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(The FASEB Journal. 2002;16:1537-1549.)
© 2002 FASEB

Testosterone, cytochrome P450, and cardiac hypertrophy

THOMAS THUM and JÜRGEN BORLAK1

Center of Drug Research and Medical Biotechnology, Fraunhofer Institute of Toxicology and Aerosol Research, Hannover, Germany

1Correspondence: Fraunhofer Institute of Toxicology and Aerosol Research, Center for Drug Research and Medical Biotechnology, Nicolai-Fuchs-Str. 1, D-30659 Hannover, Germany. E-mail: Borlak{at}ita.fhg.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cytochrome P450 mono-oxygenases (CYP) play an essential role in steroid metabolism, and there is speculation that sex hormones might influence cardiac mass and physiology. As CYP mono-oxygenases activity is frequently altered during disease, we tested our hypothesis that CYP mono-oxygenase expression and testosterone metabolism are altered in cardiac hypertrophy. We investigate major CYP mono-oxygenase isoforms and other steroid-metabolizing enzymes and the androgen receptor in normal, hypertrophic, and assist device-supported human hearts and in spontaneously hypertensive rats (SHR). We show increased and idiosyncratic metabolism of testosterone in hypertrophic heart and link these changes to altered CYP mono-oxygenase expression. We show significant induction of 5-alpha steroid reductase and P450 aromatase gene expression and enhanced production of dihydrotestosterone, which can be inhibited by the 5-alpha reductase inhibitor finasteride. We show increased gene expression of the androgen receptor and increased levels of lipid peroxidation in diseased hearts, the latter being markedly inhibited by CYP mono-oxygenase inactivation. We show alpha-MHC to be significantly repressed in cardiac hypertrophy and restored to normal on testosterone supplementation. We conclude that heart-specific steroid metabolism is of critical importance in cardiac hypertrophy.—Thum, T., Borlak, J. Testosterone, cytochrome P450, and cardiac hypertrophy.


Key Words: CYP • cytochrome P450 mono-oxygenases • testosterone metabolism • cardiac hypertrophy


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CYTOCHROME P450 MONO-OXYGENASES catalyze oxidation of a wide range of drugs and other xenobiotics and are responsible for the metabolism of endogenous compounds, including steroids (1 , 2) , cholesterol, bile, fatty acids (3 , 4) , bradykinin (5) , and certain vitamins (6) .

Expression of CYP mono-oxygenases is reported for extrahepatic tissues including lung, kidney, gastrointestinal tract, endothelium, and heart (7 8 9 10) . Tissue-specific metabolism of endogenous compounds may lead to the production of biologically active molecules, as observed with arachidonic acid, which is a substrate for several CYP isoforms (11) . Arachidonic acid is preferentially catalyzed by CYP2J to regioisomeric cis-epoxyeicosatrienoic acids, midchain cis-trans-conjugated dienols, and omega/omega-1 alcohols (12 , 13) , all of which are signaling molecules of vascular tonus and cardiac contractility. EETs have been reported to exacerbate ischemia and reperfusion injury in isolated guinea pig hearts (14) , but are also a key factor in the kidney for the onset and development of spontaneous hypertension in rats (15 , 16) . EETs are implicated in excitation-contraction coupling via modulation of intracellular cAMP levels (17) , and this provides additional evidence for an important role of CYP2J in cardiac physiology. Irreversible inhibition of cytochrome P450 reduces significantly the activity of the cardiac L-type calcium channel, the level of intracellular free calcium, and, in general, cell shortening in rat single ventricular myocytes, which points to a novel function for CYP mono-oxygenases in the regulation of cardiac contractility (18) . Besides its role in the activation of biologically active compounds, the cytochrome P450 system generates superoxide anions, hydrogen peroxide, and hydroxyl radicals (19) ; although inconclusive, oxygen-derived free radicals may insult intracellular proteins, which might contribute to the development of heart failure and cardiac hypertrophy (20 21 22 23) .

We recently reported the expression of P450 isoforms in explanted human hearts (10) and in cultures of cardiomyocytes of adult rats, and demonstrated the ability of cultured cardiomyocytes to metabolize steroid hormones (1) . It is suggested that testosterone is a risk factor for cardiac hypertrophy as abundant binding to the androgen receptor (24) and its translocation into the nucleus may lead to exaggerated cardiac-specific gene expression (25 , 26) . Tissue-specific metabolism of testosterone could also lead to the production of high-affinity ligands of the androgen receptor, i.e., dihydro-testosterone (DHT) (27) ; thus, enhanced production of DHT may accelerate cardiac hypertrophy via the androgen receptor signaling pathway.

