoid receptors (MRs), which mediate the action of aldosterone, are present in various nonepithelial tissues including the heart (2). Activation of these receptors by aldosterone in the context of inappropriate salt status has been increasingly reported, and clinical studies on aldosterone and MR antagonists have pointed out their role in heart failure and other cardiovascular disorders (3). In the Randomized Aldactone Evaluation Study (4), and the Eplerenone Postacute myocardial infarction Heart Failure Efficacy and Survival Study (5), the MR antagonists spironolactone and eplerenone were shown to reduce mortality (by 30 and 15%, respectively) in patients with congestive heart failure without affecting blood pressure. In rat models of primary and secondary hypertension, aldosterone receptor antagonism with either spironolactone or eplerenone prevents aortic and myocardial fibrosis even in the absence of effect on blood pressure (6, 7, 8). Furthermore, during high salt intake, prolonged aldosterone administration causes myocardial fibrosis and ventricular hypertrophy in rats (9, 10).

Aldosterone was also shown to be involved in inflammatory responses. For instance, studies in rats indicate that aldosterone plays a major role in angiotensin II-induced vascular inflammation in the heart (11). Furthermore, aldosteronism is associated with an activation of peripheral blood mononuclear cells and with oxidative and nitrosative stress, responses that precede coronary vascular lesions (12, 13). In a similar context, aldosterone/salt treatment was shown to induce coronary inflammation, characterized by monocyte and macrophage infiltration and increased expression of the inflammatory markers cyclooxygenase-2 (COX-2), osteopontin, macrophage chemoattractant protein-1, and intracellular adhesion molecule-1 (14). All these findings suggest a potential link between aldosterone and inflammatory molecules in the physiopathology of the heart. However, the potential contribution of cardiac myocytes and fibroblasts in the production of proinflammatory markers in response to direct aldosterone stimulation has not been studied.

Inflammatory responses are involved in the complex repairing process after myocardial infarction (15). In this context, myocardial expression of cytokines may contribute to the pathogenesis of heart failure (16). Interestingly, aldosterone production is increased in left ventricle of rats after myocardial infarction (17). Similarly, aldosterone levels have been found to increase in some patients with acute myocardial infarction and to be implicated in left ventricular remodeling (18). Although increased cardiac expression of the proinflammatory markers COX-2 (19) and IL-6 (20) has been reported after myocardial infarction, the possible role of aldosterone in these responses has not been evaluated. To better understand the proinflammatory responses induced by aldosterone in the heart, the purpose of this study was, on the one hand, to determine whether aldosterone directly promotes COX-2 and IL-6 expression in cultured cardiac myocytes and/or fibroblasts. On the other hand, the potential contribution of aldosterone in the expression of these proinflammatory markers after myocardial infarction was assessed.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
Neonatal cardiac cells were isolated from 1- to 2-d-old Wistar rat ventricles by digestion with trypsin-EDTA and type 2 collagenase as previously described (21). Once the sequential digestions were terminated, the cells were pooled in DMEM (Invitrogen, Groningen, The Netherlands) supplemented with 10% fetal calf serum (Life Technologies, Inc., Basel, Switzerland), penicillin (100 U/ml), and streptomycin (10 µg/ml) and seeded in 90-mm petri dishes to allow selective adhesion of cardiac fibroblasts (22). Thereafter, cardiomyocytes were decanted from the plates and seeded in petri dishes (60 or 90 mm) or six-well culture plates (Costar, Cambridge, MA). The majority of cultured cardiomyocytes (>90%) began to contract spontaneously within 24–48 h of plating (30–50 beats/min). Cells were used on the third day of culture for all experiments described herein. In parallel, the adherent cardiac fibroblasts were grown in DMEM and used as confluent monolayers. Before stimulation, cardiomyocytes and fibroblasts were starved for 18–24 h in serum-free DMEM.

Animals
Myocardial infarction was induced in adult male Wistar rats (250–274 g) by left anterior coronary artery ligation, as previously described (23). Age-matched sham-operated rats were submitted to the same surgery except the coronary artery ligation. When appropriate, an osmotic minipump (Alzet 2ML4) was implanted sc at the time of the surgical anesthesia to deliver 50 µg/h of RU28318 a specific mineralocorticoid receptor antagonist (24). Three weeks after surgery, rats were killed and isolated cardiac ventricular myocytes were prepared using an enzymatic perfusion method previously described (25). To discard any modification due to the ischemia, only right ventricle that underwent hypertrophy (26) was finally dissected. This procedure yielded 80–90% quiescent rod-shaped myocytes. Cells were resuspended in 1 ml RNA Later (Sigma, St. Louis, MO) and stored at –80 C.

