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(Circulation. 1995;92:175-182.)
© 1995 American Heart Association, Inc.

Articles

Prerequisite for Cardiac Aldosterone Action

Mineralocorticoid Receptor and 11ß-Hydroxysteroid Dehydrogenase in the Human Heart

Marc Lombès, MD, PhD; Nadia Alfaidy, MSc; Emmanuel Eugene, BSc; Alessandro Lessana, MD; Nicolette Farman, MD, PhD; Jean-Pierre Bonvalet, MD, PhD

From INSERM U246, Institut Fédératif de Recherche "Cellules épithéliales," Faculté de Médecine X. Bichat, Paris, France.

Correspondence to Marc Lombès, INSERM U246, Faculté de Médecine X. Bichat, 16, rue H. Huchard, BP 416, 75870 Paris, CEDEX 18, France.

	Abstract

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Background It has been proposed that aldosterone exerts direct effects on heart function, most notably on the development of myocardial fibrosis during ventricular hypertrophy in rat. Initial events in aldosterone action entail its binding to mineralocorticoid receptor (MR). Because MR displays similar affinities for aldosterone and glucocorticoids, the in vivo aldosterone selectivity of MR requires the presence of an enzyme, 11ß-hydroxysteroid dehydrogenase (11-HSD), which metabolizes glucocorticoids into inactive derivatives. Although evidence exists for the presence of MR in rodent heart, no data are available for humans; moreover, the existence of cardiac 11-HSD is controversial.

Methods and Results The heart samples used originated from tissue removed during cardiac surgery in nontransplant patients or from endocavitary biopsies done for the follow-up of heart transplantation. The expression of MR was examined at the mRNA and protein level by in situ hybridization with cRNA probes specific for human MR mRNA and by immunodetection with two specific anti-MR antibodies. 11-HSD catalytic activity was determined by measurement of the metabolic rate of tritiated corticosteroids by cardiac samples. In nontransplanted hearts, an in situ hybridization signal equivalent to that found in the whole kidney was present on cardiomyocytes. Specific immunolabeling of cardiomyocytes with anti-MR antibodies demonstrated the presence of the MR protein. Cardiac 11-HSD activity was detected (243±26 fmol · 30 min^-1 · mg protein^-1) and was dependent on the cofactor NAD, not NADP, suggesting that it corresponds to the form of the enzyme specifically responsible for MR protection. In transplanted hearts that presented severe alterations, MR immunodetection was weaker and irregular, with no specific hybridization signal.

Conclusions Our results demonstrate that MR is coexpressed with 11-HSD in human heart, which thus possesses the cellular machinery required for direct aldosterone action.

Key Words: adrenal cortex hormones • glucocorticoids • transplantation • antibodies • enzymes

	Introduction

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The mineralocorticoid hormone aldosterone exerts its known effects in epithelial cells via an intracellular receptor, the mineralocorticoid receptor (MR). The main effect of the hormone is to promote renal sodium reabsorption in exchange for potassium.¹ In addition to its classic effects on epithelial cells, such as those of the kidney and the colon, it has been shown more recently that aldosterone binds to specific receptors in nonepithelial tissues, such as the heart^{2 3 4 5} or the hippocampus of the brain.⁶ It has been established that elevated plasma aldosterone is associated with the pathogenesis and progression of human heart failure.⁷ In addition to the indirect effects of aldosterone on cardiac function during heart failure resulting from sodium retention, expansion of extravascular space, and hypervolemia, some experimental evidence suggests that this hormone may exert direct effects on the heart. Some reports^{8 9 10} favor a role of aldosterone on rat muscle contractility and membrane potential, although its steroid specificity is unclear. In cultured rat cardiomyocytes, aldosterone stimulates the biosynthesis of the Na⁺,K⁺-ATPase ₁ subunit.¹¹ Recently, Brilla et al^{12 13 14} demonstrated that in rats with experimentally induced left ventricular hypertrophy, elevated levels of plasma aldosterone (and/or renin-angiotensin) led to the development of cardiac fibrosis. It was proposed that this effect represents a direct mineralocorticoid action on the heart, since administration of the aldosterone antagonist spironolactone suppressed it at doses that did not reduce arterial hypertension.¹⁴ The mineralocorticoid specificity of this effect was confirmed in another recent study.¹⁵ The mechanism involved could be an aldosterone-induced stimulation of collagen synthesis, possibly by the cardiofibroblasts.^{12 15 16} The direct demonstration of an increase in rat cardiac types I and III collagen mRNAs in aldosterone-salt hypertension¹⁷ supports this hypothesis.

