The Aldosterone Synthase (CYP11B2) and 11-Hydroxylase (CYP11B1) Genes Are Not Expressed in the Rat Heart
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《内分泌学杂志》
Division of Cardiovascular and Medical Sciences (P.Y., S.M.M., A.S.J., C.M., M.W.M., D.G., R.F., J.M.C.C., E.D.), Western Infirmary, Glasgow G11 6NT, United Kingdom
Molecular Endocrinology (C.J.K.), Molecular Medicine Centre, Western General Hospital, Edinburgh EH4 2XU, United Kingdom
Centre for Cardiovascular Science (G.A.G., A.W.), University of Edinburgh, Edinburgh EH8 9J2, United Kingdom
Molecular Physiology Laboratory (A.S.R., J.J.M.), University of Edinburgh Medical School, Edinburgh EH8 9AG, United Kingdom
Abstract
Aldosterone synthase (CYP11B2) and 11-hydroxylase (CYP11B1) catalyze the production of aldosterone and corticosterone, respectively, in the rat adrenal cortex. Recently, there has been some debate as to whether these corticosteroids are also produced in the hearts of rodents and humans, possibly contributing to the development of hypertrophy and myocardial fibrosis. To investigate this, we have used our established, highly sensitive real-time quantitative RT-PCR method to measure CYP11B1 and CYP11B2 mRNA levels in adrenal and cardiac tissue from several rat models of cardiovascular pathology. We have also studied isolated adult rat ventricular myocytes treated with angiotensin II and ACTH. Total RNA was isolated from the adrenal and cardiac tissue of 1) male Wistar rats with heart failure induced by coronary artery ligation and sham-operated controls; 2) stroke-prone spontaneously hypertensive rats and Wistar Kyoto rats as controls; 3) cyp1a1Ren-2 transgenic rats and Fischer controls; 4) isolated adult Sprague-Dawley ventricular myocytes incubated with 11-deoxycorticosterone (DOC), DOC plus angiotensin II, or DOC plus ACTH. Adrenal CYP11B2 expression was significantly increased in transgenic rats compared with Fischer controls (1.3 x 109± 1.2 x 109 vs. 2.1 x 107± 7.0 x 106 copies/μg RNA; P < 0.05). There were no other significant differences in adrenal CYP11B2 or CYP11B1 expression between the model animals and their respective controls. Cardiac CYP11B1 and CYP11B2 mRNA transcript levels from all in vivo and in vitro groups were never greater than 100 copies per microgram total RNA and therefore too low to be detected reproducibly. This suggests that cardiac corticosteroid production is unlikely to be of any physiological or pathological significance.
Introduction
THE CLASSICAL ACTION of aldosterone on kidney epithelial cells is well characterized, but there are many other tissues over which aldosterone exerts significant effects, including the heart. In vitro and in vivo studies confirm that aldosterone stimulates collagen synthesis by cardiac fibroblasts, promoting hypertrophy and fibrosis through direct effects on the heart and vessels (1). Clinical studies describe a correlation between serum aldosterone levels and left ventricular (LV) mass in healthy populations and in patients with essential hypertension, suggesting that aldosterone plays an important role in cardiac remodeling (2, 3). In addition, administration of aldosterone receptor antagonists to patients with congestive heart failure (CHF) reduces cardiovascular-related mortality and morbidity (4, 5).
The main source of circulating aldosterone is the adrenal zona glomerulosa, but tissues other than the adrenal cortex express enzymes required for corticosteroid synthesis, such as aldosterone synthase and 11-hydroxylase, and it is possible that locally produced aldosterone has an important physiological role (6, 7, 8). Although there is strong evidence to support production of aldosterone and the major human glucocorticoid, cortisol (corticosterone in rodents), in the brain (9, 10), research on the heart has proved to be less conclusive, and published studies fail to agree on whether cardiac production of the corticosteroids actually occurs (8, 11, 12, 13, 14, 15). Given the potential effects of locally produced aldosterone on cardiac function, and the suggestion by some that aldosterone synthase expression may be increased in heart failure (16), it is obviously important that this question is resolved.
The discrepancies between previous studies might be explained, in part, by differences in the methodologies and experimental material used (11, 12, 13, 17, 18, 19). Because expression rates are extremely low in comparison with the adrenal cortex, not only must the method of measuring it be ultrasensitive, but also the performance criteria must be firmly established to allow evaluation of the reliability of small differences. Previously, we developed a fully quantitative RT-PCR method for the detection of rat CYP11B1 and CYP11B2 transcripts using the Roche LightCycler system in combination with homologous RNA standards. This system has been used successfully to investigate these genes’ expression in the rat adrenal gland and central nervous system (10). Therefore, we have used this carefully evaluated method in an attempt to identify cardiac expression and any evidence of physiological and pathophysiological variation. To this end, we investigated cardiac 11-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) gene expression in three different rat models of cardiovascular pathology, exemplifying CHF, genetic hypertension with cardiac hypertrophy, and genetic hypertension with cardiac fibrosis. In addition, we have examined ventricular myocytes isolated from adult rats in vitro.
Materials and Methods
Experimental animals
All animal experimentation was conducted in accordance with accepted standards of humane animal care and the Animal (Scientific Procedures) Act 1986.
In study I, CHF was induced in male Wistar rats (n = 3) by means of coronary artery ligation. Sham-operated animals (n = 3) were used as controls.
Male Wistar Kyoto (WKY) rats were anesthetized, intubated, and mechanically ventilated with oxygen-enriched air. A left thoracotomy was performed and the pericardium opened. The heart was then rapidly excised and a ligature passed around the proximal portion of the left main coronary artery. After returning the heart to its position in the thorax, the suture was securely tied to produce the CHF animals but pulled through in sham-operated rats (20). Subsequently, the thorax and skin were closed and the animal placed in recovery. Buprenorphine was administered after surgery for analgesia. At 15 wk after surgery, the rats were anesthetized and a cannula placed in the right carotid artery for measurement of mean arterial blood pressure and LV end-diastolic pressure (LVEDP). Animals with LVEDP at least 15 mm Hg were considered to have CHF. The infarct size in this model is typically around 40% of the LV wall. Therefore, this is not a severe infarct model, and we do not observe any deaths before 2 months after infarction.
