THE CIRCULATING renin-angiotensin system (RAS)

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1 /02/$15.00/0 The Journal of Clinical Endocrinology & Metabolism 87(8): Printed in U.S.A. Copyright 2002 by The Endocrine Society Expression of Aldosterone Synthase Gene in Failing Human Heart: Quantitative Analysis Using Modified Real-Time Polymerase Chain Reaction MICHIHIRO YOSHIMURA, SHOTA NAKAMURA, TERUHIKO ITO, MASAFUMI NAKAYAMA, EISAKU HARADA, YUJI MIZUNO, TOMOHIRO SAKAMOTO, MEGUMI YAMAMURO, YOSHIHIKO SAITO, KAZUWA NAKAO, HIROFUMI YASUE, AND HISAO OGAWA Department of Cardiovascular Medicine, Kumamoto University School of Medicine, Kumamoto , Japan; Division of Cardiology, Kumamoto Aging Research Institute (Y.M., H.Y.), Kumamoto , Japan; First Department of Internal Medicine, Nara Medical University (Y.S.), Kashihara, Nara , Japan; and Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine (K.N.), Shogoin, Sakyou-ku, Kyoto , Japan We have shown that aldosterone, previously thought to be synthesized solely in the adrenal cortex, is also produced in the failing human heart. One way to induce aldosterone synthesis in the heart would be to increase the expression of CYP11B2, the enzyme catalyzing the terminal step in aldosterone synthesis. However, CYP11B2 expression has never been examined in cardiac tissue from patients with heart failure. We assayed CYP11B2 expression in left ventricular tissue obtained at autopsy from seven patients. Total RNA was extracted from frozen samples. CYP11B2 gene expression was then quantitatively analyzed using a modified real-time PCR method that enabled assay of samples containing very small THE CIRCULATING renin-angiotensin system (RAS) plays a key role in the regulation of blood pressure and body fluid volume (1). Renin secreted from the kidney cleaves angiotensinogen synthesized in the liver into angiotensin I, which, in turn, is converted to angiotensin II by angiotensin-converting enzyme (ACE). Angiotensin II, the active hormone of the system, then enters the systemic circulation and is carried to its target organs: heart, blood vessels, kidneys, and adrenal glands (2, 3). Angiotensin II has a positive inotropic effect on the heart, but it also stimulates cardiac myocyte hypertrophy and interstitial fibrosis (2, 4, 5). Thus, the RAS is crucially involved in the pathophysiology of cardiac hypertrophy and the remodeling that leads to heart failure. In addition to formation in the circulation, recent evidence indicates that angiotensin II is synthesized locally in various tissues, including the heart and blood vessels, where it acts as an autocrine/paracrine factor (1). Moreover, along with others we have shown that cardiac ACE activity and gene expression are increased in the failing hearts of animals and humans (6). Aldosterone is a component of the renin-angiotensinaldosterone system (RAAS), which promotes the retention of Na and the loss of K, activates the sympathetic nervous Abbreviations: ACE, Angiotensin-converting enzyme; BNP, brain natriuretic peptide; Ct, critical threshold cycle; GAPDH, glyceraldehyde-3- phosphate dehydrogenase; RAAS, renin-angiotensin-aldosterone system; RAS, renin-angiotensin system. amounts of template DNA. The template DNA was initially amplified 1024-fold by subjecting it to 10 PCR cycles in the absence of the TaqMan probe. Thereafter, conventional realtime PCR was simultaneously performed on both target and standard samples. We measured the small quantities of CYP11B2 gene transcript and found the levels to be significantly higher in samples from heart failure patients than in those from cardiovascular disease-free patients. Our modified real-time PCR method enables quantitative analysis of gene expression using very small amounts of template DNA. CYP11B2 expression is up-regulated in the failing human heart. (J Clin Endocrinol Metab 87: , 2002) system, stimulates myocardial and vascular fibrosis, and causes baroreceptor dysfunction (5). Recent animal studies have shown that aldosterone, previously thought to be synthesized solely in the adrenal cortex, is also produced in such extraadrenal tissues as heart and blood vessels (7, 8). In addition, by measuring its levels in plasma sampled from the anterior interventricular vein, the coronary sinus, and the aortic root during cardiac catheterization, we were recently able to show that aldosterone synthesis is elevated in failing human ventricles (9). Because the coronary sinus drains blood from the heart as a whole, whereas the anterior interventricular vein drains blood from only the anterior left ventricle, one can conclude that the