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Endocrinology Vol. 144, No. 5 1861-1868
Copyright © 2003 by The Endocrine Society

Expression and Modulation of Steroidogenic Acute Regulatory Protein Messenger Ribonucleic Acid in Rat Cardiocytes and after Myocardial Infarction

Andrés J. Casal, Jean-Sébastien Silvestre, Claude Delcayre and Alessandro M. Capponi

Division of Endocrinology and Diabetology, University Hospital, CH-1211 Geneva 14, Switzerland; and Institut National de la Santé et de la Recherche Médicale, Unité-127, Hôpital Lariboisière (J.S.S., C.D.), 75475 Paris, France

Address all correspondence and requests for reprints to: Prof. A. Capponi, Division of Endocrinology and Diabetology, University Hospital, 24 rue Micheli du Crest, CH-1211 Geneva 14, Switzerland. E-mail: alessandro.capponi{at}medecine.unige.ch.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We examined whether the mRNA for steroidogenic acute regulatory (StAR) protein, a crucial factor in the rate-limiting step of aldosterone biosynthesis, is expressed and regulated in rat heart. We performed quantitative RT-PCR for StAR mRNA in an in vitro and an in vivo model: purified rat neonatal cardiomyocytes in primary culture and myocardial infarction (MI) in the rat. StAR mRNA was expressed in cultured cardiomyocytes, and angiotensin II (10 nM) increased it in a time-dependent fashion (132 ± 2.7% of controls after 24 h; n = 3; P < 0.05). Concomitantly, angiotensin II stimulated aldosterone production in the culture medium from 32.6 ± 6.1 to 54 ± 12.7 fmol/mg protein after 24 h (n = 8; P < 0.05). StAR mRNA levels in cardiomyocytes were dramatically reduced after 24-h treatment with dexamethasone in a concentration-dependent manner (50% inhibitory concentration, 10 nM); maximal inhibition (to 15 ± 6% of control; P < 0.001; n = 6) was achieved with 100 nM dexamethasone. This inhibition was prevented by RU486. In the rat MI model, StAR mRNA was also present in control heart tissue and was increased 2.4-fold in the noninfarcted area of the left ventricle after MI (n = 6; P < 0.01). This effect was completely prevented by treatment with losartan (8 mg/kg·d) and spironolactone (80 mg/kg·d), which reduced StAR mRNA levels to values not different from those in non-MI controls. Thus, the mRNA for an indispensable factor in aldosterone biosynthesis, the StAR protein, is expressed in the rat heart and is up-regulated after MI. These results support the view of a local synthesis of aldosterone in the heart and of intracrine/paracrine deleterious effects of the mineralocorticoid in heart failure.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE MAIN mineralocorticoid, aldosterone, is synthesized primarily in the zona glomerulosa of the adrenal cortex under the control of two major physiological stimuli, the octapeptide hormone angiotensin II (Ang II) and extracellular potassium (K+) (1). Aldosterone is synthesized from cholesterol, the common precursor of all steroid hormones, which is stored within intracellular lipid droplets and mobilized to the mitochondrion upon stimulation. The rate-limiting step in the activation of steroidogenesis is the delivery of cholesterol from the mitochondrial outer membrane to the inner membrane, where the cytochrome P450 side-chain cleavage (P450ssc) enzyme is located (2). Within the mitochondria, cholesterol undergoes an enzymatic cascade, leading eventually to the formation of aldosterone.

This crucial step of intramitochondrial cholesterol transfer is facilitated by the 30-kDa steroidogenic acute regulatory (StAR) protein (3, 4). Confirmation of the indispensable role of the StAR protein came from congenital lipoid adrenal hyperplasia, an inherited disease that leads to a dramatic deficiency in all steroid hormones and in which loss of function mutations have been discovered in the StAR gene (5).