In study II, 12-wk-old male stroke-prone spontaneously hypertensive (SHRSP) rats (n = 6) and age-matched normotensive WKY rats (n = 6) were used to investigate whether cardiac aldosterone synthesis is a factor in the phenotype of this genetic model of hypertension and cardiac fibrosis.
In study III, transgenic cyp1a1Ren-2 (TG) rats (n = 4) and Fischer rat controls (n = 4) were used to examine hypertension and LV hypertrophy in rats with renin-induced aldosterone excess. TG rats are inbred transgenic rats with inducible hypertension caused by the coupling of the mouse Ren-2 gene to the cytochrome P450 promoter, Cyp1a1, which drives expression primarily in the liver (21).
In study IV, three groups (n = 6 per group) of adult rat ventricular myocyte (ARVM) cultures were isolated from four adult male Sprague-Dawley rats (200–225 g) and treated with ACTH (1 μM) or angiotensin (Ang) II (1 μM) for an in vitro study in which CYP11B1 and CYP11B2 gene expression and steroid synthesis, using 11-deoxycorticosterone (DOC) as a substrate (2 μM), were measured.
All animals were maintained on a normal diet throughout these studies with the exception of the TG rats, which were fed 0.15% indole-3 carbinol (wt/wt) for 4 months to induce chronic hypertension and LV hypertrophy.
Typical systolic blood pressure (tail-cuff) in SHRSP rats of this age was 195 ± 5 compared with 138 ± 3 in WKY controls (P < 0.001). In TG animals of this age, typical blood pressure (telemetry) was 189 ± 7 over 144 ± 6 compared with 124 ± 7 over 92 ± 7 in Fischer controls (P < 0.001).
Blood was collected for assay of plasma steroid levels and plasma renin activity (PRA) by RIA using previously reported methods (10, 22). The adrenal glands and whole heart or cardiac tissue (right and left ventricles and septum) were removed, cleaned, and frozen at –70 C.
Isolation and culture of ARVMs
ARVMs were isolated by a previously published method (23). Briefly, each male Sprague-Dawley rat (200–225 g) was anesthetized by injection of a 0.2-ml ketamine/xylazine (3:1 ratio) cocktail to the liver. The hearts of the rats were removed quickly and perfused in a retrograde manner through the aorta (Langendorff technique) with Ca2+-free Krebs-Henseleit buffer (118 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, and 25 mM NaHCO3) for approximately 5 min until blood was removed. Digestion was then performed by perfusing enzyme-1 (0.36% collagenase; 357 U/mg (Worthington Biochemical Corp., Freehold, NJ) and 0.35% hyaluronidase (577 U/mg) (ICN, Aurora, OH) for 20 min. All solutions were kept at 37 C. The left ventricle was cut into several pieces and shaken with enzyme-2 (enzyme-1 plus 0.03 mg/ml trypsin) (Sigma Chemical Co., St. Louis, MO) and 0.03 mg/ml DNase (Worthington) in a water bath (37 C) for 25–30 min to facilitate cell dispersion. Tissue was filtered through an 80-μm nylon mesh gauze using wash medium containing 1:1 DMEM (Life Technologies, Inc./Invitrogen, Carlsbad, CA) and Krebs-Henseleit buffer and centrifuged at 530 rpm for 3 min, and the pellet was resuspended with wash medium. The resuspended cells were washed once with BSA (64 mg/ml), layered onto laminin-coated (10 μg/ml) (Invitrogen) culture dishes and maintained in DMEM/1% penicillin-streptomycin medium containing 0.2% BSA, 2 mM carnitine, 5 mM creatine, and 2 mM taurine for 24 h before the addition of ACTH or Ang II.
Myocyte treatments
Three groups (n = 6 per group) of ARVMs isolated from four rat hearts (1.5 x 106 ventricular myocytes with a viability of 80%) were incubated for 24 h with culture medium containing either DOC (2 μM), DOC (2 μM) plus ACTH (1 μM), or DOC (2 μM) plus Ang II (1 μM). The cells were then harvested, total RNA was isolated, and the cell medium was retained for steroid analysis.
RNA isolation and quality analysis
The methods of tissue homogenization, total RNA isolation, and the normalization of mRNA against total RNA have already been described (10). RNA was treated with DNase (DNA-free; Ambion, Austin, TX) to remove contaminating genomic DNA. The quality of each isolated RNA sample was verified by electrophoresis of 1 μg total RNA on a 1% agarose gel and also by RT-PCR amplification of mRNA from -actin, a housekeeping gene. The rat -actin primers (Promega, Madison, WI) had the following sequences: 5' forward primer, 5'-TCATGAAGTGTGACGTTGACATCCGT-3', and 3' reverse primer, 5'-CTTAGAAGCATTTGCGGTGCACGATG-3'.
RT-PCR was performed on the LightCycler apparatus (Roche Diagnostics, Mannheim, Germany) using the LightCycler RNA Master SYBR Green I kit (Roche) to obtain a product of 285 bp. The protocol was as follows: RT (61 C for 20 min), denaturation (95 C for 30 sec), amplification (95 C for 1 sec, annealing temperature 55 C for 5 sec, fluorescence measurement, 72 C for 13 sec) for up to 45 cycles or until all positive samples reached the plateau phase, melting curve analysis (incubation at 95 C for 5 sec, then 45 C slowly rising to 95 C), and cooling (40 C for 30 sec).
Real-time quantitative RT-PCR
A real-time quantitative RT-PCR in combination with two homologous RNA standards was used to detect and quantify the transcripts of CYP11B1 and CYP11B2 in adrenal glands and heart tissues. Details of this method, including the standard construction, inter- and intraassay coefficients of variation, and the primer and hybridization probe sequences have been described elsewhere. The detection limit of this assay for CYP11B1 and CYP11B2 is 100 copies/μg total RNA (10).