step-up in plasma aldosterone levels occurring between the aortic root and anterior interventricular vein is indicative of its increased synthesis in the failing ventricle (9). The enzyme catalyzing the terminal or key step in the synthesis of aldosterone is CYP11B2 (aldosterone synthase) (10). The CYPs are a large family of oxidative enzymes involved in a variety of biological functions, including adrenal steroid biosynthesis (10 12). Steroidogenic CYPs act on various ring carbons of cholesterol. For example, CYP11A1, present in mitochondria, cleaves the side-chain from C 21, CYP21A2 (21-hydroxylase), located in smooth endoplasmic reticulum, catalyzes hydroxylations at C 21, and another mitochondrial enzyme, CYP11B1 (11 -hydroxylase), catalyzes -hydroxylation at C 11. Sharing about 90% amino acid sequence identity with CYP11B1 in humans is CYP11B2, which 3936

2 Yoshimura et al. Aldosterone Synthase Gene Expression in Failing Human Heart J Clin Endocrinol Metab, August 2002, 87(8): is encoded by a gene on the long arm of chromosome 8 and has a molecular mass of 49 kda (12). The aforementioned increase in plasma aldosterone occurring within the coronary circulation means that enzymes indispensable to the hormone s synthesis must be present in the failing human heart; at least CYP11B2 must be present. Whether the increased aldosterone synthesis can be attributed to the induction of CYP11B2 expression in the failing heart is not yet known. Therefore, in the present study CYP11B2 gene expression was compared in tissue samples from failing and healthy hearts using real-time RT-PCR. Materials and Methods Left ventricle samples and total RNA extraction Samples of left ventricular tissue were obtained at autopsy from seven patients, four of whom had been diagnosed with heart failure due to old myocardial infarction (n 1, a woman), dilated cardiomyopathy (n 2, a man and a woman), or myocarditis (n 1, a man). The remaining three patients were free of cardiovascular disease, but died of leukemia (n 2, a man and a woman) or lung cancer (n 1, a man). In addition, adrenal glandular tissue was obtained from one patient without cardiovascular disease. All samples were stored 80 C until the assay. Total RNA was extracted from the frozen tissues using TRIzol reagent (Life Technologies, Inc., Tokyo, Japan) and cleaned using an RNeasy mini kit along with an ribonuclease free deoxyribonuclease kit (QIAGEN, Tokyo, Japan). Design of primers, probes for real-time PCR and standard samples Oligonucleotide primers and TaqMan probes for human CYP11B2 were designed from the GenBank databases using Primer Express version 1.0 (Perkin-Elmer, PE Applied Biosystems, Tokyo, Japan), as previously described (13, 14). The primers for human CYP11B2 used for RT-PCR were located in two different exons of each gene to avoid amplification of any contaminating genomic DNA: the forward primer was 5 -CGCAGCCAGCATCAGTGAAC-3 (exon 6), and the reverse primer was 5 -GCTCACCACTCGCTCCAAAA-3 (exon 7). The TaqMan probe was 5 -TCCCCAGAAGGCAACCACCGAG-3 (exon 7) and had a fluorescent reporter dye (FAM) covalently linked to its 5 -end and a downstream quencher dye (TAMRA) linked to its 3 -end. Fluorescence quenching depends on the spatial proximity of the reporter and quencher dyes. The primers for human B-type or brain natriuretic peptide (BNP) used for RT-PCR were located in two different exons of each gene: the forward primer was 5 -TCCTGCTCTTCTTGCATCTGG-3 (exon 1), and the reverse primer was 5 -TTTGCCCTGCAAATGGTTG-3 (exon 2). The TaqMan probe was 5 -CCCGGTTCAGCCTCGGACTTG- GAA-3 (exon 1). In addition, primers and the TaqMan probe for rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from Perkin-Elmer, PE Applied Biosystems and served as an internal control. Amplification and detection of specific products was carried out in an ABI PRISM 7700 sequence detection system (PE Applied Biosystems) using an amplification protocol consisting of 1 cycle at 50 C for 2 min, 1 cycle at 95 C for 10 min, 50 cycles at 95 C for 15 sec, and 60 C for 1 min (13, 14). To prepare template DNA standards, a DNA fragment spanning nucleotide positions 1048 to 1168 of the CYP11B2 gene was fused into plasmid, amplified, and refined. The amount of construct per well was adjusted to 50 pg and then serially diluted, yielding samples containing 50, 5, ,5 10 2,5 10 3, and pg, which were then used to construct standard plots. With this system, the intra- and interassay coefficients of variation were 19.5% (n 10) and 16.9% (n 8), respectively (15). RT-PCR and modification of conventional real-time RT-PCR Samples of total RNA (500 ng) obtained