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Baker Medical Research Institute (M.J.Y., T.J.C., J.W.F.), Prahran Victoria 3181; and Prince Henry’s Medical Research Institute (C.D.C.), Clayton Victoria 3168, Australia
Address all correspondence and requests for reprints to: Dr. Morag J. Young, Department of Molecular Physiology and Molecular Genetics, Baker Medical Research Institute, P.O. Box 6492, St. Kilda Road Central, Melbourne 8008, Australia. E-mail: morag.young{at}baker.edu.au
Abstract
The present study explores the possibility of local de novo aldosterone production in normal and failing hearts (human and mouse) and the regulation of such putative cardiac steroidogenesis. Total RNA was isolated from human tissue from failing hearts taken at the time of cardiac transplantation, from normal hearts obtained at autopsy, and from normal and pressure-overloaded mouse hearts. Vascular smooth muscle cells from human artery and vein were also analyzed. RNA was reverse transcribed and probed with specific primers for side-chain cleavage enzyme (CYP11A), 3ß-hydroxysteroid dehydrogenase, aldosterone synthase (CYP11B2), 11ß-hydroxylase (CYP11B1), steroidogenic factor-1, and steroid acute regulatory protein. CYP11A, 3ß-hydroxysteroid dehydrogenase-2, and steroid acute regulatory protein were expressed at modest levels in all tissues examined in both mouse and human. In failing human heart, CYP11B1 and CYP11B2 were detected in some samples, in contrast with normal hearts, which expressed neither; in the mouse heart steroidogenic factor-1 was detected, but neither CYP11B1 nor CYP11B2 was found. Steroidogenic factor-1 was detected in no human heart sample tested after 40 cycles of PCR. Although the expression of some steroidogenic genes can be detected in the heart, the likelihood of physiologically relevant levels of aldosterone production by the normal heart is very low. The exact cellular location of steroid synthesis in the failing human heart remains to be established.
THE STEROID HORMONE aldosterone is classically synthesized in the outer cortex of the adrenal gland by sequential enzymatic modification of cholesterol and acts via epithelial MR to promote unidirectional sodium and water transport. Recently, however, aldosterone has been shown to have additional effects in nonepithelial tissues such as the heart and vessel wall, where marked vasculitis, tissue remodeling, and fibrosis have been shown when aldosterone levels are inappropriately high for sodium status (1, 2, 3). This extraepithelial role for aldosterone is underscored by the recent Randomized Aldactone Evaluation Study trial, which showed that modest doses of the aldosterone antagonist, spironolactone, in addition to angiotensin-converting enzyme inhibitors, diuretics, and/or ß-blockers, reduce CV related mortality by 30% and morbidity by 35% (4). Although MR antagonists lower blood pressure in both clinical and experimental (5) hypertension, the dose of spironolactone used did not affect blood pressure, and no mechanism has yet been documented for this beneficial effect on mortality and morbidity.
The final enzymatic step in aldosterone synthesis requires the action of the enzyme aldosterone synthase, or CYP11B2. Recently there have been several reports of CYP11B2 mRNA expression and activity in rat heart (6, 7, 8) and in rat (9, 10) and human vessel wall (11, 12). Silvestre et al. (6) postulated the existence of a cardiac steroidogenic system in rat heart, reporting the presence of both 11ß-hydroxylase (CYP11B1, the enzyme responsible for the final step of glucocorticoid synthesis) and CYP11B2 mRNA and enzymatic activity. As in the adrenal, cardiac CYP11B2 was independently regulated by low sodium/high potassium diets, angiotensin II infusion increased levels of both enzymes, and ACTH elevated CYP11B1 exclusively. In another report, Takeda et al. (13) demonstrated an unanticipated up-regulation of CYP11B2 in rat heart after 8 wk of a high salt diet, whereas adrenal levels remained substantially suppressed, suggesting the presence of an independently regulated cardiac steroidogenic. Others have shown differential regulation between strains, with Wistar-Kyoto, but not Sprague Dawley, rats positive for cardiac CYP11B1 and CYP11B2 expression by PCR; after angiotensin II (100 µg/d) infusion, however, both strains were positive (7).
The human vascular wall has also been reported to express CYP11B1 and CYP11B2 (11, 12), and in another study, 21-hydroxylase (CYP21) (14), which is required for the conversion of progesterone to deoxycorticosterone. A study by Kayes-Wandover and White (15) on the human heart, surveying by PCR the steroidogenic machinery necessary for aldosterone and cortisol synthesis, has recently been published, in which enzymes necessary for de novo corticosterone, but not aldosterone, synthesis were found in all chambers; CYP11B2 expression was restricted to aortic wall and fetal tissue. Levels of steroidogenic enzyme gene expression were approximately 0.1% of those in the adrenal, which was interpreted as being consistent with an autocrine/paracrine action, but not as contributing to circulating plasma levels.