High glucocorticoid steroid (dexamethasone) therapy of preterm and newborn infants and in animal models leads to cardiac hypertrophy (28 , 29) , and confirmation of a causal relationship between dexamethasone mediated CYP mono-oxygenase induction and altered steroid (testosterone) metabolism come from a wide range of studies (30 31 32) .

Recently, Hayward and colleges published a medical hypothesis where increased left ventricular mass is linked to endogenous sex hormone concentrations (33) , and we propose cytochrome P450s to play an essential role in cardiac physiology and tissue-specific steroid metabolism. We thus investigated the gene expression and enzyme activity of major CYP isoforms and key steroid-metabolizing enzymes: 17beta- and 3beta- hydroxy-steroid-dehydrogenases, aromatase, and 5alpha-reductase in normal and hypertrophic human and rat (SHR) heart tissue. We tested our hypothesis that altered steroid hydroxylation is linked to cardiac hypertrophy. We correlate gene expression and enzyme activity of CYP mono-oxygenases with expression of the androgen receptor and investigated the metabolism of testosterone in normal, hypertrophic, and assist device-supported human hearts and in normotensive and SHRs, the latter being an accepted animal model of cardiac hypertrophy (34 , 35) .

We investigated the gene expression of alpha-myosin heavy chain (MHC), a marker for cellular differentiation and cardiac remodeling, and the amount of lipid peroxidation products in heart tissue of hypertrophic and assist device-supported left ventricles to assess the level of lipid peroxidation products in diseased cardiac tissue.

This study aims to explore CYP mono-oxygenase activity and steroid hydroxylation in cardiac hypertrophy.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
All animal procedures described were approved by the ethical committee of the local government of Hannover, Germany, and the investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996).

Male Sprague Dawley and SHRs weighing ~200 g were obtained from Charles River (Sulzfeld, Germany). Food and water was given ad libitum.

Rats were anesthetized with ketamin (anesthetic) and xylazin-hydrochloride (muscle relaxant) with 0.1 mL of ketamin/100 g body weight and 0.05 mL of xylazin-hydrochloride/100 g body weight; 2000 international units of heparin were given intraperitoneally before surgery.

Human heart tissue
Approval for the use of tissue material was obtained from the ethical committee of the Medical School of Hannover, Germany.

Immediately after explantation, biopsy material was removed from the left ventricle; excised tissue was shock-frozen in liquid nitrogen and stored at -80°C until analyzed. Patients that suffered form dilatative cardiomyopathy (DCM; n=8), of which five received implantation of a left ventricular assist device for >6 months, and patients with combined DCM and left ventricular hypertrophy (LVH; n=3) were included. Wall thickness of the hypertrophic cardiac tissue (combined DCM+LVH) was significantly higher than in assist device-supported hearts (1.65±0.25 cm vs. 1.1±0.1 cm). Biopsies from LV of normal human hearts (n=2) originally intended for transplantation were used as controls.

Isolation of microsomal membranes from ventricular tissue
Tissue from human and rat left ventricles were cut into small pieces and homogenized with an ultraturrax (IKA, Germany) in ice-cold KCl-buffer (0.15 M, pH 7.4). Isolation of microsomal membranes was done as described previously (10) . The resultant microsomal fraction was transferred into TRIS-sucrose buffer (0.25 M sucrose, 20 mM TRIS-buffer, 5 mM EDTA), shock-frozen in liquid nitrogen, and stored at -80°C until further use. All experimental results are obtained from at least n = 3 individual animals.

Determination of microsomal protein
Microsomal protein concentrations were determined according to Smith et al. (36) and adjusted to 1 mg protein/mL.

Metabolism of testosterone
Heart microsomal protein (500 µg) and 50 µg of liver microsomal protein (positive control) were incubated with 100 µM testosterone and 1 mg/mL NADPH in a final volume of 1 mL TRIS buffer (20 mM, Sigma, Deisenhofen, Germany) for 4 h at 37°C in a shaking water bath. Samples were shock-frozen in liquid nitrogen and stored at -80°C until further analysis. Assay controls included CO-treated microsomal suspensions and boiled microsomes. Finasteride (25 µM) was used as a 5-alpha-steroid reductase inhibitor to quantify the contribution of CYP mono-oxygenases in the production of DHT.