Direct effects of aldosterone on heart indeed require the presence in cardiac cells of its specific receptor, the MR. Several experimental studies show that this is the case. Specific binding of aldosterone to MR (or high-affinity type I sites) has been reported in rat^{2 3 4} and rabbit⁵ heart. Autoradiographic localization of aldosterone binding sites has been shown in the rat heart.¹⁸ Immunodetection of MR was evidenced in the four cavities of rabbit heart by use of H10E, a specific anti-idiotypic antibody against MR.⁵ In addition, the presence of MR mRNA has been established by Northern blot analysis in the rat heart.^{19 20}

An increasing body of evidence indicates that the specific expression of aldosterone effects in target tissues depends on the enzyme 11ß-hydroxysteroid dehydrogenase (11-HSD). Indeed, natural glucocorticoid hormones (corticosterone, cortisol) display an affinity for the MR similar to that of aldosterone itself,²¹ and their circulating levels are much higher than that of aldosterone. By locally metabolizing glucocorticoids into derivatives (cortisone, 11-dehydrocorticosterone) with low affinity for MR, 11-HSD allows MR to remain free for aldosterone binding and specific action.^{21 22} The demonstration of coexpression of MR and 11-HSD in the typical aldosterone target cells of the distal nephron of the kidney^{23 24} argues in favor of the MR-protecting role of this enzyme. The presence of 11-HSD activity in the rat heart is controversial, since some authors did not detect it^{21 22} and others did.^{25 26 27 28} In addition, it has recently been demonstrated that at least two forms of the enzyme exist, with different enzymatic properties, especially concerning their dependence on either the cofactor nicotinamide-adenine dinucleotide (NAD) or nicotinamide-adenine dinucleotide phosphate (NADP).^{29 30 31}

In contrast to animal studies that report on MR expression in the heart likely to mediate direct action of aldosterone on this organ, such information is not yet available for humans.

The aims of the present study were (1) to determine whether MR is expressed in human heart at the mRNA and protein levels and (2) to examine whether 11-HSD activity could be detected and to determine its cofactor dependence. Our results demonstrate that both MR and an NAD-dependent 11-HSD activity are present in cardiomyocytes of human heart.

	Methods

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Myocardial biopsies have been obtained during cardiac surgery or from endocavitary catheterization. In an initial series of investigations, it was possible to obtain fragments of left ventricular biopsies performed in patients with heart transplants for the systematic follow-up of transplant tolerance. Biopsies were fixed in Zamboni fixative (as previously described in Reference 5) and processed either for in situ hybridization or for immunodetection of MR, in addition to the microscopic examination of the biopsy. In some cases, small fragments of these biopsies were obtained for direct assessment of the catalytic activity of the enzyme 11-HSD. Because MR expression was variable in these conditions, another series of investigations was conducted on heart tissue from nontransplanted patients undergoing cardiac surgery for valve repair (essentially mitral valve surgery). Small pieces of atrium or left ventricle were processed as described above either for MR detection or for measurements of 11-HSD activity.