These findings prompted us to determine whether CYP11B1 and CYP11B2 are expressed in normal and failing human heart by PCR after RT, a rapid and sensitive method to detect specific RNAs. Similarly, mouse models of heart failure (coronary ligation) and elevated ACTH (GR-null mice) (16) were also studied for the expression of steroid hydroxylases potentially contributing in a paracrine or autocrine fashion to local tissue remodeling. Previously, cardiac failure and high ACTH have been shown to increase the expression of CYP11B1 and CYP11B2 in rat heart (6, 11). To further explore the possibility of a cardiac system of adrenal steroidogenesis, expression levels of key regulators of steroid hydroxylase expression, steroidogenic factor-1 (SF-1) (reviewed in Ref. 17) and the cholesterol transport protein, steroid acute regulatory protein (StAR) (18), were also determined.
Materials and Methods
Materials
Oligonucleotides were synthesized by Geneworks (The Barton,
Australia). Random hexanucleotide primers, Moloney murine leukemia
virus H- reverse transcriptase, and T4
polynucleotide kinase were obtained from Promega Corp.
(Madison, WI). Taq DNA polymerase was purchased from Biotech
(Belmont, Australia), GeneScreen Plus was obtained from NEN Life Science Products (Boston, MA), autoradiography film was
purchased from Kodak (Rochester, NY), and
[
-32P]ATP was obtained from Bresatec
(Adelaide, Australia).
Tissue samples
All human heart tissue used was approved for use by the Alfred Hospital discarded tissue committee and/or the Victorian Institute of Forensic Medicine ethics committee, and use of mouse tissue was approved by the Baker Medical Research Institute animal ethics committee. Failing human heart samples were obtained from all four chambers of hearts removed at the time of cardiac transplant surgery. Normal human left ventricle samples were obtained from autopsies where cause of death was not cardiovascular related. Four hearts were sampled for "failing" tissue and three for normal human tissue. The ventricular apex was obtained from three normal and three pressure-overloaded (coronary-ligated) mouse hearts and from two hearts each of wild-type, heterozygote, and GR-null mice (C57BL6/129sv background). All human and mouse tissues were immediately frozen in liquid nitrogen at the time of collection and stored at -80 C until analysis. Vascular smooth muscle cells (VSMC) from the internal mammary artery and vein were grown in FCS, to passages 8 and 7, respectively, before use in these studies.
RNA isolation and detection by RT-PCR methods
Frozen human and mouse tissue samples (100–200 mg) were homogenized in a guanadine isothiocyanate solution (TRIzol, Life Technologies, Inc., Gaithersburg, MD), and total RNA was isolated by phenol/chloroform extraction and ethanol/salt precipitation. The integrity of the RNA was monitored by agarose gel electrophoresis and 18S rRNA PCR analysis. Total RNA (500 ng) was reverse transcribed by Moloney murine virus reverse transcriptase in a reaction volume of 25 µl containing 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, 1 mM of each deoxy-NTP, and 200 ng random hexanucleotide primers and was incubated at 25 C for 10 min and then at 42 C for 60 min. PCR of 18S RNA used 2 mM MgCl2, 67 mM Tris-HCl (pH 8.8), 16.6 mM [NH4]2SO4, 4.5% Triton X-100, 2 mg/ml gelatin, 0.5 mM deoxy-NTPs, and 0.5 µM of each primer in a 30 µl reaction volume. Denaturation at 94 C was performed for an initial 2 min, then for 30 sec at 94 C for each cycle, annealing was performed at 60 C for 30 sec, and elongation was performed at 72 C for 30 sec for 18 cycles, followed by a soak at 72 C for 5 min. PCR products were visualized on a 1.3% agarose gel.
Primers specific for each steroid hydroxylase gene were located in
adjacent exons to differentiate between RNA products and any
contaminating genomic DNA. cDNA (1 µl) was used for each 30-µl PCR
reaction; cycle number and annealing temperatures are detailed in Table 1
. Positive and negative controls were
included with each run. RT-PCR products were subjected to
electrophoresis in 1.3% agarose and visualized under fluorescent
light. The specificity of the products was checked either by direct
sequencing of gel-purified bands (mouse) or by Southern blot analysis
(human).