Recent in vitro and in vivo studies have reported that extraadrenal tissues, such as blood vessels (6), brain (7), and, in particular, heart (8, 9, 10), are able to produce aldosterone. An increase in cardiac aldosterone production as well as in aldosterone synthase mRNA expression has been observed after experimental myocardial infarction (MI) in the rat, and this increase is mediated by Ang II acting via the cardiac AT1 receptor subtype (9, 11). Moreover, in the rat subjected to high sodium intake, cardiac aldosterone production and activity of aldosterone synthase are increased (12). In man, recent studies have shown that plasma aldosterone levels are higher in interventricular vein and coronary sinus than in aortic root in patients with failing ventricles (13), and that some steroidogenic genes can be detected in the heart (14, 15), suggesting possible endogenous aldosterone synthesis, at least in the failing heart. However, whether the aldosterone produced in cardiac tissue results from active cholesterol transformation as in the adrenal cortex or is merely the result of extraction from blood of aldosterone and/or some of its precursor(s) (16), such as pregnenolone, progesterone, or corticosterone, remains to be determined.

In addition to being a potential source of aldosterone, the heart appears to act as a target for the mineralocorticoid. Indeed, mineralocorticoid receptors are present in cardiac myocytes and fibroblasts (17). Aldosterone treatment, for example, induces left ventricular (LV) hypertrophy (18), fibrosis, and an increase in ventricular collagen I and III mRNAs within 2 wk (19). This delay may be shortened, however, because a bolus injection of deoxycorticosterone induces an increase in collagen III in only 2 d, probably due to the high hormone concentration acutely reached under those conditions (19, 20). Moreover, the expression of some genes of the renin-angiotensin system is also modulated by aldosterone in the heart, such as genes for the AT1 receptor (19) and angiotensin-converting enzyme (21). All of these effects can be prevented by simultaneous administration of spironolactone, an aldosterone antagonist. Finally, strong indirect evidence for an active involvement of aldosterone in heart failure was recently provided by the RALES trial, in which a 30% improvement in mortality risk was observed in patients with severe heart failure who received spironolactone in addition to classical treatment (22).

In view of the possibility of local cardiac aldosterone biosynthesis and potential intracrine/paracrine deleterious actions of the mineralocorticoid in states of heart failure, it is of crucial importance to identify the pathway leading to aldosterone. In this context, we examined in the present work whether the rat heart expresses the rate-limiting and regulated step of cholesterol transfer to the mitochondria, which involves the StAR protein, in an in vitro system, rat neonatal cardiomyocytes in primary culture, and an in vivo model, MI in the adult rat obtained by ligature of the left anterior descending coronary artery.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
LV infarction was produced in 3-month-old male Wistar rats (Iffa Credo, Lyon, France) by ligation of the left anterior descending coronary artery as described previously (11). Sham-operated animals were treated similarly, except that the ligature around the coronary artery was not tied. Seven days after coronary ligation, animals were randomly divided into three groups (n = 6/group); each group received one of the following treatments as previously described (11): 1) untreated MI, 2) losartan (Ang II receptor blockade, 8 mg/kg·d in drinking water; Merck \|[amp ]\| Co., Inc., Rahway, NJ), and 3) spironolactone (aldosterone receptor blockade, 80 mg/kg·d in drinking water; Sigma-Aldrich Corp., St. Louis, MO). Noninfarcted and nontreated sham-operated animals served as a control group.

After 25 d of treatment, hearts were excised and immediately dropped into an ice-cold NaCl 0.9% buffer to wash out plasma components. The infarct scar, including the border zone, was then removed from the heart. In sham-operated animals, corresponding parts of the heart were discarded. The LV, including the septum, was separated from the right ventricle in the remaining heart and stored at -70 C until use.

Cell culture
Primary cultures of spontaneously beating, neonatal rat ventricular cardiomyocytes were obtained from 1–2-d-old Wistar rats by the trypsin/deoxyribonuclease sequential digestion method as previously described (23). Briefly, ventricular heart tissue was washed with 40 ml Hanks’ Balanced Salt Solution, cut into small pieces, further washed with 10 ml Hanks’ Balanced Salt Solution, and enzymatically digested for 8 min with 10 ml trypsin/deoxyribonuclease (2. and 0.3 mg/ml, respectively) at 37 C in a 50-ml sterile conical tube subjected to constant stirring. The supernatant from the first incubation was discarded, 10 ml fresh enzyme solution were added, and the incubation procedure was repeated. Subsequent supernatants were collected and centrifuged at 1200 rpm for 4 min, and the resulting cell pellets were resuspended in McCoy’s medium containing 10% fetal calf serum at 37 C. Once the sequential digestions were terminated, the cells were pooled in McCoy’s modified 5A medium containing 10% fetal calf serum, 1% insulin/transferrin/sodium selenite medium supplement, 100 IU/ml penicillin, and 10 mg/ml streptomycin and seeded in 90-mm petri dishes. After 3 h of incubation, the petri dishes were shaken, and the supernatants containing the cardiomyocytes were pooled and seeded in 90-mm petri dishes or six-well culture plates (Costar, Cambridge, MA). Most of the cultured cells (i.e. >90%) began to contract spontaneously within 24–48 h of plating (30–60 beats/min). Confluent, spontaneously beating cells were used on d 3 of culture for all experiments described herein.