Data analysis
mRNA expression rates and plasma hormone concentration data were analyzed by the Mann-Whitney nonparametric test. For all analyses, P < 0.05 was required for statistical significance. Data are expressed as the mean ± SEM.
Results
Table 1 shows hemodynamic values from CHF and sham-operated control rats, which act as indices of LV dysfunction. LVEDP (P < 0.01) was significantly higher, and the maximal rate of pressure rise (dP/dtmax) (P < 0.01) was significantly lower in CHF animals than in sham-operated rats. As a result of chronic left coronary artery occlusion, a collagen-rich scar developed in the infarcted zone of the left ventricle. The atrial natriuretic peptide mRNA content was also increased, supporting the onset of heart failure (24).
In the TG animals, there was a significant increase in LV mass index (2.53 ± 0.19 vs. 1.52 ± 0.03; P < 0.01) and heart weight to body weight ratio (3.19 ± 0.27 vs. 2.11 ± 0.03, P < 0.05) compared with the Fischer controls.
RNA quality was assessed by agarose gel electrophoresis of total RNA, which showed undegraded rRNA (Fig. 1), and by real-time RT-PCR of -actin mRNA. Amplification curves for the -actin RT-PCR showed amplification in all samples including, at a low level, water controls (Fig. 2A). However, the SYBR Green method of real-time RT-PCR used for -actin detection (as distinct from the hybridization probes method used for the quantitative RT-PCR, below) measures all double-stranded DNA within the reaction vessel, including nonspecific products. Melting curve analysis of these samples demonstrated a single, specific product in all positive samples and only nonspecific product in water controls (Fig. 2B). Subsequent agarose gel electrophoresis of RT-PCR product revealed a single specific band of 285 bp, with no visible product in water controls (Fig. 3). These results confirmed that the isolated RNA was of good quality.
PRA (P < 0.05) and plasma aldosterone (P < 0.05) were elevated in the TG rats (Table 2). There was no difference in plasma corticosterone levels between the TG rats and their Fischer controls (P = 0.46). There was no significant difference in plasma aldosterone and PRA between sham and CHF rats, although the values were high because of the effect of the anesthetic administered to enable hemodynamic measurements before blood collection. Plasma from SHRSP and WKY rats was not available for analysis, but PRA and aldosterone have been reported by other groups (25).
Expression of CYP11B1 and CYP11B2 was detected in the adrenal glands of all animal groups (Table 3 and Figs. 4 and 5). Adrenal gene expression did not differ significantly between CHF or SHRSP animals and their respective controls. Adrenal CYP11B2 mRNA levels were greater in TG rats than their Fischer controls (1.3 x 109± 1.2 x 109 and 2.1 x 107± 7.0 x 106 copies/μg RNA, respectively; P = 0.03), but there was no significant difference in CYP11B1 expression.
Representative amplification curves of CYP11B1 and CYP11B2 standards and heart samples from TG rats and Fischer controls and from SHRSP rats and WKY controls are shown in Fig. 6. The standard curves and the lack of amplification of CYP11B1 and CYP11B2 were similar in all animal groups. The number of cardiac CYP11B1 and CYP11B2 mRNA transcripts from all in vivo and in vitro groups was never greater than 100 copies/μg RNA and therefore too low to be detected (Table 3). Treatment of ARVMs with ACTH (1 μM) and Ang II (1 μM) had no stimulatory effect on CYP11B1 and CYP11B2 expression (Table 3). In addition, there was no conversion of DOC to corticosterone or aldosterone.
Discussion
We have attempted to detect cardiac expression of the CYP11B1 and CYP11B2 genes using in vivo rat models of CHF, genetic hypertension with cardiac fibrosis and cardiac hypertrophy as well as an in vitro cardiac myocyte preparation. In no case were we able to detect CYP11B1 or CYP11B2 mRNAs. Previous experiments have shown that the lower detection limit of our technique is approximately 100 copies/μg total RNA for CYP11B1 and CYP11B2 (10). It follows, therefore, that if the genes are expressed at all in these tissues, the level must be lower than this, i.e. vanishingly small. Moreover, in vitro, the specific agonists ACTH and Ang II failed to induce detectable expression of their respective target genes in isolated cardiac myocytes.
In contrast, some other groups have found that the expression of the CYP11B1 and CYP11B2 genes and local steroid production in cardiac tissue is not only detectable but is also substantial and responsive to agonists (26). There are several possible explanations for this discrepancy.
It is possible that local cardiac aldosterone production is to an extent strain, age, and pathophysiology dependent and that this explains the divergent results of the various studies. For example, Kayes-Wandover and White (13) detected CYP11B2 mRNA in the human fetal heart but not in any region of the adult heart, whereas Takeda et al. (18) reported that cardiac levels of CYP11B2 mRNA were higher in 2- and 4-wk-old SHRSP than in age-matched WKY rats. Others, including Yoshimura et al. (16), suggested that CYP11B2 expression may increase in the failing heart. We have attempted to address this variability by examining a number of very different rat models. Despite this diversity, however, none demonstrated significant cardiac expression.
Where analyte levels are at the limits of reliable quantitation, a probable cause of discrepancies between studies is the performance of the method. It is obviously important that performance is carefully optimized, but the limits of this performance should also be carefully established. Several factors must be considered. In the current study, DNase treatment of RNA samples was routine because DNA contamination may influence the amplification efficiency of competitive PCR, leading to erroneous results. Furthermore, mRNA concentrations are best normalized against total RNA, and any significant DNA contamination results in inaccurate quantification (27). However, not all previous studies ensured that contaminating genomic DNA was absent from RNA samples (28, 29).