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), for 12 h. Membranes were
washed twice for 20 min each time in 5 x SSC/0.1% SDS at 42 C
and exposed to Kodak BioMax film overnight at -70 C. Results
Human heart
Expression of the first two enzymes in the synthesis of both
aldosterone and corticosterone, CYP11A (cholesterol side-chain
cleavage) and 3ß-hydroxysteroid dehydrogenase (3ßHSD), was detected
in normal and failing human hearts and in VSMC from artery and vein
(Fig. 1). When normalized to 18S rRNA,
levels detected in the hearts were significantly lower than those in
the positive control of unstimulated H295R cells, a human adrenal
cortical cell line. In contrast CYP17 (17-hydroxylase), which is
required for the conversion of pregnenolone to 17-hydroxypregnenolone
and of progesterone to 17-hydroxyprogesterone, was not found in any
heart sample tested. CYP11B2, which is required for the final three
oxidation steps of 11-deoxycorticosterone to aldosterone, and CYP11B1,
which is required for the conversion of 11-deoxycorticosterone to
corticosterone and of 11-deoxycortisol to cortisol, were found in some,
but not all, chambers of failing heart and not at all in normal heart
or VSMC. Once again, the levels detected were considerably lower than
those in H295R cells. Qualitatively, the atria, particularly the left
atria, showed the highest level of expression of all the chambers; the
variance between chambers, between hearts, may reflect at best the
semiquantitative nature of the PCR/Southern blot method used.
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Mouse heart
Two genetic mouse models were used in this study, one
characterized by high circulating ACTH due to a null mutation in the GR
gene and one of induced cardiac failure caused by coronary artery
ligation. These models were investigated given evidence for the
stimulatory effects of both on the expression of steroid hydroxylases
(6, 8). As in human hearts, CYP11A and 3ßHSD2 were
expressed in all mouse heart samples, again at a markedly lower level
than in the mouse adrenal when normalized to 18S RNA (Fig. 2). Between-sample variation was
minimal, suggesting that there was little stimulation of expression by
elevated ACTH and coronary ligation. In contrast to human hearts, no
expression of CYP11B1 or CYP11B2 was detected (data not shown) even
after Southern blot analysis of PCR products (>40 cycles PCR). StAR,
however, was detected, at a relatively low number of cycles (n =
28–30) as in the human heart, and SF-1 was also detected, but at a
higher (35 cycles of PCR) cycle number. All products were normalized to
18S RNA and were markedly lower than in the mouse adrenal, as was the
case for the steroid hydroxylases.
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The results of this study substantially extend those on localization of possible sites of adrenal steroidogenesis in the human heart recently published by Kayes-Wandover and White (15) and those from a variety of laboratories (6, 7, 8, 9, 10) on rat models.
Human studies
In the paper from White’s laboratory previously cited (15), the investigators examined pooled commercial samples of a variety of anatomical regions of healthy adult heart, pooled adult and fetal whole heart samples, and aorta; in addition, they report negative results for CYP11B2 in three individual samples of cardiac infundibulum removed at surgery for correction of Fallot’s tetralogy. Our samples came from individual normal human heart, over a more limited anatomical range (right and left atria, and right and left ventricle) and from failing hearts removed in the process of cardiac transplantation. Although the studies address overlapping, rather than identical, subject groups, the between-laboratory parallels in terms of results are considerable, with only minor areas of difference.
In both studies all tissues examined were found to express low levels of the enzymes responsible for the first two steps of adrenal steroid biosynthesis, CYP11A and 3ßHSD. In neither study could CYP17 be detected, and in neither study could CYP11B2 be shown, even at high PCR cycle number, in normal cardiac tissue. In both studies atrial levels tended to be higher than those in the ventricles.
As noted above, the studies were not coterminous in terms of either tissues examined or markers examined. Kayes-Wandover and White (15) found fetal levels to often be higher (3ßHSD2 and CYP21) than those in adults and in addition found low levels of CYP11B2 expression in fetal, but not adult, heart. We did not probe for CYP21, but found CYP11B1 and CYP11B2 to be expressed in some, but not all, chambers of the failing human heart; in addition, we found both normal and failing human heart to express StAR, but not SF-1. The only antithetical finding between the two studies, in fact, is that whereas White’s laboratory reports low levels of CYP11B1 expression in normal human heart, with fetal heart not determined, we found CYP11B1 in failing, but not in normal, heart samples. Our finding of CYP11B2 expression in failing, but not normal, heart may reflect the range of drugs to which the pretransplant patients were exposed (i.e. diuretics and angiotensin-converting enzyme inhibitors) and to which the normal hearts were not.