In Situ Hybridization
Heart sections (5 to 7 µm) were cut and mounted on subbed slides and processed for in situ hybridization as previously described.³² Briefly, after removal of paraffin with xylene, sections were rehydrated in graded alcohols followed by PBS. Sections were postfixed again in 4% paraformaldehyde for 20 minutes and rinsed in PBS. Next, treatment with proteinase K (20 µg/mL) was followed by an acetylation step (0.1 mol/L triethanolamine, pH 8, 0.025% acetic anhydride). After they were rinsed in PBS and 0.9% NaCl, sections were dehydrated and dried. Hybridization mix was then spread over the sections, and the slides were covered with paraffin film. Sections were hybridized for 16 to 18 hours at 50°C. Posthybridization treatment consisted of an initial wash in 5x saline sodium citrate (SSC; 150 mmol/L NaCl, 15 mmol/L sodium citrate), 10 mmol/L dithiothreitol (DTT) at 50°C, a high-stringency wash in 50% formamide, 2x SSC, 0.1 mol/L DTT at 65°C for 20 minutes, and two 10-minute washes in NaCl-Tris-EDTA (0.5 mol/L NaCl, 10 mmol/L Tris-HCl, 5 mmol/L EDTA) at 37°C. Ribonuclease A treatment (20 µg/mL) was then performed at 37°C for 10 minutes. After being rinsed with 0.1x SSC for 15 minutes, sections were dehydrated in graded alcohols (containing ammonium acetate 0.3 mol/L) and dried. Kodak NTB2 film (melted at 42°C) was applied to the slides, dried, and exposed at -20°C for 3 to 6 weeks. The film was developed (Kodak D19) and fixed (Kodak Unifix). At the end of the autoradiographic process, the sections were stained with toluidine blue.

Human MR (hMR) cDNA, kindly provided by Dr R. Evans (Salk Institute, Howard Hughes Medical Center, La Jolla, Calif), was cut by Sca I, and the fragment (nt398-1205) was subcloned into PGEM (Promega) to synthesize antisense (complementary to the tissue hMR mRNA) or sense (similar to the hMR mRNA) cRNA probes. This region of the cDNA, corresponding to the N-terminal region of the receptor, is not homologous to the glucocorticoid cDNA sequence (46.7% identity at the nucleotide level). After linearization, ³⁵S-labeled RNA probes were synthesized by use of SP6 or T7 polymerase. ³⁵S-labeled UTP 5'-triphosphate (>37 TBq/mmol) was from Amersham, and the other reagents (ATP, GTP, CTP, ribonucleasin, DTT, and RNA polymerases) were from Promega. The hybridization mix was 50% formamide (Fluka), 1 mmol/L DTT (Boehringer), 2x SSC, 10% dextran sulfate (Pharmacia), 1 mg/mL salmon sperm DNA (Sigma), and the ³⁵S-labeled cRNA. Immediately before hybridization, this mix was denatured at 80°C for 5 minutes. Approximately 1x10⁶ to 2x10⁶ cpm was applied to each slide.

Quantification of the hybridization signal was performed by image analysis (Optilab, Graftek). Results are given as arbitrary units per surface area. Background was measured on the slide on a zone without tissue and deducted from tissue labeling. Similar analysis was done in sections from the same tissue hybridized with antisense and with sense probe. Specific signal is the difference between these two conditions.

In addition to heart biopsies, human kidney tissue obtained from the nontumoral portion of a surgically removed kidney was hybridized on the same slides in parallel with heart sections.

Immunodetection of MR
Five- or 7-µm sections were cut, mounted on slides, and processed for immunohistochemistry. A routine procedure of indirect immunostaining was applied essentially as described.^{2 33 34} Briefly, incubation with the primary antibodies was followed by a horse-biotinylated anti-mouse or anti-rabbit antibody human adsorbed (Vector Laboratories Inc), and an avidin-biotin-peroxidase complex (ABC-Elite from Vector Laboratories) was used as a detection system.

The following antibodies were used: H10E, a monoclonal anti-idiotypic antibody of the IgG1 class that interacts with the steroid binding domain of the MR,³⁵ and A4, a rabbit polyclonal antibody directed against a peptide corresponding to the hMR412-422,³⁶ ie, in the N-terminal region of the receptor molecule. The control of the specificity of the immunolabeling was performed by presaturating the anti-peptide antibodies A4 with the free peptide at a concentration of 100 µg/mL. Because H10E is an internal image of aldosterone,³⁵ in situ competition studies could be performed to assess the specificity of immunolabeling. This was achieved by preincubating the slides with 1 to 5 µmol/L aldosterone in PBS for 30 minutes before incubation with H10E.