This study used two in vitro-transcribed synthetic homologous mRNA standards combined with gene sequence-specific hybridization probes in a one-step RT-PCR. RT and PCR efficiencies should therefore be identical or very similar between standard and target, allowing precise quantification of amplicons. Most of the previous studies involved two-step RT-PCR, with DNA standards widely employed for the quantification of RNA. A two-step approach can increase the sensitivity of RT-PCR (30), but it reflects only cDNA concentration and ignores the efficiency of the RT stage of the reaction, which may vary from 5–90%. This is probably the major source of variability encountered in quantitative RT-PCR (31). The number of PCR cycles is another source of error. Yoshimura et al. (16) subjected their RNA samples to 10 PCR cycles before performing real-time quantitative PCR. This large number of cycles (up to 65) will amplify inaccurately and will be responsible for potentially large errors.
Our method enabled us to measure CYP11B1 and CYP11B2 transcript concentration in absolute terms, expressible as mRNA copy number, with a detection limit of 100 copies/μg total RNA for each mRNA. In all of our studies, CYP11B1 and CYP11B2 expression in cardiac tissue was never greater than 100 copies/μg RNA and was therefore too low to be detected consistently and reproducibly. This is in contrast to the central nervous system, where we previously established the presence of both mRNAs by this method. We were also able to detect the two enzymes by immunohistochemistry despite their low levels of expression and demonstrate that CYP11B2 expression in the central nervous system is regulated by dietary sodium (9, 10, 32, 33). To date, the aldosterone synthase and 11-hydroxylase enzymes have never been detected in heart tissue.
There is no simple relationship between adrenal CYP11B2 mRNA levels and plasma aldosterone levels, nor would we expect this to be the case given that plasma aldosterone levels are not a direct measure of aldosterone production at any given moment and that there are various other factors that also determine aldosterone production such as translation efficiency of mRNA, the mRNA and enzyme half-lives, deoxycorticosterone availability, and so on. However, it is apparent that a large increase in adrenal CYP11B2 mRNA associates with a proportionally smaller increase in plasma aldosterone. In this study, a 62-fold increase in adrenal CYP11B2 mRNA in TG rats compared with Fischer controls was observed alongside a 1.7-fold increase in plasma aldosterone. This is broadly in line with our previous study examining the effects of dietary sodium on adrenal CYP11B2 expression where we observed that a low sodium diet resulted in a 57-fold increase in adrenal CYP11B2 mRNA and a 2.3-fold increase in plasma aldosterone compared with animals on a normal diet (10).
our in vitro study of ARVMs, we have also been unable to detect any up-regulation in CYP11B1 and CYP11B2 expression or conversion of DOC to corticosterone or aldosterone in response to ACTH or Ang II. Previous work by Delcayre’s group reported that ACTH increased the levels of aldosterone, corticosterone, and CYP11B1 mRNA within the rat heart, as did Ang II, which also raised CYP11B2 expression (26). We also studied (Ye, P., C. J. Kenyon, S. M. Mackenzie, R. Fraser, J. M. C. Connell, and E. Davies, unpublished observations) the effects of low and high sodium intake and Ang II treatment on the heart as an adjunct to our previous studies of the central nervous system and again were unable to detect gene expression (10).
Cardiac aldosterone, if it exists, seems unlikely to be of any physiological significance. Rather, the available data suggest a dominant role in myocardial damage for adrenal aldosterone, as opposed to locally produced steroid. Rocha et al. (34) found that removal of circulating aldosterone by adrenalectomy or through administration of the selective aldosterone receptor antagonist eplerenone in a rat model of cardiac injury markedly reduced the attendant cardiac and renal damage caused by NaCl and Ang II infusion without significantly altering blood pressure.
For cardiac fibrosis to result from the direct action of aldosterone on the heart, the type 2 11-hydroxysteroid dehydrogenase enzyme would also have to be present to allow aldosterone access to the mineralocorticoid receptor. Type 2 11-hydroxysteroid dehydrogenase has never been detected in rat cardiac fibroblasts (35). Although direct effects cannot be excluded, it seems more likely that aldosterone plays its major role in the development of myocardial fibrosis indirectly, through its actions on systemic sodium retention, expansion of extravascular space, and hypervolemia. Rapid nongenomic actions of aldosterone have also been reported, and it is possible that some of the effects in the cardiovascular system may be mediated by these mechanisms (36).
Instead of cardiac aldosterone being generated locally, it is more likely that it derives from the adrenal cortex via the circulation. It has been suggested that the high concentrations of aldosterone that have been reported in heart tissue (up to 16 nM) (26) could be a result of extraction of aldosterone from the circulation rather than local synthesis (14). We conclude that the CYP11B1 and CYP11B2 genes are not expressed in rat cardiac tissue, or that expression is at such low levels that reliable detection has yet to be achieved and, therefore, that any physiological significance is extremely unlikely. Furthermore, having failed to observe any increase in CYP11B2 expression in several models of cardiac pathology, we can provide no evidence that cardiac aldosterone production is of any physiological or pathological relevance.
Acknowledgments
We thank Professor Wilson Colucci and his colleagues (Boston, MA) for their assistance with the isolated cardiomyocyte studies.
Footnotes
P.Y. is supported by a UK Overseas Research Student’s Award and a Graham Wilson Traveling Scholarship. C.A.M. is supported by the Foulkes Foundation. S.M.M. is supported by Wellcome Trust Project Grant 060362. R.F., J.M.C.C., and E.D. are supported by Medical Research Council Program Grant G9317119. C.J.K. is supported by Medical Research Council Program Grant G9306883. A.W. is supported by British Heart Foundation Grant FS/99037. A.R. is funded by a Wellcome Trust Cardiovascular Research Initiative Clinical Training Fellowship. The Roche LightCycler was financed through a grant from the National Heart Research Fund.
First Published Online September 22, 2005
Abbreviations: Ang II, Angiotensin II; ARVM, adult rat ventricular myocyte; CHF, congestive heart failure; DOC, 11-deoxycorticosterone; LV, left ventricular; LVEDP, LV end-diastolic pressure; PRA, plasma renin activity; SHRSP, stroke-prone spontaneously hypertensive; TG, transgenic cyp1a1Ren-2; WKY, Wistar Kyoto.