Total RNA extraction
Total RNA from hearts was extracted according to the TRIzol reagent protocol (Life Technologies, Inc., Basel, Switzerland). The yields of total RNA extracted were similar in sham-operated rats and in treated and untreated MI rats. Total RNA from cultured neonatal cardiomyocytes was extracted according to the RNAgents reagent protocol (Promega Corp., Madison, WI).

Quantitative RT-PCR
To determine the relative abundance of StAR mRNA in heart tissue as well as in neonatal cardiomyocytes, we used quantitative RT-PCR. Total RNA (500 ng) was amplified by one-step RT-PCR using the following gene-specific primers: sense, 5'-AGCTCCTACAGACATATGCGG-3' (position 93–113, exon I); and antisense, 5'-CACAGGTGGAACCTCTACGC-3' (position 648–629, exon V). Primers, selected according to the sequence published by Ariyoshi et al. (24), amplified a 563-bp PCR product. PCR conditions were such that the reaction was performed within the linear range of amplification. One-step RT-PCR was carried out in 20 mM Tris-HCl, pH 7.5, containing 100 mM KCl, 1.5 mM MgCl2, 5 mM dithiothreitol, 0.2 mM deoxy-NTPs, 0.4 µM of each primer, 10 U ribonuclease inhibitor (Roche, Mannheim, Germany), and 1 µl avian myeloblastosis virus reverse transcriptase/Taq/Pwo blend (Roche). RT was performed at 55 C for 30 min, followed by 30 PCR cycles according to the following scheme: 1 min of denaturing at 94 C, 1 min of annealing at 57 C, and extension for 30 sec at 68 C. After the last cycle, a final extension was performed at 68 C for 10 min. PCR products were analyzed on 1.2% agarose gel and quantitated by densitometry using a Molecular Dynamics, Inc. (Sunnyvale, CA) computing densitometer. All mRNA levels were normalized to glyceraldehyde-3-phosphate dehydrogenase mRNA, and results were expressed as a percentage of the control. All PCR products were checked by sequencing.

Southern blot analysis
For Southern hybridization, PCR products were separated on 1.2% agarose gel. After migration, the gel was treated for 30 min with 250 mM HCl, then with denaturing solution (1.5 M NaCl and 0.5 M NaOH) for an additional 30 min. The treated gel was then vacuum-transferred onto a Hybond-N+ nylon membrane (Amersham Pharmacia Biotech, Dubendorf, Switzerland) and fixed by UV cross-linking. Hybridization was performed using the previously cloned 1.5-kb mouse StAR cDNA (3). The cDNA was labeled with [32P]deoxy-CTP using the Rediprime random primer labeling kit from Amersham Pharmacia Biotech. Southern blots were prehybridized in Rapid Hybridization Buffer (Amersham Pharmacia Biotech) at 65 C for 30 min. The 32P-labeled probe (specific activity, 2 x 106 cpm/ng DNA) was then added, and the incubation was continued for 2 h at 65 C. Blots were washed for 5 and 15 min successively at room temperature in 2x saline sodium citrate (SSC)/0.1% sodium dodecyl sulfate, and then for 15 min in 1x SSC/0.1% sodium dodecyl sulfate. The final wash was performed at 65 C for 15 min in 1x SSC/0.1% sodium dodecyl sulfate. Hybridization was visualized on Hyperfilm (Amersham Pharmacia Biotech) and quantitated by densitometry as described above.