Determination of 11-HSD Catalytic Activity
Enzyme activity was measured by determining the rate of transformation of tritiated corticosterone into 11-dehydrocorticosterone as described previously.^{23 37} Small pieces of heart (<3 mg each) were incubated in 100 µL of solution (in mmol/L: NaCl 137, KCl 5, MgSO₄ 0.8, Na₂HPO₄ 0.33, KH₂PO₄ 0.44, MgCl₂ 1, CaCl₂ 1, D-glucose 5, and Tris-HCl 10; pH 7.4) with 10 nmol/L ³H-corticosterone (1,2,6,7-³H-corticosterone, 2.22 TBq/mmol, Amersham) at 37°C. Samples of supernatant (5 µL) were carefully removed at various time intervals and transferred into 95 µL methanol, to which 10^-4 mol/L unlabeled corticosterone and 11-dehydrocorticosterone were added to serve as internal standards for high-performance liquid chromatographic analysis (Beckman Gold HPLC system). Samples (100 µL) were injected onto a reverse-phase column (Novapak C18, Waters Associates) with a precolumn (C18, 5 µm; Waters) and eluted isocratically with methanol-H₂O (1:1) at 1 mL/min. UV absorbance was recorded at 240 nm to monitor the elution time of internal standards for each sample. Eluate from the column was collected at 30-second intervals, and radioactivity was counted (LKB Rackbeta) after the addition of 3 mL Optiphase (LKB). The eventual spontaneous metabolism in the incubation medium was evaluated by its incubation with steroids in the absence of tissue (blanks) and appeared to be very low (generally <0.5%). In some cases, an inhibitor of 11-HSD (carbenoxolone, 10^-4 mol/L, Sigma) was added to the incubation medium. To test the cofactor specificity of the heart 11-HSD, the enzyme was measured after permeabilization of the tissue (three consecutive freezing-thawing procedures) in the absence and in the presence of 1 mmol/L NAD or NADP.

Results are expressed as femtomoles of 11-dehydrometabolites produced per milligram wet weight or per milligram protein. The protein content of each sample was measured by the method of Bradford³⁸ at the end of the incubation and after solubilization in NaOH.

	Results

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In Situ Hybridization
Figs 1 and 2 illustrate the results obtained after hybridization of heart from nontransplant patients and kidney sections with antisense and sense hMR specific probes. A clear labeling by the antisense probe is present over atrial and left ventricular myocytes (Fig 1), contrasting with a very low signal over intramyocardial vascular structures. The control or sense probe clearly gave a lower signal. For purposes of comparison, the labeling obtained in the kidney with these same two probes is shown in Fig 2. The antisense probe strongly labeled the typically mineralocorticoid-sensitive cortical collecting duct and distal tubules,^{1 33} with low nonspecific labeling of proximal tubules and glomeruli, thus establishing its adequacy to detect hMR mRNA. The sense probe gave a low and homogeneous signal all over the sections. As for human heart, a positive signal associated with the antisense hMR probe was found in all heart biopsies originating from seven patients undergoing mitral valve replacement.

View larger version (0K): [in this window] [in a new window]   Figure 1. Photographs of in situ hybridization of human mineralocorticoid receptor mRNA in human heart. Hybridization was performed with 35S-cRNA antisense (A, B) or sense (C, D) probes. A and C are bright fields (phase contrast) and B and D are dark fields of the same tissue sections from left ventricle. The antisense probe (B) showed a specific signal over myocytes and not over small intraventricular vessels. Nonspecific homogeneous low signal was observed with the sense probe (D). Magnification x200.

View larger version (0K): [in this window] [in a new window]   Figure 2. Photographs of in situ hybridization of human mineralocorticoid receptor mRNA in human kidney. Hybridization was performed with 35S-cRNA antisense (A, B) or sense (C, D) probes. A and C are bright fields and B and D are dark fields of the same tissue sections from kidney cortex. The antisense probe (B) showed a high specific signal of the distal tubules and cortical collecting ducts (* in A and C) and not over proximal tubules (P) or glomeruli (G). Nonspecific-signal homogeneous low signal was observed with the sense probe (D). Magnification x200.