HPLC assay for testosterone and its metabolites
Testosterone and its metabolites were analyzed by HPLC according to Arlotto et al. (37) with slight modifications and with 11{alpha}-hydroxyprogesterone as an internal standard. 11-{alpha}-hydroxyprogesterone (1 µg) was added to 1 mL of cell culture supernatant. After addition of 100 µL isopropanol, the samples were extracted with 5 mL ethyl acetate by gentle shaking for 20 min. Extracts were evaporated to dryness, the residues were reconstituted in 100 µL of the mobile phase (water/methanol/acetonitrile, 60/25/15, v/v/v), and 80 µL of the sample was injected in the HPLC system (Hewlett Packard HP1100). The mobile phase was delivered at a flow rate of 1 mL min-1 using the HP 1100 Quaternary Pump. Chromatographic separation of metabolites was achieved on a C18 Nucleosil column, 250 x 4 mm and a particle size of 5 µm (Macherey-Nagel, Germany). At a temperature of 30°C testosterone metabolites were detected by UV absorption at 246 and 285 nm (for detection of DHT) using synthetic reference standards. The mobile phase consisted of water (solvent A), methanol (solvent B), and acetonitrile (solvent C); analysis was done with an isocratic elution of 60% A, 25% B, and 15% C for 12 min, followed by 45% A, 40% B, and 15% C for 3 min, and finally, 45% A, 45% B, and 10% C thereafter. The total run time was 45 min per sample.

Isolation and culture of cardiomyocytes of adult rats
This was done as described previously (1) . Twenty-four hours after isolation, cells were treated with 100 µM testosterone for 8 h. Cardiomyocytes were removed, centrifuged for 5 min at 1200 RPM, and the pellet was shock-frozen until further RNA analysis.

RNA and cDNA
RNA was isolated from heart tissue using a total RNA Isolation kit (Macherey-Nagel, Germany) according to the manufacturer’s recommendation. Quality of isolated RNA was checked using a 1.0% agarose gel. Total RNA (2 µg) from each sample was used for reverse transcription. RNA and random primer (Roche, Mannheim, Germany) were preheated for 10 min at 70°C, then 5x RT-AMV-buffer, dNTPs (1 mM, Roche, Germany), 40 U RNase inhibitor (Stratagene, Amsterdam, Netherlands), 20 U AMV-RT (Promega, Mannheim, Germany) were added and diethyl pyrocarbonate (Sigma, Deisenhofen, Germany) -treated water was added to a final volume of 20 µL. Reverse transcription was carried out for 60 min at 42°C and stopped by heating to 95°C for 5 min. The resulting cDNA was frozen at -20°C until further experimentation.

Thermocycler RT-PCR
PCR reactions were carried out in a thermal cycler (T3, Biometra, Germany) using the following melting, annealing, and extension cycling conditions: denaturation for 30 s at 94°C, annealing for 60 s at 57°C, and extension for 60 s at 72°C for CYP isoforms (33 cycles), alpha-MHC, and cyclophilin (26 cycles). Annealing temperature for the androgen receptor, 17beta HSD I-IV, 3beta HSD I, P450 aromatase, renin, c-jun, and 5-alpha-reductase was 55°C for 60 s and 30–35 cycles were carried out. DNA contamination was checked for by direct amplification of RNA extracts before conversion of RNA to cDNA and any possible contamination could be excluded. PCR reactions were done within the linear range of amplification, separated using a 1.5% agarose gel, stained with ethidium bromide, and photographed on a transilluminator (Kodak image station 440; see Fig. 1 ).



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  		Figure 1. mRNA expression of cytochrome P450 mono-oxygenases in rat heart and liver tissue. LV = left ventricle; SD = Sprague Dawley; SHR = spontaneously hypertensive rat.

Real-time semiquantitative PCR
cDNA was diluted 1:10 with nuclease-free water and 1 µL of the diluted cDNA was added to the Lightcycler-Mastermix (0.5 µM of specific primer, 4 mM MgCl2, and 2 µL Master-SYBR-Green, Roche, Mannheim, Germany) and adjusted to a final volume of 20 µL. PCR reactions were carried out with the Lightcycler (Roche, Mannheim, Germany). After an initial denaturation step at 95°C for 30 s, the PCR reaction was initiated with an annealing temperature of 55–58°C for 7 s, followed by an extension phase for 12–20 s at 72°C and a denaturation cycle at 95°C for 1 s (for further details and oligonucleotide sequences, see Table 1 ). The PCR reaction was stopped after a total of 30 to 50 cycles; at the end of each extension phase, fluorescence was observed and used for quantitative measurements. To exclude unspecific product formation, fluorescence was measured above the primer dimer melting temperature. A melting point analysis was carried out by heating the DNA synthesis product from 65°C to 95°C and a characteristic melting point curve was obtained (data not shown). Exact quantification was achieved by a serial dilution with cDNA produced from heart total RNA extracts using 1:10 dilution steps. Control samples contained water instead of cDNA; occasionally dimer production was seen but could easily be distinguished by melting point analysis.