Western blotting
After incubation in serum-free DMEM, cells were washed with ice-cold PBS and lysed with 50 µl of the following buffer: Tris-HCl [50 mM (pH 7.4)], NaCl (150 mM), glycerol (10%), EDTA (2 mM), EGTA (2 mM), Triton X-100 (1%), ß-glycerophosphate (40 mM), Na3VO4 (200 µM), and NaF (50 mM) containing a cocktail of protease inhibitors (Roche, Mannheim, Germany). Proteins (20 µg) were separated by SDS-PAGE on an 8% acrylamide gel and blotted onto nitrocellulose membrane. Afterward, membranes were incubated for 2 h at room temperature with an antibody raised against COX-2 (Cayman Chemicals, Ann Arbor, MI). The blots were then washed and incubated for 1 h with a horseradish peroxidase-labeled antirabbit antibody (CovalAb, Oullins, France). Immunoreactive bands were visualized with a chemiluminescence kit (Amersham, Zürich, Switzerland) and quantified by densitometry.

Total RNA isolation and mRNA quantification
Total RNA from adult rat ventricular cardiomyocytes was extracted according to the TRIzol reagent protocol (Life Technologies, Inc.). Total RNA from cultured neonatal cardiac myocytes or fibroblasts was extracted using the RNeasy minikit (QIAGEN, Basel, Switzerland) according to the manufacturer’s instructions.

Total RNA (5 µg) was reverse transcribed using 800 U of Moloney murine leukemia virus reverse transcriptase (Invitrogen) in the presence of 0.3 U/µl RNAsin (Promega, Madison, WI), 7.5 µM of random primers ]oligo(dN)6[, 1.2 mM deoxynucleotide triphosphate, and 12 mM dithiothreitol.

The expression of RNAs for rat cyclophilin A (cyclo), COX-2, MR, IL-6, and TNF{alpha} was determined by quantitative real-time RT-PCR using a LightCycler (Roche Diagnostic, Rotkreuz, Switzerland) with the DNA Master SYBR Green I or Fast Start DNA Master SYBR Green I kits (Roche Molecular Biochemicals) as appropriate. Primers, used at a final concentration of 0.25 µM, were as follows: cyclo, sense 5'-AGCACTGGGGAGAAAGGATT-3', antisense 5'-CATGCCTTCTTTCACCTTCC-3', product size 291 bp; COX-2, sense 5'-CTGAGGGGTTACCACTTCCA-3', antisense 5'-TGAGCAAGTCCGTGTTCAAG-3', product size 209 bp; MR, sense 5'-GAGTTCCTTCCCACCTGTCA-3', antisense 5'-GTGACACCCAGAAGCCTCAT-3', product size 202 bp; IL-6, sense 5'-ACCACTTCACAAGTCGGAGG-3', antisense 5'-ACAGTGCATCATCGCTGTTC-3', product size 112 bp; and TNF{alpha}, sense 5'-GGTGATCGGTCCCAACAAGGA-3', antisense 5'-CACGCTGGCTCAGCCACTC-3', product size 173 bp. The adequacy of the different PCR products was verified by nucleic acid sequencing and agarose gel electrophoresis. All mRNA levels were normalized to cyclophilin A mRNA amounts, and results were expressed as a percentage of the control.

Prostaglandin E2 (PGE2) measurements
After cardiomyocytes’ stimulation in serum-free DMEM, the culture medium was collected and centrifuged at 10,000 rpm for 5 min. Supernatants (100 µl) were assayed by PGE2 high sensitivity immunoassay kit (R&D Systems Europe, Abingdon, UK) according to the manufacturer’s instructions. All measurements were made in duplicate and repeated in three separate experiments.