Quantification of in situ hybridization experiments is shown in Fig 3. The antisense signal was clearly much higher than the sense labeling in each biopsy, although absolute levels vary among patients. The Table gives the mean values of specific hMR mRNA signals (antisense minus sense labeling) in heart and kidney. With respect to the whole organ, hMR mRNA levels are roughly equivalent in heart and kidney cortex. In contrast to the heart, however, the signal is quite heterogeneous within the kidney, since only a small proportion of the tubular cells express hMR mRNA. As shown in Fig 2, the messenger is indeed restricted to the distal tubule and cortical collecting duct, which represent a small percentage (<10%) of the total mass of the kidney cortex. In these particular aldosterone-sensitive cells, the labeling per unit surface area was 4 to 5 times higher than over myocytes.

View larger version (17K): [in this window] [in a new window]   Figure 3. Graph showing quantification of hybridization signal on human heart. The signal was quantified by image analysis (arbitrary units) on heart sections hybridized with 35S-cRNA antisense (AS) or sense (S) probes encoding for human mineralocorticoid receptor (hMR). For each patient, symbols corresponding to values obtained with AS and S probes in the same heart sample are joined by a line. Vertical bars indicate the mean±SEM. The specific hMR mRNA signal corresponds to the difference between results obtained with AS and S probes.

View this table: [in this window] [in a new window]   Table 1. Human Mineralocorticoid Receptor mRNA Signal

The same hybridization protocol was performed on sections of ventricular biopsies from nine different heart transplant patients undergoing polytherapy, including corticosteroids. In each case, the hybridization signals were similar with the antisense and the sense probe. Thus, no reproducible specific signal could be detected in this group of patients.

Immunolocalization of MR
Fig 4 shows the immunolabeling obtained with the antipeptide antibody A4 and the monoclonal anti-idiotypic anti-MR antibody H10E on sections of cardiac tissue from nontransplant patients. A clear immunostaining was observed with both antibodies on myocytes, whereas small intraventricular vessels lack specific labeling. The extinction of labeling achieved by preincubation with the peptide (for A4) or aldosterone (for H10E) results in a background signal similar to that obtained with unrelated primary antibodies, thus attesting to the specificity of MR detection. Similar results were obtained in samples from eight nontransplant patients.

View larger version (0K): [in this window] [in a new window]   Figure 4. Photographs of immunolocalization of human mineralocorticoid receptor (hMR) with specific anti-hMR antibodies. Left photographs are phase-contrast and right are bright-field views of the same section of left ventricle. Immunolabeling with A4 anti-peptide antibody (A, B) showed staining over myocytes, which was abolished by previous incubation of the tissue with the immunizing peptide (C, D). When H10E antibody was used, immunostaining of the tissue was also found over myocytes (E, F), and extinction of the signal was observed by preincubation of the tissue with aldosterone (G, H). No signal was observed over small intraventricular vessels with both antibodies. Magnification x200.

Experiments performed on fragments of biopsies from transplanted heart did not yield clear-cut and reproducible results. Profound disturbance of heart tissue was often observed in these biopsies, with edema and presence of fibrosis and inflammatory cells. Whereas a specific signal could be observed with H10E for some samples, immunolabeling was widely variable from one zone to another within one single biopsy and from one patient to another (data not shown).

11-HSD Catalytic Activity
11-HSD catalytic activity was assayed on heart tissue from nontransplant patients. Fig 5 shows the time dependence of 11-HSD activity in permeabilized tissue incubated in the presence of the cofactor NAD or NADP. Enzyme activity increased with incubation time. Values were clearly higher in the presence of NAD than NADP. The enzyme inhibitor carbenoxolone strongly reduced the NAD-dependent enzyme activity. This cofactor dependence was further assessed in three different patients tested, as illustrated in Fig 6. Values in the presence of NAD (243±26 fmol · 30 min^-1 · mg protein^-1, mean±SEM, n=3) were twice as high as those observed in permeabilized tissue in the presence and in the absence of NADP (99±13, n=3), as well as in intact tissue. Thus, it appears clear that the human heart 11-HSD is NAD- and not NADP-dependent.