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  		Table 1A. Used oligonucleotide primers in human studies


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  		Table 1B. Used oligonucleotide primers in rat studies

Thiobarbituric acid-reactive substances in microsomes of explanted heart tissue
Microsomal extracts (each 500 µg) from heart tissue of LVAD patients (n=2) and hypertrophied left ventricles (n=3) were mixed with 25 µL of 4% butylated hydroxytoluene, 250 µL of 0.7% thiobarbituric acid, and 500 µL of an aqueous solution containing 10% phosphotungstic acid in 0.5 M sulfuric acid. The reaction mixture was heated for 50 min at 95°C, then 1 mL n-butanol was added. The mixture was then centrifuged at 4000 rpm for 5 min, the supernatant was taken, and the absorbance was measured photometrically at 535 nm against a reaction mixture blank. The amounts of TBARS were calculated by using tetraethoxypropane in 1% sulfuric acid as a standard.

Additional incubation studies with microsomes from hypertrophic human left ventricles (n=3) were carried out where microsomes were heat inactivated, pretreated with carbon monoxide (CO) for 2 min, or not treated. Incubation solutions consisted of 500 µg microsomal protein and 1 mg/mL NADPH in 1 mL TRIS buffer (20 mM, pH 7.4) and were incubated for 60 min at 37°C in a shaking water bath. Thereafter, 400 µL from each sample was taken and TBARS content was determined as described above.

Androgen-responsive elements (AREs) in the alpha-MHC gene promotor
We used GEMS Launcher Release 3.0 software (Genomatix, Germany) to construct a matrix for the ARE using sequence information from the transcription factor database TRANSFAC 4.0 (http://transfac.gbf.de/TRANSFAC/), as well as from published data (38 39 40) . We developed a matrix with highly conserved bases for the androgen-responsive element (5'-XGXXCXXXXAGTTCT-3') and searched for possible binding sites in the human, rat, and murine alpha-MHC promotor.

Statistical analysis
Results are expressed as mean ± SD. The Wilcoxon signed rank test was used, where control and diseased heart tissue was studied in parallel and paired in a given experiment. Differences were considered significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Human and rat cardiac gene expression studies (qualitative analysis)
With human healthy, hypertrophic, and assist device-supported left ventricle CYP mono-oxygenase gene expression was confined to CYP1A1, CYP2A6/7, CYP2B6/7, CYP2C8, CYP2E1, CYP2J2, and CYP4A11. For some individuals, expression of CYP1A1, CYP2B6/7, and CYP2C8 transcript levels was low or could not be detected (see Table 2 ). Expression of CYP1A2, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2F1, CYP3A4, CYP3A5, CYP3A7, and CYP4B1 was below the limit of detection.


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  		Table 2. Cytochrome P450 monooxygenase gene expression in human and rat heart tissue (qualitative analysis)

With rat cardiac tissue (left ventricles) gene expression was confined to CYP1A1, CYP1B1, CYP2A1/2, CYP2B6/7, CYP2E1, and CYP2J3 whereas expression of CYP1A2, CYP2C11, CYP3A1, CYP3A2, or CYP4A1 was below the limit of detection.

Cyclophilin and GAPDH served as housekeeping genes and were uniformly expressed in all tissues investigated (see Table 2 ); rat and human liver cDNA was used as positive control for CYP gene expression. Figure 1 depicts a representative ethidium bromide stained agarose gel of RT-PCR amplified genes.

Semiquantitative gene expression analysis in ventricular tissue
CYP mono-oxygenases
We examined CYP gene expression in human normal, hypertrophic, and assist device-supported left ventricular tissue and found CYP2J2 gene expression to be four- to fivefold increased when explanted normal and hypertrophic human left ventricles were compared with LVAD-supported hearts. In hypertrophic tissue, CYP2A6/7 and CYP4A11 gene expression was increased two- to threefold but CYP2E1 gene expression remained basically unchanged (see Fig. 2 ).



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  		Figure 2. Semiquantitative real-time RT-PCR of cytochrome P450 mono-oxygenases, P450 aromatase, renin, and c-jun with human heart cDNA. Results obtained with normal healthy human left ventricular tissue were set to 100%. The ratio of gene expression of interest (nominator) vs. cyclophilin (housekeeping gene, denominator) is shown. Data represent mean ± SD (*P<0.05).

We compared CYP mono-oxygenase gene expression in left ventricular tissue of normotensive (n=3) and SHRs (n=3); expression of CYP1B1, CYP2A1/2, CYP2B1/2, CYP2E1, and CYP2J3 was 8-, 50-, 6-, 6-, and 4-fold increased in left ventricular tissue of SHRs (see Fig. 3 ).