Accepted for publication September 12, 2005.
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Molecular Endocrinology (C.J.K.), Molecular Medicine Centre, Western General Hospital, Edinburgh EH4 2XU, United Kingdom
Centre for Cardiovascular Science (G.A.G., A.W.), University of Edinburgh, Edinburgh EH8 9J2, United Kingdom
Molecular Physiology Laboratory (A.S.R., J.J.M.), University of Edinburgh Medical School, Edinburgh EH8 9AG, United Kingdom
Abstract
Aldosterone synthase (CYP11B2) and 11-hydroxylase (CYP11B1) catalyze the production of aldosterone and corticosterone, respectively, in the rat adrenal cortex. Recently, there has been some debate as to whether these corticosteroids are also produced in the hearts of rodents and humans, possibly contributing to the development of hypertrophy and myocardial fibrosis. To investigate this, we have used our established, highly sensitive real-time quantitative RT-PCR method to measure CYP11B1 and CYP11B2 mRNA levels in adrenal and cardiac tissue from several rat models of cardiovascular pathology. We have also studied isolated adult rat ventricular myocytes treated with angiotensin II and ACTH. Total RNA was isolated from the adrenal and cardiac tissue of 1) male Wistar rats with heart failure induced by coronary artery ligation and sham-operated controls; 2) stroke-prone spontaneously hypertensive rats and Wistar Kyoto rats as controls; 3) cyp1a1Ren-2 transgenic rats and Fischer controls; 4) isolated adult Sprague-Dawley ventricular myocytes incubated with 11-deoxycorticosterone (DOC), DOC plus angiotensin II, or DOC plus ACTH. Adrenal CYP11B2 expression was significantly increased in transgenic rats compared with Fischer controls (1.3 x 109± 1.2 x 109 vs. 2.1 x 107± 7.0 x 106 copies/μg RNA; P < 0.05). There were no other significant differences in adrenal CYP11B2 or CYP11B1 expression between the model animals and their respective controls. Cardiac CYP11B1 and CYP11B2 mRNA transcript levels from all in vivo and in vitro groups were never greater than 100 copies per microgram total RNA and therefore too low to be detected reproducibly. This suggests that cardiac corticosteroid production is unlikely to be of any physiological or pathological significance.
Introduction
THE CLASSICAL ACTION of aldosterone on kidney epithelial cells is well characterized, but there are many other tissues over which aldosterone exerts significant effects, including the heart. In vitro and in vivo studies confirm that aldosterone stimulates collagen synthesis by cardiac fibroblasts, promoting hypertrophy and fibrosis through direct effects on the heart and vessels (1). Clinical studies describe a correlation between serum aldosterone levels and left ventricular (LV) mass in healthy populations and in patients with essential hypertension, suggesting that aldosterone plays an important role in cardiac remodeling (2, 3). In addition, administration of aldosterone receptor antagonists to patients with congestive heart failure (CHF) reduces cardiovascular-related mortality and morbidity (4, 5).
The main source of circulating aldosterone is the adrenal zona glomerulosa, but tissues other than the adrenal cortex express enzymes required for corticosteroid synthesis, such as aldosterone synthase and 11-hydroxylase, and it is possible that locally produced aldosterone has an important physiological role (6, 7, 8). Although there is strong evidence to support production of aldosterone and the major human glucocorticoid, cortisol (corticosterone in rodents), in the brain (9, 10), research on the heart has proved to be less conclusive, and published studies fail to agree on whether cardiac production of the corticosteroids actually occurs (8, 11, 12, 13, 14, 15). Given the potential effects of locally produced aldosterone on cardiac function, and the suggestion by some that aldosterone synthase expression may be increased in heart failure (16), it is obviously important that this question is resolved.
The discrepancies between previous studies might be explained, in part, by differences in the methodologies and experimental material used (11, 12, 13, 17, 18, 19). Because expression rates are extremely low in comparison with the adrenal cortex, not only must the method of measuring it be ultrasensitive, but also the performance criteria must be firmly established to allow evaluation of the reliability of small differences. Previously, we developed a fully quantitative RT-PCR method for the detection of rat CYP11B1 and CYP11B2 transcripts using the Roche LightCycler system in combination with homologous RNA standards. This system has been used successfully to investigate these genes’ expression in the rat adrenal gland and central nervous system (10). Therefore, we have used this carefully evaluated method in an attempt to identify cardiac expression and any evidence of physiological and pathophysiological variation. To this end, we investigated cardiac 11-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) gene expression in three different rat models of cardiovascular pathology, exemplifying CHF, genetic hypertension with cardiac hypertrophy, and genetic hypertension with cardiac fibrosis. In addition, we have examined ventricular myocytes isolated from adult rats in vitro.
Materials and Methods
Experimental animals
All animal experimentation was conducted in accordance with accepted standards of humane animal care and the Animal (Scientific Procedures) Act 1986.
In study I, CHF was induced in male Wistar rats (n = 3) by means of coronary artery ligation. Sham-operated animals (n = 3) were used as controls.
Male Wistar Kyoto (WKY) rats were anesthetized, intubated, and mechanically ventilated with oxygen-enriched air. A left thoracotomy was performed and the pericardium opened. The heart was then rapidly excised and a ligature passed around the proximal portion of the left main coronary artery. After returning the heart to its position in the thorax, the suture was securely tied to produce the CHF animals but pulled through in sham-operated rats (20). Subsequently, the thorax and skin were closed and the animal placed in recovery. Buprenorphine was administered after surgery for analgesia. At 15 wk after surgery, the rats were anesthetized and a cannula placed in the right carotid artery for measurement of mean arterial blood pressure and LV end-diastolic pressure (LVEDP). Animals with LVEDP at least 15 mm Hg were considered to have CHF. The infarct size in this model is typically around 40% of the LV wall. Therefore, this is not a severe infarct model, and we do not observe any deaths before 2 months after infarction.