Western blot analysis
Spontaneously beating, ventricular cardiomyocytes were exposed to various agonists for the indicated periods of time. The cell monolayers were then washed twice with cold PBS (137 mM NaCl, 1.47 mM KH2PO4, and 8.9 mM Na2HPO4, pH 7.4) and scraped into 500 µl lysis buffer containing 50 mM Tris-HCl, 1% Triton X-100, 150 mM NaCl, 10% glycerol, 2 mM EDTA, 2 mM EGTA, 40 mM ß-glycerophosphate, 50 mM NaF, 10 mM sodium pyrophosphate, 200 mM Na3VO4, 0.3 mM leupeptin, 1 mM pepstatin A, 1 mM phenylmethylsulfonylfluoride, and 100 nM okadaic acid, pH 7.4. The homogenates were centrifuged for 10 min at 10,000 x g at 4 C, and supernatants were collected. Cell lysates (20 mg) were analyzed by SDS-PAGE at 150 V for 1 h. After transfer, the nitrocellulose membranes were incubated in a blocking buffer (50 mM Tris-HCl, 200 mM NaCl, 0.2% Tween 20, and 5% nonfat dried milk) for 1 h at room temperature, then for 2 h in the same buffer containing 1% nonfat dried milk with a polyclonal antibody raised against phosphorylated p42/44 MAPK (New England Biolabs, Inc., Beverly, MA) or total p42/44 MAPK (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The membranes were washed with the same buffer without milk and then incubated for 1 h with horseradish peroxidase-labeled goat antirabbit (CovalAb, Oullins, France) or rabbit antigoat (Sigma-Aldrich Corp.) antibodies. After washing six times for 10 min each time, the immunoreactive bands were visualized by enhanced chemiluminescent detection reagent (Amersham Pharmacia Biotech) and quantified by densitometry as described above.

Determination of aldosterone production
Steroids were extracted from the incubation medium on Sep-Pak C18 cartridges (Waters Corp., Milford, MA) according to the manufacturer’s protocol. The aldosterone content in the extracts was measured by direct RIA using a commercially available kit (Diagnostic Systems Laboratories, Inc., Webster, TX).

Analysis of data
Results are expressed as the mean ± SEM. The mean values were compared by one-way ANOVA using Fisher’s test. A value of P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
StAR mRNA levels in cardiomyocytes and modulation by Ang II and aldosterone
We first examined whether neonatal rat cardiomyocytes in primary culture express StAR mRNA. Indeed, as shown in Fig. 1AGo, StAR mRNA was detected after RT-PCR amplification of RNA from control rat cardiomyocytes. Moreover, as shown in Fig. 1Go, A and B, treatment with 10 nM Ang II led to a time-dependent increase in the StAR mRNA level, reaching 132 ± 2.7% of the control value after 24 h (n = 3; P < 0.05). Cultured cardiomyocytes produced detectable amounts of aldosterone in the incubation medium (32.6 ± 6.1 fmol/mg protein·24 h), and Ang II challenge for 24 h increased aldosterone production 1.6-fold to 54 ± 12.7 fmol/mg·24 h (n = 8; P < 0.05; Fig. 1CGo). Interestingly, after simultaneous treatment with 1 µM spironolactone, a specific aldosterone receptor antagonist, there was a consistent trend toward a more pronounced increase in StAR mRNA levels (Fig. 1BGo). The latter result suggested that in cultured cardiomyocytes, aldosterone might exert a negative control over StAR mRNA expression.



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  		Figure 1. Effect of a Ang II (10 nM) treatment on StAR mRNA expression in cultured neonatal rat cardiomyocytes. A, Representative ethidium bromide staining of an agarose gel after 30 cycles of RT-PCR. Cells were treated with 10 nM Ang II for the indicated periods of time. B, Effect of Ang II (10 nM, 24 h), in the absence or presence of spironolactone (spiro; 1 µM), on StAR mRNA levels in cultured neonatal rat cardiomyocytes (n = 3). C, Aldosterone production by cultured cardiomyocytes in response to Ang II (10 nM, 24 h). Values are the mean ± SEM (n = 8 separate experiments). *, P < 0.05; **, P < 0.01 (vs. controls).