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  		Figure 3. Semiquantitative real-time RT-PCR of cytochrome P450 mono-oxygenases, renin, and c-jun with rat heart cDNA. Results obtained with normotensive rat left ventricular tissue were set to 100%. The ratio of gene expression of interest (nominator) vs. cyclophilin (housekeeping gene, denominator) is shown. Data represent mean ± SD of n = 3 different rats. (*P<0.05).

5-Alpha-steroid reductase
5-Alpha-steroid reductase gene expression was up to fourfold increased in human hypertrophic left ventricles compared with healthy or assist device-supported cardiac tissue. Similarly, 5-alpha-steroid reductase gene expression was 2.5-fold increased in left ventricles of SHRs vs. normotensive rats (see Fig. 4 ).



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  		Figure 4. Semiquantitative real-time RT-PCR of 5-alpha-steroid reductase, 17-beta-hydroxy-steroid-dehydrogenase isoforms I, II, and IV with human and rat heart cDNA. Results obtained with normal healthy human left ventricular tissue of normotensive rat heart tissue were set to 100%. The ratio of gene expression of interest (nominator) vs. cyclophilin (housekeeping gene, denominator) is shown. Data represent mean ± SD (*P<0.05).

17-Beta-hydroxysteroid dehydrogenases type I-IV
Isoform I of 17-beta-hydroxy-steroid-dehydrogenase (17-beta-HSD) was decreased by 50% in left ventricles of human hypertrophic hearts, but we observed no change in the expression of isoform II of this gene (see Fig. 4 ). Isoforms III and IV were not detected in any of the human heart tissues examined by us, but were expressed in human liver.

In strong contrast, expression of 17-beta-HSD isoform I and IV was increased five- and twofold in SHR rats, whereas expression of 17-beta-HSD isoforms II and III was below the limit of detection.

3-Beta-hydroxysteroid dehydrogenase type I
Transcript copies of this gene were not detected in human or rat heart tissue but were identified in human and rat liver tissue (data not shown).

P450 aromatase
Gene expression of P450 aromatase was increased up to 20-fold in hypertrophic and assist device-supported hearts, but due to high interindividual differences, no statistical significance could be calculated (3- to 20-fold; see Fig. 2 ). In strong contrast, P450 aromatase was only detected in rat liver tissue.

Androgen receptor
Gene expression of the androgen receptor was 4- and 12-fold increased in LVAD and human hypertrophic left ventricles (see Fig. 5 A). Similarly, gene expression of the rat androgen receptor was increased 27-fold in left ventricular tissue of SHRs (Fig. 5B ).



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  		Figure 5. A, B) Semiquantitative real-time RT-PCR the androgen receptor with human (A) and rat (B) heart cDNA. Results obtained with normal human hearts or normotensive rat left ventricular tissue were set to 100%. The ratio of gene expression of interest (nominator) vs. cyclophilin (housekeeping gene, denominator) is shown. Data represent mean ± SD (*P<0.05). C) mRNA expression of the androgen receptor in control and treated (100 µM testosterone for 8h) cultures of cardiomyocytes of adult rats. Data represent mean ± SD of n = 3 individual cell culture experiments with ~ 2 million cells per culture dish (*P<0.05).

Gene expression of the androgen receptor was 50% of controls upon treatment of rat cardiomyocyte cultures with 100 µM testosterone for 24h (see Fig. 5C ).

Renin and c-jun
Renin and c-jun gene expression is up-regulated in hearts of SHRs (41 , 42) and thus served as marker genes.

Renin and c-jun gene expression was increased 12- and 2-fold in left ventricular tissue of SHRs (see Fig. 3 ), and our data are in accordance with previous findings (41 , 42) . Expression of the latter genes was also investigated in normal and diseased human ventricular tissue; we found c-jun to be basically unchanged in hypertrophic left ventricles, but its expression was 14% of controls in LVADs. In contrast, renin expression was increased ~ fourfold in hypertrophic left ventricular tissue (see Fig. 2 ) but was absent in LVADs.

Alpha-myosin heavy chain
Alpha-MHC was ~20% of controls in hypertrophic cardiac tissue and with LVADs ~70% of controls. Rat alpha-MHC gene expression was 50% of normotensive controls (see Fig. 6 A, B).



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  		Figure 6. A, B) Semiquantitative real-time RT-PCR of alpha myosin heavy chain with human (A) and rat (B) heart cDNA. Results obtained with normal human hearts or normotensive rat left ventricular tissue was set to 100%. The relative expression gene of interest vs. cyclophilin is shown. (SHR=spontaneously hypertensive rat). Data represent mean ± SD (*P<0.05). C) mRNA expression of alpha myosin heavy chain in control and treated (100 µM testosterone for 8h) cultures of cardiomyocytes of adult rats. Data represent mean ± SD of 3 individual cell culture experiments with ~ 2 million cells per culture dish (*P<0.05).