In study II, 12-wk-old male stroke-prone spontaneously hypertensive (SHRSP) rats (n = 6) and age-matched normotensive WKY rats (n = 6) were used to investigate whether cardiac aldosterone synthesis is a factor in the phenotype of this genetic model of hypertension and cardiac fibrosis.
In study III, transgenic cyp1a1Ren-2 (TG) rats (n = 4) and Fischer rat controls (n = 4) were used to examine hypertension and LV hypertrophy in rats with renin-induced aldosterone excess. TG rats are inbred transgenic rats with inducible hypertension caused by the coupling of the mouse Ren-2 gene to the cytochrome P450 promoter, Cyp1a1, which drives expression primarily in the liver (21).
In study IV, three groups (n = 6 per group) of adult rat ventricular myocyte (ARVM) cultures were isolated from four adult male Sprague-Dawley rats (200–225 g) and treated with ACTH (1 μM) or angiotensin (Ang) II (1 μM) for an in vitro study in which CYP11B1 and CYP11B2 gene expression and steroid synthesis, using 11-deoxycorticosterone (DOC) as a substrate (2 μM), were measured.
All animals were maintained on a normal diet throughout these studies with the exception of the TG rats, which were fed 0.15% indole-3 carbinol (wt/wt) for 4 months to induce chronic hypertension and LV hypertrophy.
Typical systolic blood pressure (tail-cuff) in SHRSP rats of this age was 195 ± 5 compared with 138 ± 3 in WKY controls (P < 0.001). In TG animals of this age, typical blood pressure (telemetry) was 189 ± 7 over 144 ± 6 compared with 124 ± 7 over 92 ± 7 in Fischer controls (P < 0.001).
Blood was collected for assay of plasma steroid levels and plasma renin activity (PRA) by RIA using previously reported methods (10, 22). The adrenal glands and whole heart or cardiac tissue (right and left ventricles and septum) were removed, cleaned, and frozen at –70 C.
Isolation and culture of ARVMs
ARVMs were isolated by a previously published method (23). Briefly, each male Sprague-Dawley rat (200–225 g) was anesthetized by injection of a 0.2-ml ketamine/xylazine (3:1 ratio) cocktail to the liver. The hearts of the rats were removed quickly and perfused in a retrograde manner through the aorta (Langendorff technique) with Ca2+-free Krebs-Henseleit buffer (118 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, and 25 mM NaHCO3) for approximately 5 min until blood was removed. Digestion was then performed by perfusing enzyme-1 (0.36% collagenase; 357 U/mg (Worthington Biochemical Corp., Freehold, NJ) and 0.35% hyaluronidase (577 U/mg) (ICN, Aurora, OH) for 20 min. All solutions were kept at 37 C. The left ventricle was cut into several pieces and shaken with enzyme-2 (enzyme-1 plus 0.03 mg/ml trypsin) (Sigma Chemical Co., St. Louis, MO) and 0.03 mg/ml DNase (Worthington) in a water bath (37 C) for 25–30 min to facilitate cell dispersion. Tissue was filtered through an 80-μm nylon mesh gauze using wash medium containing 1:1 DMEM (Life Technologies, Inc./Invitrogen, Carlsbad, CA) and Krebs-Henseleit buffer and centrifuged at 530 rpm for 3 min, and the pellet was resuspended with wash medium. The resuspended cells were washed once with BSA (64 mg/ml), layered onto laminin-coated (10 μg/ml) (Invitrogen) culture dishes and maintained in DMEM/1% penicillin-streptomycin medium containing 0.2% BSA, 2 mM carnitine, 5 mM creatine, and 2 mM taurine for 24 h before the addition of ACTH or Ang II.
Myocyte treatments
Three groups (n = 6 per group) of ARVMs isolated from four rat hearts (1.5 x 106 ventricular myocytes with a viability of 80%) were incubated for 24 h with culture medium containing either DOC (2 μM), DOC (2 μM) plus ACTH (1 μM), or DOC (2 μM) plus Ang II (1 μM). The cells were then harvested, total RNA was isolated, and the cell medium was retained for steroid analysis.
RNA isolation and quality analysis
The methods of tissue homogenization, total RNA isolation, and the normalization of mRNA against total RNA have already been described (10). RNA was treated with DNase (DNA-free; Ambion, Austin, TX) to remove contaminating genomic DNA. The quality of each isolated RNA sample was verified by electrophoresis of 1 μg total RNA on a 1% agarose gel and also by RT-PCR amplification of mRNA from -actin, a housekeeping gene. The rat -actin primers (Promega, Madison, WI) had the following sequences: 5' forward primer, 5'-TCATGAAGTGTGACGTTGACATCCGT-3', and 3' reverse primer, 5'-CTTAGAAGCATTTGCGGTGCACGATG-3'.
RT-PCR was performed on the LightCycler apparatus (Roche Diagnostics, Mannheim, Germany) using the LightCycler RNA Master SYBR Green I kit (Roche) to obtain a product of 285 bp. The protocol was as follows: RT (61 C for 20 min), denaturation (95 C for 30 sec), amplification (95 C for 1 sec, annealing temperature 55 C for 5 sec, fluorescence measurement, 72 C for 13 sec) for up to 45 cycles or until all positive samples reached the plateau phase, melting curve analysis (incubation at 95 C for 5 sec, then 45 C slowly rising to 95 C), and cooling (40 C for 30 sec).
Real-time quantitative RT-PCR
A real-time quantitative RT-PCR in combination with two homologous RNA standards was used to detect and quantify the transcripts of CYP11B1 and CYP11B2 in adrenal glands and heart tissues. Details of this method, including the standard construction, inter- and intraassay coefficients of variation, and the primer and hybridization probe sequences have been described elsewhere. The detection limit of this assay for CYP11B1 and CYP11B2 is 100 copies/μg total RNA (10).