View larger version (14K): [in this window] [in a new window]   Figure 5. Graph showing 11ß-hydroxysteroid dehydrogenase (11-HSD) catalytic activity in human heart tissue. Heart samples were permeabilized and incubated with 10 nmol/L 3H-corticosterone in the presence of cofactor NAD, NADP, or NAD plus the enzyme inhibitor carbenoxolone (CBX). The amount of metabolite produced (3H-11-dehydrocorticosterone) is given on the y axis as a function of incubation time. The time-dependent increase in 11-HSD activity was much higher in the presence of NAD than NADP. CBX inhibited this increase.

View larger version (46K): [in this window] [in a new window]   Figure 6. Bar graph showing cofactor dependency of 11ß-hydroxysteroid dehydrogenase (11-HSD) catalytic activity in human heart. 11-HSD was measured in intact (I) or permeabilized (P) small tissue samples from three different patients: patient 1, stippled bars; patient 2, hatched bars; and patient 3, open bars. Enzymatic activity was NAD- and not NADP-dependent in each case.

In addition, it was possible to confirm the presence of 11-HSD on small fragments of left ventricle biopsies from some transplant patients. These experiments demonstrate a time-dependent, carbenoxolone-inhibitable 11-HSD activity in these transplanted heart samples (data not shown). The enzyme activity of intact tissue from five different patients reached a mean value (±SEM) of 13.8±3.6 fmol · mg wet wt^-1 · 120 min^-1 incubation with ³H-corticosterone, which was reduced to 1.2±0.8 in the presence of carbenoxolone.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The present study shows that the MR is expressed in human heart at both the mRNA and protein levels. When a specific cRNA probe encoding for the N-terminal portion of hMR was used, a clear-cut signal was observed on cardiomyocytes. This finding is coherent with a previous demonstration of MR mRNA by Northern blot in rat heart.^{19 20} The adequacy of the probe used was established by the specific labeling observed in the human distal nephron. Although the overall abundance of hMR mRNA was of comparable magnitude in heart and whole kidney cortex, the intensity of the hybridization signal was fivefold lower in cardiomyocytes than in the specific aldosterone target cells of the renal distal tubule.³³ The MR was also found on cardiomyocytes at the protein level by immunohistochemistry using two different antibodies: H10E, which is specific to the steroid-binding domain of the receptor, and A4, which recognizes an N-terminal portion of hMR. No apparent variation in hMR mRNA or protein levels was found among tissue samples originating from the left ventricle, the papillary muscle of the mitral apparatus, or the right auricle. Neither mRNA nor immunoreactive hMR was detected over small intramyocardial blood vessels. This is in agreement with previous observations made on the rabbit heart.⁵ Whereas hMR was constantly and reproducibly found by both approaches in heart samples from nontransplant patients, this was not the case in transplanted hearts. Although positive immunostaining could be observed in some biopsies, wide variations in intensity and heterogeneous staining were observed from one fragment to another. Messenger RNA detection was weak and inconsistent. The reason for the variability of results in transplanted hearts is unclear. One might invoke the tissue alterations (edema, fibrosis), which affected, to a variable extent, the heart sections examined. In addition, the patients had received various polytherapies, including immunosuppressive agents as well as glucocorticoids, that might have modified MR expression.

Results of immunolabeling and in situ hybridization clearly indicate that MR is expressed in the whole population of cardiomyocytes. This is in accordance with previous data obtained with the anti-MR antibody H10E in rabbit heart.⁵ This previous report indicated that, in addition to cardiomyocytes, endothelial cells and other cell types (possibly cardiofibroblasts) also expressed MR. The present study neither confirms nor contradicts this observation. Moreover, no conclusions concerning endothelial cells could be reached because of the limited size of the samples. In addition, the available material did not permit us to examine precisely the possibility of a specific labeling on cell types other than cardiomyocytes, except for the negative results on intramyocardial small vessels. The MR localization in cardiomyocytes deserves discussion with regard to the reported aldosterone-induced cardiac fibrosis.^{12 13} Indeed, an enhanced rate of intracardiac collagen gene expression has been demonstrated in hypertrophied hearts of aldosterone-treated rats.¹⁷ On the other hand, aldosterone has been shown to increase collagen synthesis in rat cardiac cultured fibroblasts.¹⁶ The aldosterone-induced stimulation of collagen synthesis could depend on a direct effect of the hormone on cardiofibroblasts. Alternatively, aldosterone could modulate the functions of cardiomyocytes, leading to changes in the cell environment. In this case, the cardiac fibrosis should be considered a paracrinelike effect of this hormone.