Drugs
PGE2, d-aldosterone, spironolactone, cycloheximide, actinomycin D, and RU486 were purchased from Sigma. NS-398 was from Cayman. RU28318 was generously provided by Aventis (France). PGE2, d-aldosterone, spironolactone, and RU486 were dissolved in ethanol, whereas cycloheximide, actinomycin D, and NS-398 were dissolved in dimethylsulfoxide. In each case we compared the effect of the agonist with its corresponding vehicle, and final concentrations are indicated in Results.

Statistical analysis
All values are expressed as mean ± SEM. Differences between groups were determined using two-tailed unpaired Student’s t tests. P < 0.05 was accepted as statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Aldosterone induces COX-2 expression in cultured cardiomyocytes
The effect of aldosterone on COX-2 expression was assessed by Western blotting as well as by quantitative real-time RT-PCR. The incubation of cultured neonatal rat ventricular cardiomyocytes with aldosterone induced a time-dependent increase in COX-2 amount. Indeed, after 4 h incubation with 1 µM aldosterone, the COX-2 protein level was increased by 220% over control, and the expression remained sustained for 18 h (P < 0.05, n = 3) (Fig. 1AGo). The increase in COX-2 protein expression was concentration dependent, and significant responses were observed using 100 nM aldosterone (P < 0.05, n = 3) (Fig. 1BGo).



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  		FIG. 1. Aldosterone induces COX-2 protein expression in cultured cardiomyocytes. A, Time-dependent increase in COX-2 expression induced by aldosterone (Aldo, 1 µM), compared with unstimulated cells (control). Shown are a representative Western blot and data generated from three separate experiments. B, Cells were stimulated for 18 h with increasing concentrations of aldosterone. C, Cardiomyocytes were stimulated for 18 h with 1 µM aldosterone (Aldo). Protein synthesis, transcription, and MR activity were inhibited preincubating cells for 30 min with cycloheximide (CHX, 5 µg/ml), actinomycin D (Actino, 2.5 µg/ml), or spironolactone (Spiro, 10 µM), respectively. D, Cells were stimulated for 18 h with 1 µM of aldosterone (Aldo). GR activity was inhibited preincubating cells for 30 min with RU486 (1 µM). A–D, COX-2 protein level was detected by Western blotting, and protein loading was determined by nonspecific bands (NS). Specific bands corresponding to COX-2 were quantified by densitometry. Experiments were performed three times, and results are expressed as a percentage of the control. *, P < 0.05, compared with control values.

 
To determine whether aldosterone-induced COX-2 expression involves protein and/or mRNA synthesis, cardiomyocytes were treated with 5 µg/ml cycloheximide or 2.5 µg/ml actinomycin D, respectively, 30 min before and during aldosterone stimulation. Cycloheximide and actinomycin D prevented aldosterone-induced COX-2 expression, indicating that gene transcription is involved in this response (P < 0.05, n = 3) (Fig. 1CGo). Consistent with these results, COX-2 mRNA expression was increased by aldosterone in a time- and concentration-dependent manner (Fig. 2Go). Indeed, a 5-fold increase in COX-2 mRNA was observed after 2 h stimulation (Fig. 2AGo), and a significant response was obtained using 100 nM aldosterone (P < 0.05, n = 3) (Fig. 2BGo). As expected, actinomycin D abolished aldosterone-induced COX-2 expression (Fig. 2CGo). The MR antagonist spironolactone prevented COX-2 protein (Fig. 1CGo) and mRNA (Fig. 2CGo) induction by aldosterone, indicating a MR-dependent pathway (P < 0.05, n = 3). Confirming the specificity of the response and eliminating a possible implication of the glucocorticoid receptor (GR), the specific GR blocker RU486 did not affect aldosterone-induced COX-2 protein expression (Fig. 1DGo) (P < 0.05, n = 3).



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  		FIG. 2. Aldosterone induces COX-2 mRNA expression in cardiomyocytes. A, Time-dependent increase in COX-2 expression induced by aldosterone (Aldo, 1 µM), compared with unstimulated cells (control). B, Cells were stimulated for 8 h with increasing concentrations of aldosterone. C, Cardiomyocytes were stimulated for 4 h with 1 µM of aldosterone (Aldo). Transcription or MR activity was inhibited in preincubating cells for 30 min with actinomycin D (Actino, 2.5 µg/ml) or spironolactone (Spiro, 10 µM), respectively. A–C, COX-2 mRNA level was determined by quantitative real-time PCR and normalized to cyclophilin A (cyclo) mRNA. Experiments were performed three times, and results are expressed as a percentage of the control. *, P < 0.05, compared with control values.