from hearts and adrenal gland were reverse transcribed according to the SuperScript First-Strand Synthesis System protocol (Life Technologies, Inc.) using oligo(deoxythymidine) as primers. In the conventional TaqMan PCR system, the reaction mixture contained 500 ng cdna template, 800 nm of each primer, 200 nm TaqMan probe, and 2 TaqMan universal master mix (Perkin-Elmer, PE Applied Biosystems) (13, 14). Target and standard samples were assayed simultaneously. The critical threshold cycle (Ct), which is defined as the cycle at which the fluorescence from the TaqMan probe becomes detectable above background, is inversely proportional to the logarithm of the initial number of template molecules. In conventional real-time RT-PCR, the Ct is cycles, and measurement is difficult if Ct is over 35 (13, 14). Consequently, very small amounts of DNA template requiring Ct to be more than 35 cannot be measured accurately using this approach. To measure the very small amounts of DNA template for human CYP11B2 obtained from our cardiac tissue samples, we modified the conventional real-time PCR method. Target gene samples, but not standard samples, were subjected to 10 PCR cycles in the absence of the TaqMan probe (protocol: 1 cycle at 50 C for 2 min, 1 cycle at 95 C for 10 min, 10 cycles at 95 C for 15 sec, and 60 C for 1 min). After this preliminary PCR step, the TaqMan probe was added to the target samples, and the conventional real-time PCR used with standard samples was applied (protocol: 1 cycle at 50 C for 2 min, 1 cycle at 95 C for 10 min, 50 cycles at 95 C for 15 sec, and 60 C for 1 min). Statistical analysis Data were expressed as the mean sd. Statistical analysis was performed using unpaired Mann-Whitney U test, as appropriate. P value less than 0.05 was considered significant. Results Assay using conventional real-time PCR For conventional real-time PCR, the CYP11B2 template content of 96 samples, including the standards, was measured simultaneously in each assay. The assays were run according to the manufacturer s protocol and normalized with GAPDH mrna. Figure 1A shows the conventional PCR amplification plot of the target samples (CYP11B2). Note that PCR was successfully applied to the target samples, but because of the small amounts of template DNA used, the Ct values were quite large ( 34 35) and were unevenly distributed in the linear range, with some positioned nearly outside the range spanned by the standard plots (Fig. 1B). This assay system was therefore deemed unsuitable for our purposes. Assay using modified real-time PCR To improve the reliability of our measurements, target gene (CYP11B2) samples were initially subjected to 10 PCR cycles without the TaqMan probe, after which the protocol for conventional real-time PCR was applied. This initial PCR step increased the amount of template DNA in the target samples (Fig. 1C) and shifted the Ct values into the linear range of the standard curve, making them suitable for quantitative analysis (Fig. 1D). Quantitative analysis of CYP11B2 or BNP in samples of human cardiac tissue We then compared the levels of CYP11B2 expression in samples of ventricular tissue from heart failure patients and patients free of cardiovascular disease by our modified real-time PCR method. Whereas the respective CYP11B2/GAPDH ratios for the three patients free of cardiovascular disease were 1.43, 3.45, and 7.57 ( 1/1024), they were 8.56, 9.60, 26.15, and ( 1/

3 3938 J Clin Endocrinol Metab, August 2002, 87(8): Yoshimura et al. Aldosterone Synthase Gene Expression in Failing Human Heart FIG. 1. Conventional or modified real-time RT-PCR. A, This figure shows the conventional PCR amplification plot of the target samples. PCR was successfully applied; however, Ct was about because of the small amounts of template DNA. Rn, Normalization is accomplished by dividing the emission intensity of the reporter dye by the emission intensity of the passive reference to obtain a ration defined as the Rn (normalized reporter) for a given reaction tube. Rn is the Rn value of a reaction containing all components including the template. Rn is the Rn value of an unreacted sample. This value may be obtained from the early cycles of a real-time run, those cycles before a detectable increase in fluorescence. This value may also be obtained from a reaction not containing the template. Rn is the difference between the Rn value and the Rn value. The magnitude of the signal generated by the given set of PCR conditions was reliably indicated. B, This figure shows the typical amplification pattern of the CYP11B2 template standards obtained by serial