Data analysis
mRNA expression rates and plasma hormone concentration data were analyzed by the Mann-Whitney nonparametric test. For all analyses, P < 0.05 was required for statistical significance. Data are expressed as the mean ± SEM.
Results
Table 1 shows hemodynamic values from CHF and sham-operated control rats, which act as indices of LV dysfunction. LVEDP (P < 0.01) was significantly higher, and the maximal rate of pressure rise (dP/dtmax) (P < 0.01) was significantly lower in CHF animals than in sham-operated rats. As a result of chronic left coronary artery occlusion, a collagen-rich scar developed in the infarcted zone of the left ventricle. The atrial natriuretic peptide mRNA content was also increased, supporting the onset of heart failure (24).
In the TG animals, there was a significant increase in LV mass index (2.53 ± 0.19 vs. 1.52 ± 0.03; P < 0.01) and heart weight to body weight ratio (3.19 ± 0.27 vs. 2.11 ± 0.03, P < 0.05) compared with the Fischer controls.
RNA quality was assessed by agarose gel electrophoresis of total RNA, which showed undegraded rRNA (Fig. 1), and by real-time RT-PCR of -actin mRNA. Amplification curves for the -actin RT-PCR showed amplification in all samples including, at a low level, water controls (Fig. 2A). However, the SYBR Green method of real-time RT-PCR used for -actin detection (as distinct from the hybridization probes method used for the quantitative RT-PCR, below) measures all double-stranded DNA within the reaction vessel, including nonspecific products. Melting curve analysis of these samples demonstrated a single, specific product in all positive samples and only nonspecific product in water controls (Fig. 2B). Subsequent agarose gel electrophoresis of RT-PCR product revealed a single specific band of 285 bp, with no visible product in water controls (Fig. 3). These results confirmed that the isolated RNA was of good quality.
PRA (P < 0.05) and plasma aldosterone (P < 0.05) were elevated in the TG rats (Table 2). There was no difference in plasma corticosterone levels between the TG rats and their Fischer controls (P = 0.46). There was no significant difference in plasma aldosterone and PRA between sham and CHF rats, although the values were high because of the effect of the anesthetic administered to enable hemodynamic measurements before blood collection. Plasma from SHRSP and WKY rats was not available for analysis, but PRA and aldosterone have been reported by other groups (25).
Expression of CYP11B1 and CYP11B2 was detected in the adrenal glands of all animal groups (Table 3 and Figs. 4 and 5). Adrenal gene expression did not differ significantly between CHF or SHRSP animals and their respective controls. Adrenal CYP11B2 mRNA levels were greater in TG rats than their Fischer controls (1.3 x 109± 1.2 x 109 and 2.1 x 107± 7.0 x 106 copies/μg RNA, respectively; P = 0.03), but there was no significant difference in CYP11B1 expression.
Representative amplification curves of CYP11B1 and CYP11B2 standards and heart samples from TG rats and Fischer controls and from SHRSP rats and WKY controls are shown in Fig. 6. The standard curves and the lack of amplification of CYP11B1 and CYP11B2 were similar in all animal groups. The number of cardiac CYP11B1 and CYP11B2 mRNA transcripts from all in vivo and in vitro groups was never greater than 100 copies/μg RNA and therefore too low to be detected (Table 3). Treatment of ARVMs with ACTH (1 μM) and Ang II (1 μM) had no stimulatory effect on CYP11B1 and CYP11B2 expression (Table 3). In addition, there was no conversion of DOC to corticosterone or aldosterone.
Discussion
We have attempted to detect cardiac expression of the CYP11B1 and CYP11B2 genes using in vivo rat models of CHF, genetic hypertension with cardiac fibrosis and cardiac hypertrophy as well as an in vitro cardiac myocyte preparation. In no case were we able to detect CYP11B1 or CYP11B2 mRNAs. Previous experiments have shown that the lower detection limit of our technique is approximately 100 copies/μg total RNA for CYP11B1 and CYP11B2 (10). It follows, therefore, that if the genes are expressed at all in these tissues, the level must be lower than this, i.e. vanishingly small. Moreover, in vitro, the specific agonists ACTH and Ang II failed to induce detectable expression of their respective target genes in isolated cardiac myocytes.
In contrast, some other groups have found that the expression of the CYP11B1 and CYP11B2 genes and local steroid production in cardiac tissue is not only detectable but is also substantial and responsive to agonists (26). There are several possible explanations for this discrepancy.
It is possible that local cardiac aldosterone production is to an extent strain, age, and pathophysiology dependent and that this explains the divergent results of the various studies. For example, Kayes-Wandover and White (13) detected CYP11B2 mRNA in the human fetal heart but not in any region of the adult heart, whereas Takeda et al. (18) reported that cardiac levels of CYP11B2 mRNA were higher in 2- and 4-wk-old SHRSP than in age-matched WKY rats. Others, including Yoshimura et al. (16), suggested that CYP11B2 expression may increase in the failing heart. We have attempted to address this variability by examining a number of very different rat models. Despite this diversity, however, none demonstrated significant cardiac expression.
Where analyte levels are at the limits of reliable quantitation, a probable cause of discrepancies between studies is the performance of the method. It is obviously important that performance is carefully optimized, but the limits of this performance should also be carefully established. Several factors must be considered. In the current study, DNase treatment of RNA samples was routine because DNA contamination may influence the amplification efficiency of competitive PCR, leading to erroneous results. Furthermore, mRNA concentrations are best normalized against total RNA, and any significant DNA contamination results in inaccurate quantification (27). However, not all previous studies ensured that contaminating genomic DNA was absent from RNA samples (28, 29).