 
In contrast to what we observed in cardiomyocytes, aldosterone (1 µM, 4–24 h) did not significantly affect COX-2 expression in cardiac fibroblasts (Fig. 3AGo). To determine the relative abundance of MR mRNA levels in cultured cardiac cells, quantitative real-time RT-PCR experiments were performed. As shown in Fig. 3BGo, the MR mRNA level was approximately 5-fold higher in cardiac myocytes than in cardiac fibroblasts (P < 0.05, n = 4).



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  		FIG. 3. A, Aldosterone does not induce COX-2 expression in cardiac fibroblasts. Cells were incubated with aldosterone (Aldo, 1 µM) for the indicated periods of time. COX-2 protein level was detected by Western blotting and protein loading was determined by nonspecific bands (NS). Specific bands corresponding to COX-2 were quantified by densitometry. Experiments were performed three times, and results are expressed as a percentage of the control. B, Relative mRNA level of MR in cardiac myocytes and fibroblasts. MR mRNA levels in untreated cardiac myocytes (CM) and fibroblasts (Fibro) were determined by quantitative real-time RT-PCR and normalized to cyclophilin A (cyclo) mRNA. MR mRNA level in cardiomyocytes was set at 100%. *, P < 0.05, compared with cardiomyocytes.

 
We next investigated the possible effect of aldosterone on IL-6 expression in cardiomyocytes. Quantitative real-time RT-PCR was performed using the same cDNA samples tested for COX-2 expression in the time-course and concentration-dependent experiments illustrated in Fig. 2Go, A and B. In contrast to COX-2, aldosterone did not increase IL-6 expression in cardiomyocytes (data not shown, n = 3). Similarly, in cardiac fibroblasts aldosterone had no effect on IL-6 expression (data not shown, n = 3). Our results indicate that aldosterone increases COX-2 but not IL-6 expression in cardiomyocytes, whereas in cardiac fibroblasts neither of these two genes is induced by this steroid.

Aldosterone stimulates PGE2 secretion in ventricular cardiomyocytes
It is well established that induction of COX-2 by cytokines and growth factors leads to the production of a variety of eicosanoids including proinflammatory prostaglandins and thromboxane A2 (27). Investigating the possible consequences of the induction of COX-2 by aldosterone in the context of proinflammatory responses, we examined whether aldosterone stimulates the production of the prostaglandin, PGE2. To test this hypothesis, cardiomyocytes were stimulated with 1 µM aldosterone for 8 h, and PGE2 secretion in the culture medium was determined by immunoassay. As shown in Fig. 4Go, PGE2 release in aldosterone-treated cells was increased by 250% over control, reaching a concentration of approximately 1.8 nM (P < 0.05, n = 3). To determine whether aldosterone-induced PGE2 secretion results from COX-2 activation, cardiomyocytes were incubated with the COX-2-specific inhibitor NS-398 1 h before and during aldosterone stimulation. NS-398 prevented aldosterone-induced PGE2 production, indicating that COX-2 activity is responsible for this response (Fig. 4Go).



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  		FIG. 4. Aldosterone increases PGE2 secretion in cardiomyocytes. Cells were stimulated for 8 h with aldosterone (Aldo, 1 µM) or left untreated (control). To inhibit COX-2 activity, cardiomyocytes were preincubated for 1 h with NS398 (1 µM). Experiments were performed three times, and results are expressed as a percentage of the control (basal PGE2 secretion = 260 ± 44 pg/ml). *, P < 0.05, compared with control values.