Treatment of calcium-tolerant adult rat cardiomyocyte cultures with 100 µM testosterone resulted in an ~ twofold increase in alpha-MHC gene expression (see Fig. 6C ).

Testosterone metabolism in cardiac tissue
Assays with human normal, hypertrophic, and assist device-supported heart tissue
Metabolite production of DHT, androstenedione, and 7-alpha-HT was increased 3-, 2.5-, and 4-fold in hypertrophic human hearts compared with LVAD-supported hearts. Production of metabolites 6-alpha-HT, 6-beta-HT, and 16-alpha-HT was observed only in assays with hypertrophic human heart microsomes. For comparison, we assayed metabolite production with microsomal preparations from normal healthy human hearts and found levels of individual metabolites to be between those of hypertrophic and LVAD-supported hearts (see Fig. 7 A–C). A typical HPLC chromatogram is given in Fig. 8 .



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  		Figure 7. A–C) Production of testosterone metabolites with microsomal suspensions of human left ventricles. Microsomes were also treated with CO (CYP inhibitor; A) or Finasteride (25 µM, 5-alpha-steroid reductase inhibitor; B). Data represent mean ± SD (*P<0.05; **not detected). D) Production of testosterone metabolites with microsomal suspensions of left ventricles of normotensive and hypertensive rats. Data represent mean ± SD of n = 3 different rats(*P<0.05; **not detected).



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  		Figure 8. Representative HPLC chromatograms of testosterone and its metabolites. 11-Alpha-hydroxy-testosterone was used as an internal standard. The metabolite DHT is not shown in this chromatogram due to differences in the absorption maxima (see Materials and Methods).

Androstenedione production decreased to 30% of controls when microsomal fractions of hypertrophic human hearts were treated with the CYP inhibitor CO (see Fig. 7A ).

Addition of the 5-alpha-steroid reductase inhibitor finasteride (25 µM) to microsomal suspensions of hypertrophic human hearts resulted in a similar significant decrease (50%) in DHT formation (see Fig. 7B ).

Assays with rat heart tissue
Incubation assays with microsomal fractions of normotensive rat heart tissue yielded an array of metabolites, of which 2-alpha-HT, 6-alpha-HT, 6-beta-HT, 7-alpha-HT, 16-alpha-HT, androstenedione, and DHT could be quantified. Noticeably, production of 7-alpha-HT, 16-alpha-HT, androstenedione, and DHT was increased 6-, 6-, 10-, and 3-fold when the assay was done with microsomes from SHR heart tissue (see Fig. 7D ). The metabolites 2-alpha-HT, 6-alpha-HT and 6-beta-HT could only be detected in assays with SHR heart tissue.

Microsomal lipid peroxidation assay
We used the thiobarbituric acid test to assay for lipid oxidation products (43) . This test estimates the amount of thiobarbituric acid reactive substance (TBARS), including hydroperoxides and endoperoxides. Figure 9 A depicts the level of TBARS in explanted human heart tissue, and we show a 10-fold increase in lipid peroxidation products in assays with hypertrophic human left ventricles.



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  		Figure 9. Production of TBARS in hypertrophic and LVAD-supported human left ventricles (A). Microsomal suspensions were either treated with CO or heat inactivated and incubated for 60 min at 37°C in a shaking water bath before quantification of TBARS (B). Controls were set to 100%.

We assayed for TBARS with heat inactivated or CO-treated microsomes. We found the total amount of lipid peroxidation products (measured as TBARS) to be significantly reduced in heat inactivated and/or CO-treated microsomes: 73% and 81% of controls (see Fig. 9B ). This suggests heart-specific CYP mono-oxygenase activity to be linked to TBARS production.

Alpha-MHC promotor analysis
We studied the promotor sequence of alpha-MHC (for further details, see Entrez from the National Center for Biotechnological Information; http://www.ncbi.nlm.nih.gov) and constructed a matrix for the androgen receptor binding site using the GenomatixTM software (see Fig. 10 ). As depicted, we identified several potential binding sites in each of the promotors tested.



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  		Figure 10. Potential cognate DNA binding sites for the androgen receptor in the promotor of the alpha myosin heavy chain gene. A matrix was developed from consensus sequences obtained from http://transfac.gbf.de/TRANSFAC/ or refs 38 39 40 .