This study used two in vitro-transcribed synthetic homologous mRNA standards combined with gene sequence-specific hybridization probes in a one-step RT-PCR. RT and PCR efficiencies should therefore be identical or very similar between standard and target, allowing precise quantification of amplicons. Most of the previous studies involved two-step RT-PCR, with DNA standards widely employed for the quantification of RNA. A two-step approach can increase the sensitivity of RT-PCR (30), but it reflects only cDNA concentration and ignores the efficiency of the RT stage of the reaction, which may vary from 5–90%. This is probably the major source of variability encountered in quantitative RT-PCR (31). The number of PCR cycles is another source of error. Yoshimura et al. (16) subjected their RNA samples to 10 PCR cycles before performing real-time quantitative PCR. This large number of cycles (up to 65) will amplify inaccurately and will be responsible for potentially large errors.
Our method enabled us to measure CYP11B1 and CYP11B2 transcript concentration in absolute terms, expressible as mRNA copy number, with a detection limit of 100 copies/μg total RNA for each mRNA. In all of our studies, CYP11B1 and CYP11B2 expression in cardiac tissue was never greater than 100 copies/μg RNA and was therefore too low to be detected consistently and reproducibly. This is in contrast to the central nervous system, where we previously established the presence of both mRNAs by this method. We were also able to detect the two enzymes by immunohistochemistry despite their low levels of expression and demonstrate that CYP11B2 expression in the central nervous system is regulated by dietary sodium (9, 10, 32, 33). To date, the aldosterone synthase and 11-hydroxylase enzymes have never been detected in heart tissue.
There is no simple relationship between adrenal CYP11B2 mRNA levels and plasma aldosterone levels, nor would we expect this to be the case given that plasma aldosterone levels are not a direct measure of aldosterone production at any given moment and that there are various other factors that also determine aldosterone production such as translation efficiency of mRNA, the mRNA and enzyme half-lives, deoxycorticosterone availability, and so on. However, it is apparent that a large increase in adrenal CYP11B2 mRNA associates with a proportionally smaller increase in plasma aldosterone. In this study, a 62-fold increase in adrenal CYP11B2 mRNA in TG rats compared with Fischer controls was observed alongside a 1.7-fold increase in plasma aldosterone. This is broadly in line with our previous study examining the effects of dietary sodium on adrenal CYP11B2 expression where we observed that a low sodium diet resulted in a 57-fold increase in adrenal CYP11B2 mRNA and a 2.3-fold increase in plasma aldosterone compared with animals on a normal diet (10).
our in vitro study of ARVMs, we have also been unable to detect any up-regulation in CYP11B1 and CYP11B2 expression or conversion of DOC to corticosterone or aldosterone in response to ACTH or Ang II. Previous work by Delcayre’s group reported that ACTH increased the levels of aldosterone, corticosterone, and CYP11B1 mRNA within the rat heart, as did Ang II, which also raised CYP11B2 expression (26). We also studied (Ye, P., C. J. Kenyon, S. M. Mackenzie, R. Fraser, J. M. C. Connell, and E. Davies, unpublished observations) the effects of low and high sodium intake and Ang II treatment on the heart as an adjunct to our previous studies of the central nervous system and again were unable to detect gene expression (10).
Cardiac aldosterone, if it exists, seems unlikely to be of any physiological significance. Rather, the available data suggest a dominant role in myocardial damage for adrenal aldosterone, as opposed to locally produced steroid. Rocha et al. (34) found that removal of circulating aldosterone by adrenalectomy or through administration of the selective aldosterone receptor antagonist eplerenone in a rat model of cardiac injury markedly reduced the attendant cardiac and renal damage caused by NaCl and Ang II infusion without significantly altering blood pressure.
For cardiac fibrosis to result from the direct action of aldosterone on the heart, the type 2 11-hydroxysteroid dehydrogenase enzyme would also have to be present to allow aldosterone access to the mineralocorticoid receptor. Type 2 11-hydroxysteroid dehydrogenase has never been detected in rat cardiac fibroblasts (35). Although direct effects cannot be excluded, it seems more likely that aldosterone plays its major role in the development of myocardial fibrosis indirectly, through its actions on systemic sodium retention, expansion of extravascular space, and hypervolemia. Rapid nongenomic actions of aldosterone have also been reported, and it is possible that some of the effects in the cardiovascular system may be mediated by these mechanisms (36).
Instead of cardiac aldosterone being generated locally, it is more likely that it derives from the adrenal cortex via the circulation. It has been suggested that the high concentrations of aldosterone that have been reported in heart tissue (up to 16 nM) (26) could be a result of extraction of aldosterone from the circulation rather than local synthesis (14). We conclude that the CYP11B1 and CYP11B2 genes are not expressed in rat cardiac tissue, or that expression is at such low levels that reliable detection has yet to be achieved and, therefore, that any physiological significance is extremely unlikely. Furthermore, having failed to observe any increase in CYP11B2 expression in several models of cardiac pathology, we can provide no evidence that cardiac aldosterone production is of any physiological or pathological relevance.
Acknowledgments
We thank Professor Wilson Colucci and his colleagues (Boston, MA) for their assistance with the isolated cardiomyocyte studies.
Footnotes
P.Y. is supported by a UK Overseas Research Student’s Award and a Graham Wilson Traveling Scholarship. C.A.M. is supported by the Foulkes Foundation. S.M.M. is supported by Wellcome Trust Project Grant 060362. R.F., J.M.C.C., and E.D. are supported by Medical Research Council Program Grant G9317119. C.J.K. is supported by Medical Research Council Program Grant G9306883. A.W. is supported by British Heart Foundation Grant FS/99037. A.R. is funded by a Wellcome Trust Cardiovascular Research Initiative Clinical Training Fellowship. The Roche LightCycler was financed through a grant from the National Heart Research Fund.
First Published Online September 22, 2005
Abbreviations: Ang II, Angiotensin II; ARVM, adult rat ventricular myocyte; CHF, congestive heart failure; DOC, 11-deoxycorticosterone; LV, left ventricular; LVEDP, LV end-diastolic pressure; PRA, plasma renin activity; SHRSP, stroke-prone spontaneously hypertensive; TG, transgenic cyp1a1Ren-2; WKY, Wistar Kyoto.
Accepted for publication September 12, 2005.
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