 
To test this hypothesis, we incubated neonatal rat cardiomyocytes with increasing concentrations of aldosterone for 24 h and measured StAR mRNA levels by RT-PCR (Fig. 2AGo). Indeed, aldosterone treatment led to a concentration-dependent decrease in StAR mRNA expression; a maximal inhibition to 30 ± 4.4% of the control value (n = 3; P < 0.01) was reached with 10 µM aldosterone. This inhibitory effect of aldosterone was completely prevented by 10 µM spironolactone (98 ± 1.7% of controls; n = 3; P = NS; Fig. 2Go, A and B), a finding consistent with a genomic effect via the mineralocorticoid receptor. However, as the glucocorticoid receptor antagonist, RU486, also blocked aldosterone-induced repression of StAR mRNA (Fig. 2Go), cross-reaction with the glucocorticoid receptor was expected.



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  		Figure 2. Effect of aldosterone treatment (24 h), with or without spironolactone (spiro; 10 µM) or RU486 (10 µM), on StAR mRNA levels in cultured neonatal rat cardiomyocytes. A, Representative ethidium bromide staining of agarose gels after 30 cycles of RT-PCR from two separate experiments in which the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) signals differed. B, Densitometric analysis. Results were normalized to GAPDH mRNA levels and expressed as a percentage of StAR mRNA levels in control cells. Values are the mean ± SEM (n = 3 separate experiments for each condition). **, P < 0.01; ***, P < 0.001 (vs. control).

 
Regulation of cardiac StAR mRNA expression by glucocorticoids
We thus incubated neonatal cardiomyocytes for 24 h with a glucocorticoid, dexamethasone, at concentrations ranging from 1–100 nM (Fig. 3Go, A and B). As expected, dexamethasone treatment led to a concentration-dependent decrease in StAR mRNA levels; a maximal inhibition to 15.5 ± 6.3% of the control value was reached with 100 nM dexamethasone (n = 6; P < 0.001). This inhibitory effect of dexamethasone on StAR mRNA levels was completely prevented by RU486, which favors a genomic effect of dexamethasone through its glucocorticoid receptor (Fig. 3Go). Thus, at the concentrations used above, the effect of aldosterone most likely also occurred via the glucocorticoid receptor.



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  		Figure 3. Effect of dexamethasone treatment (24 h) on StAR mRNA levels in cultured neonatal rat cardiomyocytes with or without RU486 (1 µM). A, Representative ethidium bromide staining of an agarose gel after 30 cycles of RT-PCR. B, Densitometric analysis. Results were expressed as described in Fig. 2Go. Values are the mean ± SEM (n = 6 separate experiments for each condition). *, P < 0.05; **, P < 0.01; ***, P < 0.001 (vs. control).

 
Kinetic experiments on StAR mRNA repression with 100 nM dexamethasone showed that the inhibition started after 60 min, was maximal after 6 h, and remained sustained thereafter for at least 24 h (data not shown).

Modulation of StAR gene expression by glucocorticoids via the p44 MAPK
We observed in separate experiments performed in the presence of actinomycin D, a transcription inhibitor, that dexamethasone did not affect StAR mRNA stability over 24 h (data not shown).

To determine whether de novo protein synthesis is required for glucocorticoid-mediated StAR repression, we treated cardiomyocytes for 24 h with 100 nM dexamethasone in the presence of 10 µg/ml cycloheximide, a protein synthesis inhibitor. As shown in Fig. 4Go, A and B, under conditions of protein synthesis blockade, the inhibitory effect of dexamethasone on StAR mRNA was dramatically suppressed, although StAR mRNA levels remained significantly lower than control values. This suggests that a newly synthesized factor is required.



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  		Figure 4. Inhibition of protein synthesis with cycloheximide (CHX) prevents dexamethasone (Dexa)-induced repression of StAR mRNA levels in rat neonatal cardiomyocytes. A, Representative ethidium bromide staining of an agarose gel after 30 cycles of RT-PCR. B, Densitometric analysis. Results were expressed as described in Fig. 2Go. Values are the mean ± SEM (n = 3 separate experiments for each condition). **, P < 0.01 vs. control; ++, P < 0.01 vs. dexamethasone alone.