dilution. Because of the small amounts of template DNA used, the Ct values proportional to the amount of DNA template were unevenly distributed in the linear range, with some positioned nearly beyond the range of the standard plots; all experimental points other than the adrenal gland were so low as to be off the standard curve. This assay system was therefore deemed unsuitable for our purposes. Black dots indicate the standard samples. An asterisk (red dot) indicates the adrenal sample. C, To improve the reliability of our measurements, target gene samples were initially subjected to 10 PCR cycles without the TaqMan probe, after which the protocol for conventional real-time PCR was applied. This initial PCR step increased the amount of template DNA in the target samples. D, The initial PCR step shifted the Ct values to the linear range of the standard curve, making all experimental points including the adrenal gland suitable for quantitative analysis. Black dots indicate the standard samples. Red dots indicate the target samples. An asterisk (red dot) indicates the adrenal sample. 1024) in the patients with heart failure, and the indicated levels of CYP11B2 expression in their failing hearts were shown to be about 6-fold higher than those in healthy hearts (Fig. 2). Also, we compared the levels of human BNP expression in samples from heart failure patients and from patients free of cardiovascular disease by conventional real-time PCR. Whereas the respective BNP/GAPDH ratios for the three patients free of cardiovascular disease were 0.25, 0.61, and 1.18, they were 3.21, 6.65, 22.25, and in the patients with heart failure, and the indicated levels of BNP expression in their failing hearts were about 16-fold higher than those in control hearts (Fig. 2). CYP11B2 was subjected to 10 cycles of PCR before the real-time PCR was run, and so the ratios vs. GAPDH were not same between BNP and CYP11B2; it was 1/1024 (1/2 10 )in the CYP11B2 gene (Fig. 2). Discussion Although previously thought to be synthesized solely in the adrenal cortex, recent animal studies have shown that aldosterone is also synthesized in such extraadrenal tissues as the heart and blood vessels (7, 8); in particular, our recent findings indicate that aldosterone is produced in the ventricles of failing human hearts (9). This means that enzymes, at

4 Yoshimura et al. Aldosterone Synthase Gene Expression in Failing Human Heart J Clin Endocrinol Metab, August 2002, 87(8): FIG. 2. Quantitative analysis of CYP11B2 of human BNP in samples of human cardiac tissue. We compared the levels of gene expression in samples of ventricular tissue from heart failure patients and patients free of cardiovascular disease. CYP11B2 gene expression was measured by modified real-time PCR, and human BNP was performed by conventional real-time PCR. We found that each gene expression was significantly higher in patients with heart failure. Heart Failure, patients with heart failure; Control, patients free of cardiovascular disease. least CYP11B2, essential for aldosterone synthesis must be present in the ventricle. We therefore used a modified realtime PCR method to quantitatively analyze CYP11B2 gene expression in samples of cardiac tissue obtained at autopsy from patients with heart failure and those free of cardiovascular disease. We first performed conventional real-time RT-PCR, but the amount of target gene mrna in the cardiac tissue proved too small to generate amounts of template DNA adequate for accurate measurement. Although real-time PCR is widely used, the inability to carry out assays using only small amounts of template DNA represents a serious limitation of this method (13 15). Therefore, we initially amplified the target samples by applying 10 PCR cycles without the TaqMan probe, which increased the amount of template DNA 1024 (2 10 )-fold; only then was the conventional real-time PCR protocol applied. Theoretically, the rate of amplification of the original and the initially amplified samples should be the same; thus, the amount of PCR product ultimately produced from the latter should reflect the original amount of template applied to the assay. Indeed, by varying the number of amplification cycles in the initial PCR step, this method should enable quantitative analysis of samples of virtually any size. Using this approach we were able to show that levels of CYP11B2 expression were significantly higher in failing heart. Using this approach we were able to show that small amounts of CYP 11B2 were present even in hearts free of cardiovascular disease, and that those levels increased significantly in failing hearts. That