In addition to the MR expression, the present study establishes the presence of 11-HSD dehydrogenase activity in the human heart. The existence of such an activity in the rat heart has been previously reported by several^{25 26 27 28 29} but not all^{21 22} authors. The question arises of evaluating the level of 11-HSD activity in heart compared with other tissues, particularly the classically aldosterone-sensitive tissues. However, such a comparison is difficult to make because of the large variations in experimental conditions from one study to another. Indeed, experiments are performed in a variety of preparations (microsomes, homogenates, intact or permeabilized cells), and results are given either as percent conversion of the glucocorticoid substrate (corticosterone or cortisol) or as the absolute value of the metabolite produced (dehydrocorticosterone or cortisone). Incubation times vary from a few minutes to several hours. This renders quantitative comparisons difficult, since substrate depletion could reduce enzyme activity after long incubation time. Finally, the glucocorticoid substrate concentration varies from the nanomolar range to above the micromolar range, a concentration generally considered as exceeding the K_m.^{24 29} However, authors generally agree that in rat, dehydrogenase activity is much lower in the heart than in the kidney. Comparison of the present results with previous data from our laboratory³⁷ obtained under similar experimental conditions in human tissue and recalculated in the same units is in accordance with this notion: 11-HSD dehydrogenase activity is about 100-fold lower in heart than in the typically aldosterone-sensitive renal collecting duct. Such a difference raises the question of the actual physiological role played by 11-HSD. This question cannot be resolved with certainty, since the answer depends on several parameters, some of which are still difficult to assess, such as the relative expression and regulation of 11-HSD and MR in a given cell type or the subcellular relative spatial distribution of the receptor and the enzyme. A growing body of evidence indicates that the MR-protecting role of 11-HSD, implied in mineralocorticoid selectivity, depends on the isoform of the enzyme.^{29 30 31 39} The prevailing notion is that at least two different isoforms of 11-HSD exist, with different enzymatic properties and different tissue and/or cell distribution. One of them exhibits low affinity for glucocorticoids, is relatively ubiquitous, but is particularly prevalent in the liver. According to the cell context, it can function as a dehydrogenase or reductase. It is thought to play little or no role in MR protection and might be involved in regulatory processes of glucocorticoid functions.³⁹ Another form displays a high affinity for the glucocorticoid substrate. It has been found in placenta⁴⁰ and in the aldosterone-sensitive renal cells^{29 30 31} and is considered to be the MR-protecting enzyme. Importantly, these two isoforms differ by their dependence on cofactors. The first one is NADP-dependent, whereas the latter, ensuring MR protection, is NAD-dependent. The present results clearly show that the form expressed in human heart is NAD- and not NADP-dependent and may therefore correspond to the MR-protecting form of the enzyme. This could explain why the natural glucocorticoid corticosterone, which displays the same affinity for the MR as aldosterone, does not induce cardiac fibrosis,¹⁵ while also supporting the claim for the mineralocorticoid specificity of the aldosterone-induced fibrosis.

In conclusion, the present study demonstrates the coexpression of MR and the MR-protecting NAD-dependent 11-HSD in the human heart. This coexpression does permit direct specific effects of aldosterone in the heart in physiological as well as pathological conditions, such as the aldosterone-induced development of fibrosis in hypertrophied left ventricle. These findings may open new therapeutic perspectives on the use of antimineralocorticoids in the prevention of cardiac fibrosis and heart failure.

	Acknowledgments

 
This work was supported by INSERM funds and by a grant from the Searle "Fondation pour l'hypertension artérielle." We wish to thank Dr A. Nitenberg for providing us with the human tissue samples. We are indebted to V. Lévêque for expert secretarial assistance, to F. Delahaye for her help in performing immunolocalization of mineralocorticoid receptor, and to T. Carlson for editing the manuscript.

Received October 18, 1994; revision received December 27, 1994; accepted January 10, 1995.

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