 
PGE2 induces COX-2 and IL-6 expression in cardiac fibroblasts and myocytes
Because aldosterone induces PGE2 secretion in cardiomyocytes, we hypothesized that this prostaglandin may amplify aldosterone proinflammatory effects by acting in a paracrine manner on cardiac fibroblasts modulating gene expression. As assessed by quantitative real-time RT-PCR, stimulation of fibroblasts for 2 h with PGE2 (100 nM) induced COX-2 and IL-6 mRNA expression by 3- and 5-fold, respectively (P < 0.05, n = 4) (Fig. 5Go, A and C). Concentration-dependent experiments revealed an increase in COX-2 and IL-6 expression using 1 nM PGE2, a concentration of prostaglandin comparable with that reached in the culture medium of aldosterone-stimulated cardiomyocytes (P < 0.05, n = 4) (Fig. 5Go, B and D). To test for a possible autocrine effect of PGE2, similar experiments were performed on cardiomyocytes. Although PGE2 increased COX-2 expression in cardiomyocytes by 350% after 4 h of stimulation, significant results were obtained using only 100 nM of the prostaglandin (P < 0.05, n = 3–4) (Fig. 6Go, A and B). Compared with COX-2, the induction of IL-6 by PGE2 in cardiomyocytes was stronger and was significant using lower concentrations of the prostaglandin. Indeed, PGE2 increased IL-6 mRNA expression by approximately 3- to 10-fold after 2–8 h of stimulation, and 10 nM PGE2 induced a significant response (P < 0.05, n = 3–4) (Fig. 6Go, C and D). All the cDNA samples used in Figs. 5Go and 6Go were assessed by quantitative real-time RT-PCR to determine the expression of another inflammatory marker, TNF{alpha}. PGE2 increased TNF{alpha} expression in neither cardiac myocytes nor fibroblasts (data not shown, n = 3), indicating that PGE2 induces specific responses. Our data indicate that PGE2 induces COX-2 and IL-6 expression in cardiac myocytes and fibroblasts, suggesting that after aldosterone-induced COX-2 expression and PGE2 secretion in cardiomyocytes, an amplification of the proinflammatory response may occur in a paracrine and/or autocrine manner.



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  		FIG. 5. Time- and concentration-dependent increase in COX-2 and IL-6 expression induced by PGE2 in cardiac fibroblasts. Cells were stimulated with PGE2 (100 nM) for the indicated periods of time (A and C) or for 4 h with increasing concentrations of PGE2 (B and D). COX-2 (A and B) and IL-6 (C and D) mRNA levels were determined by quantitative real-time PCR and were normalized to cyclophilin A (cyclo) mRNA. Experiments were performed four times and results are expressed as a percentage of the control. *, P < 0.05, compared with control values.

 


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  		FIG. 6. Time- and concentration-dependent increase in COX-2 and IL-6 expression induced by PGE2 in cardiomyocytes. Cells were stimulated with PGE2 (100 nM) for the indicated periods of time (A and C) or for 4 h with increasing concentrations of PGE2 (B and D). COX-2 (A and B) and IL-6 (C and D) mRNA levels were determined by quantitative real-time PCR and were normalized to cyclophilin A (cyclo) mRNA. Experiments were performed three to four times, and results are expressed as a percentage of the control. *, P < 0.05, compared with control values.

 
Aldosterone is involved in COX-2 and IL-6 induction after myocardial infarction
To test the hypothesis that aldosterone plays a role in the expression of proinflammatory markers in the heart, we used an in vivo model of myocardial infarction (MI). It has been shown that cardiac aldosterone production is increased in this model (17). Right ventricular cardiomyocytes were isolated 3 wk after MI, and gene expression was determined by quantitative real-time RT-PCR. Compared with sham-operated animals, the expression of COX-2, IL-6, and TNF{alpha} was increased in cardiomyocytes after MI (P < 0.05, n = 4) (Fig. 7Go). To check the involvement of aldosterone in these MI-induced effects, we treated the animals with the specific MR antagonist RU28318. This treatment blunted the MI-induced COX-2 expression, whereas it partially inhibited that of IL-6 (Fig. 7Go, A and B). No effect of RU28318 treatment was observed in sham-operated rats (data not shown). Conversely, the increase in TNF{alpha} expression was not affected by RU28318, demonstrating the specific inhibitory effect of this MR antagonist (Fig. 7CGo). Taken together, our data indicate that aldosterone directly induces COX-2 in cardiomyocytes in vitro and after MI. They further suggest that aldosterone contributes to MI-induced IL-6 expression, although it does not directly increase IL-6 expression in cultured cardiomyocytes.