 
Inhibition of MAPK (also called extracellular signal-regulated kinase) has been shown to mediate some effects of glucocorticoids (25, 26). We therefore investigated the effect of MAPK blockade on StAR mRNA. To inhibit p42/44 MAPK, we incubated cardiomyocytes for 24 h in the absence or presence of either 50 µM PD98059 (BIOMOL Research Laboratories, Inc., Plymouth Meeting, PA) or 10 µM U0126 (Alexis Corp., San Diego, CA), two inhibitors of p42/44 MAPK phosphorylation and activation, and measured StAR mRNA levels by RT-PCR. As illustrated in Fig. 5Go, the two compounds decreased StAR mRNA levels to 60 ± 8.7% and 52 ± 7% of control values (n = 3; P < 0.01 for both values), respectively, thus mimicking in part the effect of dexamethasone. This result suggested that the repressor effect of glucocorticoids might be mediated to some extent through inhibition of p42/44 MAPK.



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  		Figure 5. Effect of inhibitors of MAPK activation on StAR mRNA levels in rat neonatal cardiomyocytes. A, Representative ethidium bromide staining of an agarose gel after 30 cycles of RT-PCR. B, Densitometric analysis. Cells were treated for 24 h with PD98059 (50 µM) U0126 (10 µM), or dexamethasone (1 µM), and results were expressed as described in Fig. 2Go. Values are the mean ± SEM (n = 3 separate experiments for each condition). **, P < 0.01; ***, P < 0.001 (vs. control).

 
Indeed, Western blot analysis of the phosphorylated p42/44 MAPK showed that p44 phosphorylation diminished to 47 ± 10.8% of the control value (n = 3; P < 0.01) after dexamethasone treatment (Fig. 6Go, A and 6B). This effect was completely prevented by 1 µM RU 486.



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  		Figure 6. Effect of dexamethasone on p42/p44 MAPK phosphorylation in rat neonatal cardiomyocytes. A, Representative Western blot of phosphorylated p42/p44 MAPK (top). A Western blot of total MAPK (bottom) was performed to verify that loading was equivalent in each lane. B, Densitometric analysis. Results were expressed as a percentage of the control. Values are the mean ± SEM (n = 3 separate experiments for each condition). **, P < 0.01 vs. control.

 
In vivo regulation of StAR mRNA after MI in the rat
After MI in the adult rat, there is an increase in aldosterone content and aldosterone synthase expression in the noninfarcted area of LV tissue (11). We thus investigated whether StAR mRNA was also present in adult rat heart. As evidenced by RT-PCR and Southern blotting, we were able to detect significant levels of StAR mRNA in ventricular tissue of both control and infarcted rat hearts (Fig. 7Go). Most importantly, StAR mRNA expression was increased to 242 ± 24% of controls in the noninfarcted area of the LV after MI (n = 6; P < 0.001). This effect of MI was completely prevented by treatment with spironolactone and losartan, which returned StAR mRNA levels to values not different from those of non-MI controls (n = 6).



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  		Figure 7. Effect of MI on StAR mRNA levels in ventricular tissue. A, Representative Southern blot analysis after 30 cycles of RT-PCR. Rat adrenal cortex tissue was included as a positive control. B, Densitometric analysis. Results were expressed as a percentage of the control. Values are the mean ± SEM (n = 6 rats for each treatment). **, P < 0.01; ***, P < 0.001 (vs. controls).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The StAR protein plays a pivotal role in the rate-limiting step of steroidogenesis, intramitochondrial cholesterol transfer (4, 5, 27). Because of the accumulating evidence that heart tissue can produce aldosterone (8, 9, 10), it is of crucial importance to determine whether cholesterol substrate can indeed be supplied to the cardiac mitochondrial enzymatic cascade by the StAR protein.

Four main conclusions can be drawn from the present work: 1) StAR mRNA is present in neonatal rat cardiomyocytes and is regulated by Ang II; 2) glucocorticoids repress StAR gene expression through a pathway involving, at least in part, p44 MAPK; 3) StAR mRNA levels are increased after MI; and 4) this increase is prevented by losartan and spironolactone.