Although this is the only area in which the findings are opposed, it is one of considerable possible pathophysiological significance. Kayes-Wandover and White (15) propose, on the basis of their studies, that while the absence of CYP17 and CYP11B2 expression rules out local paracrine/autocrine roles for cortisol or aldosterone, cardiac MR and/or GR may be occupied by locally produced corticosterone and/or deoxycorticosterone. In our studies this may be true for the failing human heart, but not for the normal heart as a whole, in contrast to the previous study in which atrial, but not ventricular, expression of CYP11B1 was found. It is possible that this sole difference between the two studies reflects the differences in tissue sourcing between CLONTECH Laboratories, Inc., pooled samples, on the one hand, and individual samples from autopsy material, on the other, although ultimately the tissues derive from what would appear to be very similar sources. It may also reflect their use of mRNA and 45 cycles vs. ours (total RNA and 40 cycles); whether such a very low level of expression, or what this difference may imply, is of pathophysiological importance is thus uncertain.
Very recently, Mizuno et al. (19) published compelling data on the failing, but not the normal, human heart, producing aldosterone on the basis of consistent differences in arteriovenous concentrations in plasma aldosterone in the former, but not the latter, circumstance. Values for aortic root aldosterone levels were 61 ± 7 pg/ml for normal hearts, levels not different from those in anterior interventricular venous samples (58 ± 6 pg/ml) or coronary sinus (62 ± 7 pg/ml). In contrast, in both systolic and diastolic dysfunction, intraventricular venous and coronary sinus levels were significantly higher than control values (98 ± 10 and 97 ± 11 vs. 72 ± 0, P < 0.001, in LV systolic dysfunction; 87 ± 10 and 84 ± 10 vs. 71 ± 9 pg/ml, P < 0.01, in LV diastolic dysfunction). In addition, these researchers were able to show a significant correlation between the arteriovenous difference in aldosterone levels and the degree of end-diastolic pressure and a significant negative correlation with ejection fraction in the LV systolic dysfunction group.
These findings in three groups, each of 20 or more patients, do not provide a pointer to where in the heart aldosterone biosynthesis might occur. They do, however, provide compelling evidence that such production does occur under conditions of systolic or diastolic dysfunction, but not to any measurable level by the normal heart. The data reported by Mizuno et al. (19) are thus in agreement with those previously published by Kayes-Wandover and White (15) (no CYP11B2 expression in normal heart) and those in the present paper (CYP11B2 expression in failing, but not normal, human heart). No cortisol measurements were reported by Mizuno et al., and it is uncertain whether potential cardiac production of cortisol by normal (Kayes-Wandover and White) or failing human heart (our data) would be sufficient to be reflected in a consistent arteriovenous difference, given the much higher circulating levels of cortisol than aldosterone.
In stark contrast to the findings of Mizuno et al. (19) cited above, Tsutamoto et al. (20) reported substantial extraction across the normal (n = 15) and failing (n = 96) hearts, with such extraction abolished in patients with congestive cardiac failure receiving spironolactone therapy (n = 17). Although the absolute level of extraction (21.9 pg/ml) was higher in normal hearts than in patients with heart failure (14.5 pg/ml), consistent with some level of compensatory level of steroidogenesis in the failing heart, the fate of the extracted aldosterone in each case remains theoretically very challenging. Although spironolactone is a classical MR antagonist, it is inconceivable that receptor occupancy might account for all but a tiny fraction of the extracted steroid and for a strictly limited time. If aldosterone is truly being modified by the heart to the extent of 20–30% of arterial levels, then it is crucial to establish what these cardiac metabolites, not recognized but the antiserum used, in fact are.
The aorta/vascular smooth muscle cells represent the other area where apparently discrepant results are found between the two studies, perhaps again reflecting differences in tissue source. White’s laboratory found expression of both CYP11B1 and CYP11B2 in pooled aortic samples; we found expression of neither enzyme in VSMC cultured from internal mammary artery or vein. There may be significant differences in enzyme expression between vascular beds, with both VSMC and endothelial cells from the pulmonary artery reported to express CYP11B2 only (12), and human umbilical vein endothelial cells appear to express both CYP11B1 and CYP11B2 (11). In this latter instance, Takeda et al. (11) report that, as in the adrenal gland, both expression of CYP11B2 and conversion of deoxycorticosterone to aldosterone were increased by angiotensin II or elevated K+.