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We investigated gene expression of CYP mono-oxygenases and other steroid-metabolizing enzymes in normotensive and SHRs and in biopsy material of explanted human hearts. We examined the gene expression of the androgen receptor and of alpha-MHC, a structural and heart-specific protein, and compared its expression in normal, assist device-supported, and hypertrophic human heart tissue. We show metabolism of testosterone to be increased in hypertrophic hearts and induction of CYP mono-oxygenases to be linked to enhanced production of certain testosterone metabolites. We thus propose an important role for CYP mono-oxygenases in cardiac hypertrophy. We show increased production of lipid peroxidation products (TBARS) in assays with microsomes of hypertrophic human cardiac tissue and link CYP gene expression and enzyme activity with the production of peroxidation products. We propose derailed steroid metabolism as an essential event in the multistage development of cardiac hypertrophy.

Steroid hormones are important endogenous signaling molecules that are metabolized via several enzymes, including CYP mono-oxygenases, hydroxy-steroid-dehydrogenases/reductases, aromatases, and bind reversibly to carrier proteins in the blood. On receptor mediated endocytosis (44) , they interact specifically with steroid-hormone receptor proteins in the cytoplasm and nucleus (45) . Binding of the hormone activates the receptor, enabling high affinity to specific DNA sequences to act as transcriptional enhancers. This binding increases the level of transcription of hormone sensitive genes, including the gene coding for the myosin heavy chain (25) . The translational products of some of these genes activate other genes to produce a delayed secondary response, thereby extending the initial effect of the hormone.

It is now recognized that heart tissue is metabolically competent (1 , 10 , 46 , 47) and disease may influence the expression of metabolic enzymes, such as CYP mono-oxygenases (16 , 48) . Our study links gene expression and enzyme activity of CYP mono-oxygenases and other steroid-metabolizing enzymes with hypertrophic cardiac tissue. We extend the earlier findings of Marsh et al., who reported the androgen receptor to be involved in cardiac hypertrophy (24) but did not determine expression levels.

We demonstrate metabolism of testosterone to be altered in cardiac hypertrophy. Compared with controls, production of DHT, androstenedione and 2-alpha-HT, 16-alpha-HT, 6-beta-HT, 6-alpha-HT, and 7-alpha-HT was elevated in left ventricles of human hypertrophic and SHRs. Corroborative evidence stems from microsomal assays with heart tissue from patients supported with left ventricular assist devices, where wall thickness returned to normal and significantly lower amounts of testosterone metabolites were observed (see Fig. 7B, C ). Reduction of left ventricular cardiac hypertrophy after LVAD support was observed in the study by Zafeiridis et al. (49) .

Testosterone represses expression of the androgen receptor (50 , 51) ; increased metabolism of this particular hormone, as observed in our study, affects its transcriptional regulation. The considerable increase in gene expression of the androgen receptor can now be viewed as an adaptive response to lower hormone tissue levels in hypertrophic heart tissue. This would explain the increase in mRNA expression of the androgen receptor observed in our study (see Fig. 5 ). We also show gene expression of the androgen receptor to return to normal or to be reduced upon treatment of cardiomyocyte cultures with testosterone. We thus confirm the important role of testosterone in transcriptional regulation of the androgen receptor.

The up to fourfold increased gene expression of 5-alpha-steroid reductase in hypertrophic human and rat heart tissue correlates well with the elevated production of DHT (see Figs. 4 and 7 ), and this metabolite has been linked to the onset of cardiac hypertrophy in athlete drug abuse, i.e., anabolic steroid doping (52) . We show finasteride to successfully lower DHT production in microsomal assays; it will be of interest to further investigate whether 5-alpha-steroid reductase inhibition is beneficial in the treatment of cardiac hypertrophy.

As shown in Fig. 11 production of androstenedione can arise from different metabolic pathways. We did not find changes in the expression of 17-beta-HSD II, an androstenedione-metabolizing enzyme, but observed induction of several cytochrome P450 isoforms. We consider enhanced production of androstenedione to be due to altered CYP activity rather than activity of 17-beta-HSD II (see also Figs. 7 , 8 , 11 ). Further evidence comes from inhibition studies with CO, which leads to Fe-CO adducts and microsomal CYP mono-oxygenase inactivation (53) . Consequently, the production of testosterone metabolites was significantly lower with CO-treated microsomal assays (see Fig. 7A ). Transcripts for 3-beta-HSD I were not detected in heart, but liver tissue (and thus androstenedione production in heart tissue) is driven primarily by CYP mono-oxygenases.



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  		Figure 11. Proposed scheme of derailed steroid metabolism in cardiac hypertrophy.