an earlier study failed to detect CYP11B2 gene expression in adult heart (16) probably reflects the lower sensitivity of the assay used in that case and underscores the utility of our modified real-time PCR. Expression of CYP11B2 was found to be about 6 times higher in the heart failure group than in patients free of cardiovascular disease; however, the true difference in the levels of CYP11B2 expressed in healthy and failing hearts may actually be greater than was apparent from this experiment. One reason is that the three patients thought to be free of cardiovascular disease died of cancer or leukemia, which means they may have lapsed into heart failure occurring secondarily to their ailment just before death; healthy hearts from accident victims would have made for better comparisons. Moreover, as we discussed in our previous report (9), A-type or atrial natriuretic peptide and BNP are secreted from failing hearts (17), which would probably suppress the synthesis of cardiac CYP11B2 (18, 19). Aldosterone was originally thought to be important in the pathophysiology of heart failure only because of its ability to increase Na retention and K loss, but it is now understood that it also stimulates myocardial and vascular fibrosis and causes baroreceptor dysfunction (5). In addition, we have used a cultured neonatal rat cardiocyte model to reveal the presence of a positive feedback pathway from aldosterone to ACE within the local cardiac RAAS (15). This positive feedback reinforces the circular cascade of cardiac RAAS, exacerbating the heart failure. Consistent with the importance of aldosterone in the pathophysiology of heart failure, Pitt et al. (20) showed that blocking aldosterone action with a low dose of spironolactone substantially reduces the risk of morbidity and mortality among patients with severe heart failure (Randomized Aldactone Evaluation Study trial). In the present and our other studies we have clarified that the expression of the CYP11B2 gene is augmented in the failing human heart (21); however, the amount of expression was shown to be very small compared with that in the adrenal gland. There are still many issues to be discussed, such as the importance of cardiac aldosterone synthesis in cardiovascular pathophysiology (22). Furthermore, it has been reported that aldosterone is extracted by the heart in normal subjects and in patients with congestive heart failure and acute myocardial infarction (23, 24). They contend that the heart sequesters, rather than produces, aldosterone. These reports seem at odds with our report (9). In those reports ACE inhibitors were used during the study period in most, although not all, cases (23, 24). We suspect that ACE inhibitors are responsible for the difference, because we recently found that ACE inhibitors could reduce the synthesis of cardiac aldosterone synthesis (data not shown). However, the explanation citing ACE inhibitors may be partially correct. Further discussion regarding cardiac aldosterone synthesis is required to resolve these issues. In summary, we have modified the real-time PCR method, thus enabling the measurement of very small amounts of DNA template, and have found that the level of CYP11B2 expression in samples of cardiac tissue from patients diagnosed with heart failure is significantly higher than that in comparable tissue samples from patients free of cardiovascular disease. Considered in the context of our earlier studies (9), this finding indicates that CYP11B2 expression and aldosterone synthesis are both up-regulated in failing human hearts.

5 3940 J Clin Endocrinol Metab, August 2002, 87(8): Yoshimura et al. Aldosterone Synthase Gene Expression in Failing Human Heart Acknowledgments Received November 28, Accepted April 25, Address all correspondence and requests for reprints to: Michihiro Yoshimura, M.D., Department of Cardiovascular Medicine, Kumamoto University School of Medicine, Honjo, Kumamoto , Japan. bnp@kumamoto-u.ac.jp. This study was supported in part by a research grant for cardiovascular disease from the Ministry of Health and Welfare, Japan; a grantin-aid for science research from the Ministry of Education, Culture, Sports, Science and Technology, Japan; and a Smoking Research Foundation for Biomedical Research, Tokyo, Japan. References 1. Dzau VJ 1988 Circulating versus local renin-angiotensin system in cardiovascular homeostasis. Circulation 77: Moravec CS, Schluchter MD, Paranandi L, Czerska B, Stewart RW, Rosenkvanz E, Bond M 1990 Inotropic effects of angiotensin II on human cardiac muscle in vitro. 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