The levels of StAR mRNA in purified cardiomyocytes were approximately 1000 times lower than those observed in adrenal glomerulosa cells, in agreement with a previous report showing a similar expression ratio for 11ß-hydroxylase and aldosterone synthase mRNA in rat cardiac and adrenocortical tissue (9). Importantly, Ang II treatment of cardiomyocytes increased StAR mRNA expression, similarly to what has been shown for StAR mRNA and protein in bovine glomerulosa and human adrenocortical carcinoma cells (28, 29). In the perfused rat heart, stimulation with Ang II for 3 h led to increased aldosterone and corticosterone levels in the coronary sinus effluent and in cardiac tissue (9). Similarly, the induction of StAR mRNA expression by Ang II was accompanied by an increase in aldosterone production. The amounts of aldosterone produced in the medium were approximately 400 times lower than those measured with adrenal cells. There is evidence for Ang II generation in the heart; neonatal rat cardiomyocytes are known to produce Ang II (30). Moreover, in canine ventricular myocytes, the renin-angiotensin system is up-regulated with heart failure (31). There is also increased renin mRNA expression in the border zone of the infarcted left ventricle, with a possible role for intracardiac Ang II in infarct healing (32). Lastly, the cardiac Ang II concentration is increased 2- to 4-fold after MI; this increase is more marked in the infarcted zone than in noninfarcted tissue (11, 33, 34). In view of the accumulating evidence showing that, in addition, most components of the renin-angiotensin system (renin, angiotensin-converting enzyme, and AT1 receptor) can be overexpressed in the heart under conditions of heart failure (11, 33, 34), our results strongly suggest the presence and modulation by Ang II of a critical molecular factor indispensable for aldosterone and corticosterone synthesis in the heart.

Adrenal steroids modulated StAR mRNA expression, most likely via the glucocorticoid receptor. Both glucocorticoid and mineralocorticoid receptors have been identified in human and rodent heart (35, 36, 37, 38) and in neonatal rat cardiomyocytes (39, 40). In an experimental model of MI in the rat, the concentration of myocardial aldosterone has been reported to range from 10–100 nM (9). The inhibition of StAR mRNA levels we report here was obtained at concentrations of a similar order of magnitude, which suggests a potential physiological relevance of these intracrine effects, in which the source and the targets of the steroid hormones reside within the same cell. Interestingly, this repression of steroidogenesis by mineralocorticoids and glucocorticoids may be counterbalanced or overcome by activation of the renin-angiotensin system. Indeed, aldosterone has been recently shown to induce angiotensin-converting enzyme gene expression in neonatal rat cardiocytes (21) and to increase the ventricular density of AT1 receptors in aldosterone/salt-treated rats (19). One could thus speculate that in the healthy heart, aldosterone may play the role of an intracrine/paracrine cardioprotective regulator through a negative feedback on its own biosynthesis and that this control may be lost under pathological conditions (see below).

Glucocorticoids are known to repress numerous genes involved in immune, endocrine, developmental, and other functions (41). Many of these genes are transcriptionally regulated by nuclear factors such as nuclear factor-{kappa}B or by negative glucocorticoid response elements (41). The fact that cycloheximide prevents the effect of glucocorticoids strongly suggests the necessity of de novo protein synthesis. Taken together, these results indicate that steroids may exert an indirect, rather than a direct, action on the StAR promoter.

In this respect, several studies have shown an involvement of MAPK in the inhibitory effects of glucocorticoids (25, 26). More specifically, opposite regulatory effects of p42/44 MAPK on StAR gene expression have been recently reported depending upon cell type: an attenuation of StAR expression in ovarian granulosa-derived cell lines (42) and an increase in StAR gene transcription in mouse adrenocortical Y1 cells (43). Our data clearly show that phosphorylated, active p42/44 MAPK contributes to maintaining resting levels of StAR mRNA and that the repressor effect of steroids occurs at least in part through a preferential dephosphorylation and inactivation of the p44 MAPK. Other additional factors cannot be excluded, however.