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  		FIG. 7. Role of aldosterone in the expression of COX-2, IL-6, and TNF{alpha} induced after MI. Right ventricular cardiomyocytes were isolated from heart of sham-operated rats (control) and 3 wk after MI. To inhibit MR activity, rats were treated with RU28318 (50 µg/h delivered by a sc osmotic minipump). COX-2 (A), IL-6 (B), and TNF{alpha} (C) mRNA levels were determined by quantitative real-time PCR and normalized to cyclophilin A (cyclo) mRNA. Results are expressed as a percentage of the control (n = 4 rats for each treatment). *, #, P < 0.05, compared with control or MI values, respectively.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we showed that aldosterone modulates proinflammatory genes expression in cardiac cells. We demonstrated that aldosterone directly induces COX-2 expression in cardiac myocytes, whereas it does not affect COX-2 levels in fibroblasts. Conflicting data concerning the presence of mineralocorticoid receptors in cultured rat cardiac fibroblasts have been published (28, 29, 30). We found that MR mRNA is significantly less abundant in cardiac fibroblasts than in myocytes. Taking into account this information, two hypotheses can explain the lack of aldosterone effect on COX-2 expression that we observed in cardiac fibroblasts. On the one hand, cardiac fibroblasts might be less responsive than myocytes to aldosterone stimulation. On the other hand, the effect of aldosterone on COX-2 induction might be cell specific. Supporting this hypothesis, previous studies reported that promoter elements, signal transduction pathways, and transcription factors involved in COX-2 expression vary according to the cell type, and their relative roles are often contradictory (31, 32).

Considering globally our in vitro studies, we propose the model illustrated in Fig. 8Go. Aldosterone induces COX-2 expression in ventricular cardiomyocytes leading to the subsequent secretion of PGE2. In turn, PGE2 (1 nM) strongly induces IL-6 and COX-2 expression in cardiac fibroblasts. On the other hand, PGE2 (10 nM) also acts in an autocrine manner on cardiomyocytes inducing IL-6. An autocrine effect of PGE2 on COX-2 expression is less probable because a higher PGE2 concentration (100 nM) is required to significantly increase COX-2 amount in cardiomyocytes. This model is in agreement with and can help the interpretation of our in vivo results. Indeed, we showed that MI induces COX-2 expression and that this response was prevented by RU28318, indicating a MR-dependent mechanism. These data are consistent with previous findings showing that aldosterone production in the heart is activated after MI (17) and show for the first time that MR activation is responsible for the induction of COX-2 observed in noninfarcted ventricular cardiomyocytes after MI. A potential role of aldosterone in vascular inflammation measuring various parameters including COX-2 expression has recently been reported (11, 14). An elevation of COX-2 mRNA in left ventricular tissue was reported during aldosterone/salt treatment in uninephrectomized rats (14). Moreover, COX-2 expression was increased after angiotensin II/salt treatment in medial cells of coronary arteries and macrophages in the perivascular space (11). In both reports the vascular inflammatory responses were reduced by the aldosterone blocker eplerenone. In contrast to what we observed after MI, in these in vivo models of experimental hypertension, COX-2 expression was not detected in cardiomyocytes. Although our results together with previous reports indicate that, in the cardiovascular system, the MR is involved in COX-2 induction in vivo and in vitro, the molecular mechanism underlying this response remains to be elucidated.



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  		FIG. 8. Proposed model of aldosterone effects on proinflammatory marker expression in cardiac cells. Aldosterone induces COX-2 expression in cardiomyocytes (1 ) leading to the subsequent secretion of PGE2 (2 ). In a paracrine manner, PGE2 (low concentration) induces IL-6 and COX-2 expression in the surrounding fibroblasts (3 ). On the other hand, in an autocrine manner, PGE2 (higher concentrations) also induces IL-6 expression in cardiomyocytes (4 ). Spiro, Spironolactone, aldosterone blocker. Filled arrows, aldosterone-induced responses; unfilled arrows, PGE2-induced responses.