Mouse studies
Previously a variety of studies of cardiac and vascular expression of steroidogenic enzymes and production of aldosterone have been reported in various rat strains, but none to our knowledge in the mouse. The results for mouse heart in the present paper appear clear-cut: expression at similar levels of CYP11A and 3ßHSD in wild-type, GR knockout, and postcoronary ligation mice, with no evidence for CYP11B1 or CYP11B2 expression in normal, failing, or high ACTH (GR knockout) hearts. As in the human heart, StAR could be seen across the spectrum of hearts examined; unlike the human samples, SF-1 was also seen, albeit at higher cycle number, across the range.
In rats there are multiple reports of cardiac aldosterone synthesis. Silvestre et al. (6) reported expression of CYP11B1 and CYP11B2 in hearts from male Wistar rats, with biosynthesis of corticosterone, deoxycorticosterone, and aldosterone from endogenous precursors able to be demonstrated. Cardiac steroidogenesis was regulated by angiotensin II, ACTH, sodium restriction, and potassium supplementation in a direction identical to that in the adrenal. In contrast, Takeda et al. (13) reported that 8-wk high sodium intake increases both CYP11B2 mRNA and conversion of radiolabeled deoxycorticosterone to aldosterone, producing cardiac hypertrophy without elevation of blood pressure and suggesting a possible pathophysiological role for locally produced cardiac aldosterone quite distinct from that produced systemically. Rudolph et al. (7) similarly found cardiac expression of CYP11B1 and CYP11B2 in Wistar (but not Sprague Dawley) rats; expression could be found in either strain after 7 d of angiotensin II infusion.
There appears, then, to be a spectrum of species and strain differences in terms of cardiac aldosterone production. Wistar rats appear to enjoy cardiac aldosterone synthesis of aldosterone, with proximate stimuli a matter of some dispute; normal Sprague Dawley rat or human heart does not appear to express CYP11B2, whereas failing human heart or chronically angiotensin II-stimulated Sprague Dawley rat heart does; the mouse appears the most conservative, in that neither normal, ACTH-stimulated, nor failing mouse heart appears to express CYP11B2 (or CYP11B1). The consistency between studies (except in terms of Na+ regulation, and even here the conditions of study differed widely between the groups) and within studies (for example, between various conditions for CYP11A in the present study) suggest that the differences are real and species/strain specific, rather than reflecting vagaries of experimental design.
Kinetic considerations
Kayes-Wandover et al. (15) reported cardiac enzyme levels 0.1% or less than those in the adrenal, a finding with which the present data are in accord. Although the total mass of the heart is orders of magnitude higher than that of the adrenal, it is likely that substantial steroidogenesis is confined to relatively few cells in the heart or coronary blood vessels. If individual cells were to express steroidogenic enzymes at 1/1000th the level of a glomerulosa cell, all of the enzymes become rate-limiting, so that the biosynthetic capacity of such a cardiac cell would be 10-12 or less of that of an aldosterone-producing cell in the adrenal. If, however, aldosterone biosynthesis reflects respectable levels of enzyme expression in a tiny minority of cells on a tissue weight/total mRNA basis, then the kinetics of cardiac aldosterone biosynthesis may become orders of magnitude closer to that obtained in the adrenal. Whether such a relatively minor set of cells on an RNA content base represents vascular endothelial cells or a particular cell subpopulation among the cardiomyocytes is susceptible to experimental examination by in situ hybridization and/or immunohistochemical studies. These can thus be foreshadowed as the next major contribution to our appreciation of cardiac aldosterone synthesis and the possible pathophysiological roles that such local synthesis may play.
In summary, the present studies confirm and extend previous studies on the expression of adrenal steroidogenic enzymes in human heart and point to the potential role for cardiac aldosterone production by the failing heart. They also add an additional species (mouse) to those already studied and show that in heart failure in this species comparable expression of CYP11B2 is not seen, underlining the importance of studies of human models in this instance to explore the potential pathophysiological roles for cardiac steroidogenesis.
Footnotes
This work was supported by the Baker Medical Research Institute, a block-funded institute by the National Health and Medical Research Council of Australia, and a National Health and Medical Research Council C. J. Martin postdoctoral fellowship (to M.J.Y.).
Abbreviations: 3ßHSD, 3ß-Hydroxysteroid dehydrogenase; SF-1, steroidogenic factor-1; StAR, steroid acute regulatory protein; VSMC, vascular smooth muscle cells.
Received April 18, 2001.
Accepted July 3, 2001.
References
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