It was suggested that testosterone enhances cardiac alpha-MHC expression in castrated rats receiving testosterone substitution (25) . We show increased alpha-MHC expression in cultures of cardiomyocytes on treatment with testosterone (see Fig. 6C ). No genomic footprint data are available, and thus the role of the androgen receptor in the transcriptional regulation of the alpha-MHC gene remains speculative. We used a bioinformatic tool and constructed a specific matrix to predict ARE (androgen-responsive element) binding sites in the promotor of the human, rat, and murine alpha-MHC gene (see Fig. 10 ). We found DNA cognate ARE binding sites in all alpha-MHC promotors and propose alpha-MHC gene transcription to be regulated at least in part via ARE protein–DNA interaction. We observed reduced alpha-MHC gene expression in hypertrophic human and rat heart tissue (see Fig. 6A, B ) and propose that enhanced testosterone metabolism results in lower tissue levels of this hormone. By implication, this leads to fewer agonists available to activate the androgen receptor and, as a consequence, to impaired interaction of the receptor with consensus binding sites in the alpha-MHC promotor. Additional studies are needed to provide unequivocal evidence for our proposed mechanism. Reduced alpha-MHC expression may lead to systolic dysfunction (54 , 55) , which is often observed in cardiac hypertrophy (56) .

We observed increased expression of the proto-oncogene c-jun and renin in hypertrophic rat heart tissue, which is in accordance with published data (41 , 42) (see Fig. 3 ).

We show expression of CYP2J to be increased in hypertrophic hearts, and this isoform catalyzes the biotransformation of arachidonic acid (4 , 17) . Yu et al. demonstrated increased CYP2J expression and epoxyeicosatrienoic acid production in kidneys of spontaneously hypertensive rats (16) . In kidney, increased gene expression of this particular CYP isoform is linked to changes in arachidonic acid metabolism and tissue damage (57) . We show an 11-fold increased gene expression of CYP2J3 in hearts of SHRs and a similar 5-fold increased expression in human hypertrophic heart when compared with assist device-supported hearts (see Figs. 2 and 3 ). Elevated CYP2J expression levels may affect ventricular force and increased muscle mass in patients with cardiac hypertrophy.

CYP mono-oxygenases may produce oxygen-derived free radicals and have been implicated in cardiac ischemia/reperfusion injury and cardiac hypertrophy (20 21 22 23) . We show a 10-fold increased production of lipid peroxidation products (see Fig. 9A ) and suggest enhanced CYP expression to be linked to increased lipid peroxidation products in cardiac hypertrophy. Further evidence comes from CO inhibition studies, where the total amount of lipid peroxidation products (measured as TBARS) was significantly reduced after destruction of CYP mono-oxygenases by boiling or CO inactivation (see Fig. 9B ).

In a recently proposed medical hypothesis, Hayward, Webb, and Collins suggested increased left ventricular mass to be linked to endogenous hormone concentrations (33) . The authors suggest that normal decline in endogenous sex hormones with age has contrary effects on ventricular mass in men and women, particularly as androgens have an anabolic effect on cardiac cells, whereas estrogens have antiproliferative properties. There is speculation and circumstantial evidence that woman are protected against cardiac disease at least in part by higher estrogen hormone levels.

Our study is the first report on testosterone metabolism in human normal, hypertrophic, and assist device-supported heart tissue and we propose an important role for cytochrome P450 mono-oxygenases in cardiac physiology and heart disease. Further evidence comes from a study by Xiao et al. where modulation of cardiomyocyte contractility and L-type calcium channel was achieved with the potent CYP mono-oxygenase inhibitor and antifungal remedy imidazole (18) , and systemic therapy of patients with this antifungal has been linked to severe arrhythmias (58) . The CYP inhibitor clotrimazole was also reported to inhibit the sarcoplasmic reticulum calcium ATPase, leading to imbalance in calcium homeostasis and impaired cardiac contractile function (59) . In a recent study by Ahmad et al. negative inotropic effects are reported for patients receiving systemic treatment with itraconazole (60) ; this provides further evidence for the important role of cytochrome P450 in cardiac physiology.

In conclusion, we show metabolism of testosterone to be significantly altered in hypertrophic hearts and we propose a novel role of CYP mono-oxygenases in cardiac hypertrophy via modulation of tissue-specific testosterone metabolism and hormone-dependent gene expression. Our findings may translate into new concepts in the treatment of cardiac hypertrophy based on modulation of tissue-specific steroid metabolism.


   ACKNOWLEDGMENTS
 
We thank Prof. Haverich (Department of Cardiovascular Surgery, Medizinische Hochschule Hannover, Germany) for providing human biopsy materials.

Received for publication March 5, 2002. Revision received May 24, 2002.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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