 
Our findings further indicate that the expression of two additional proinflammatory molecules, namely IL-6 and TNF{alpha}, is induced after MI. Elevated levels of these cytokines have been reported in human heart failure (33) and the potential role of these cytokines in the development and progression of disease in the failing heart is a topic of current interest (34). However, the source of production for these cytokines in heart failure is still a matter of debate. Our results indicate that, after MI, cardiomyocytes of the noninfarcted ventricle express increased levels of IL-6 and TNF{alpha} mRNA. Although an increased mRNA expression does not always correlate with an increased protein secretion, our data suggest that cardiomyocytes may contribute to IL-6 and TNF{alpha} production after MI. However, RU28318 did not affect TNF{alpha} mRNA expression, excluding a role for MR activation in this response. Conversely, the aldosterone blocker reduced by half MI-induced IL-6 mRNA levels. These data indicate that after MI, IL-6 expression in cardiomyocytes is increased by aldosterone-dependent and -independent mechanisms. Concerning aldosterone-dependent mechanisms and taking into account our in vitro data, we propose that aldosterone indirectly modulates IL-6 expression in vivo by increasing local PGE2 production. Indeed, we showed that aldosterone does not induce IL-6 expression in cultured cardiomyocytes. However, aldosterone increases COX-2 expression and PGE2 secretion in cultured cardiomyocytes, and in turn PGE2 induces IL-6 expression in these same cells. In a recent publication, a contribution of COX-2 and PGE2 in inflammatory responses after MI was reported (35). It is therefore conceivable that an increase in prostaglandin production occurs in vivo after MI, in which aldosterone production and COX-2 expression are increased. Supporting a potential role of COX-2 products in inflammatory gene expression, previous work indicates that PGE2 stimulates the activity of the IL-6 promoter in osteoblasts (36) and that in macrophages IL-6 expression is regulated by PGE2 (37). Furthermore, Hinson et al. (38) demonstrated that PGE2 induces IL-6 production in an in vivo murine model of inflammation and suggested that the expression of COX-2 gene is involved in the regulation of this cytokine. Further in vivo studies aimed to evaluate the potential effect of selective COX-2 inhibition on MI-induced IL-6 expression will help to verify our model describing direct and indirect effects of aldosterone in the expression of proinflammatory markers after MI.

The present results also show that PGE2 induces COX-2 expression in cardiac fibroblasts and, at higher concentration, possibly also in myocytes, suggesting that IL-6 is not the unique proinflammatory gene induced by this prostaglandin. Similarly, up-regulation of COX-2 expression by PGE2 was previously described in endometrial stromal cells (39). Although we clearly demonstrate that PGE2 induced IL-6 and/or COX-2 expression in cardiac myocytes and fibroblasts, we cannot exclude that other eicosanoids are produced by cardiomyocytes in response to aldosterone and may contribute to inflammatory responses. Supporting our hypothesis that COX-2 plays a major role in initiating and amplifying inflammatory cascades, Anderson et al. (40) reported that COX-2-derived PGE2 up-regulates COX-2 and IL-6 expression at inflammatory sites in rat adjuvant arthritis. Furthermore, PGE2 was shown to play a role in responses induced by the proinflammatory mediator, bradykinin (41). In particular, the induction of COX-2 by bradykinin in human pulmonary artery smooth muscle cells was reported to be mediated by an autocrine loop involving endogenous PGE2 (42). Although in our in vivo experiments only cardiomyocytes were considered, allowing us to verify the autocrine part of our proposed model, we can expect that the paracrine responses that we suggest occur in vivo. Indeed, we showed that, compared with cardiomyocytes, in cardiac fibroblasts lower concentrations of PGE2 strongly induced IL-6 and COX-2 expression.

Overall, our study provides new insights on aldosterone effects in the heart, possibly contributing to cardiac remodeling. In particular, aldosterone induces the expression of proinflammatory markers in cardiac cells, which may mediate the development of hypertrophy and fibrosis via complex autocrine and paracrine networks. Supporting this hypothesis, IL-6 was shown to induce hypertrophic responses in cardiomyocytes and stimulate fibroblasts proliferation (43, 44).


   Acknowledgments
 
We thank M. Rey, R. Perrier, and P. Bideaux for their excellent technical assistance for myocytes isolation and animal surgery.


   Footnotes
 
This work was supported by Swiss National Science Foundation Grant 31-063799.00, the Swiss University Conference, the Foundation Prévot, and the Foundation Rentenanstalt/Swiss Life.

Abbreviations: COX, Cyclooxygenase; GR, glucocorticoid receptor; MI, myocardial infarction; MR, mineralocorticoid receptor; PGE2, prostaglandin E2.

Received November 12, 2003.

Accepted for publication March 18, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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