The pathophysiological relevance of our in vitro findings was confirmed in an in vivo model of MI in the rat. The previously reported higher cardiac aldosterone production (11) is thus associated with increased amounts of mRNA coding for the StAR protein, the critical and rate-limiting factor of steroidogenesis. This association strongly suggests that the heart is endowed with the essential factors required for making aldosterone. Because cardiac aldosterone production does not contribute significantly to circulating plasma aldosterone levels, there is a possibility that the mineralocorticoid is bound or sequestered within cardiac tissue. Interestingly, high sodium intake-induced LV hypertrophy appears to lead to increased cardiac aldosterone synthesis in the rat (12). Although, under the latter conditions, the circulating renin-angiotensin-aldosterone system is suppressed, the LV hypertrophy might well have led to increased activation of the cardiac renin-angiotensin system, thus explaining the observed rise in cardiac aldosterone synthesis. In fact, a recent study in man has shown that plasma aldosterone levels are higher in the interventricular vein and coronary sinus than in the aortic root in patients with failing ventricles (13), which corroborates our present results. Similarly, the observation of increased cardiac Ang II formation during the clinical course of heart failure (44) may lend further pathophysiological relevance to the fact that Ang II induces StAR mRNA expression in rat cardiomyocytes (our data) and activates mineralocorticoid synthesis in the isolated perfused rat heart (9). On the other hand, Rocha et al. (45) have recently shown that the deleterious effects of aldosterone treatment in the rat can be suppressed by adrenalectomy and that myocardial necrosis and renal arteriopathy are restored by exogenous aldosterone. These results would tend to rule out a role of cardiac aldosterone in cardiac dysfunction. However, the levels of aldosterone in heart tissue were not measured in this work, and it is not known whether cardiac synthesis of the steroid was stimulated under those conditions.

Finally, the increase in StAR mRNA levels observed after MI was prevented by treatment with both spironolactone, a mineralocorticoid receptor antagonist, and losartan, an AT1 receptor subtype antagonist. This result is in agreement with our earlier observation that losartan prevents MI-induced ventricular interstitial fibrosis in the rat, and that losartan blocks the enhancement of cardiac aldosterone production and Ang II content occurring after MI (11). The effect of spironolactone can be best explained on the basis of a blockade of the major action of aldosterone in vivo, i.e. the strong induction of AT1 receptors (Fig. 8Go). This blockade will then result in a suppression of Ang II-induced StAR mRNA overexpression in MI. To our knowledge, the effect of aldosterone treatment in vitro on AT1 receptors in cultured neonatal rat cardiomyocytes has not been examined.



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  		Figure 8. A working model of Ang II-induced StAR mRNA expression and aldosterone production. In neonatal cardiomyocytes (left), the synthesized aldosterone exerts a negative feedback control on StAR mRNA expression via mineralocorticoid and/or glucocorticoid receptors. In MI, increased levels of tissular Ang II up-regulate StAR mRNA expression, and higher levels of aldosterone are produced. The known positive feedback exerted by aldosterone on AT1 receptor expression in vivo overcomes the repression of StAR mRNA, and deleterious aldosterone-mediated effects can occur. A similar effect of aldosterone on AT1 receptors in vitro in cultured neonatal cardiomyocytes has not yet been reported.

 
In summary, we have shown that the rat heart expresses the mRNA of a key regulator of steroidogenesis, the StAR protein. Transcription of StAR mRNA is increased by Ang II and repressed by adrenal steroids in neonatal cardiocytes, and StAR mRNA levels are markedly increased in rat ventricular tissue after MI. In view of these results, it is tempting to speculate that under physiological conditions, the cardiac steroidogenic machinery is expressed at low noise, under adrenal steroid repression, and that, for some as yet unknown reason, this repression is no longer exerted under pathological conditions such as MI, thus allowing aldosterone, for example, to be produced at higher levels and to develop its positive intracrine and paracrine effects on elements of the renin-angiotensin cascade, among others (Fig. 8Go) (19, 21).


   Acknowledgments
 
We are grateful to Dr. Jean-François Arrighi for his advice, and to Manuela Rey and Rachel Porcelli for their excellent technical assistance.


   Footnotes
 
This work was supported by Swiss National Science Foundation Grant 31-52779.97 (to A.M.C.) and a grant from the Swiss Society of Cardiology (to A.C. and A.M.C.).

Abbreviations: Ang II, Angiotensin II; LV, left ventricle; MI, myocardial infarction; SSC, saline sodium citrate; StAR, steroidogenic acute regulatory.

Received September 6, 2002.

Accepted for publication January 29, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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