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Modulation of Cytochrome P450 Gene Expression and Arachidonic Acid Metabolism during Isoproterenol-Induced Cardiac Hypertrophy in Rats

Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada

(Received June 23, 2008; Accepted August 21, 2008)

	Abstract

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Several cytochrome P450 (P450) enzymes have been identified in the heart, and their levels have been reported to be altered during cardiac hypertrophy. Moreover, there is a strong correlation between P450-mediated arachidonic acid metabolites and the pathogenesis of cardiac hypertrophy. Therefore, we investigated the effect of isoproterenol-induced cardiac hypertrophy on the expression of several P450 genes and their associated P450-derived metabolites of arachidonic acid. Cardiac hypertrophy was induced by seven daily i.p. injections of 5 mg/kg isoproterenol. Thereafter, the heart, lung, liver, and kidney were harvested, and the expression of different genes was determined by real-time polymerase chain reaction. Heart microsomal protein from control or isoproterenol treated rats was incubated with 50 µM arachidonic acid, and arachidonic acid metabolites were determined by liquid chromatography-electron spray ionization-mass spectrometry. Our results show that isoproterenol treatment significantly increased the heart/body weight ratio and the hypertrophic markers. In addition, there was a significant induction of CYP1A1, CYP1B1, CYP4A3, and soluble epoxide hydrolase and a significant inhibition of CYP2C11 and CYP2E1 in the hypertrophied hearts as compared with the control. CYP1A1, CYP2E1, and CYP4A3 gene expression was induced in the kidney, and CYP4A3 was induced in the liver of isoproterenol-treated rats. Isoproterenol treatment significantly reduced 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acid formation and significantly increased their corresponding 8,9-, and 14,15-dihydroxyeicosatrienoic acid and the 20-hydroxyeicosatetraenoic acid metabolite. In conclusion, isoproterenol-induced cardiac hypertrophy alters arachidonic acid metabolism and its associated P450 enzymes, suggesting their role in the development and/or progression of cardiac hypertrophy.

The role of cytochrome P450 (P450) enzymes in cardiovascular health and disease is well established (Elbekai and El-Kadi, 2006; Zordoky and El-Kadi, 2008). P450 is a family of mono-oxygenases that is involved in the oxidative metabolism of a wide range of xenobiotics and endogenous substances (Elbekai and El-Kadi, 2006). Many studies have examined the expression of P450 enzymes in the heart (Elbekai and El-Kadi, 2006). In vivo, P450 enzymes have been reported in explanted human hearts (Thum and Borlak, 2000b; Delozier et al., 2007) and in the left ventricle of Sprague Dawley (SD) rats and spontaneously hypertensive rats (SHRs) (Thum and Borlak, 2002). At the in vitro level, the gene expression and protein activity of many P450 enzymes have been reported in cultured cardiomyocytes (Thum and Borlak, 2000a) and in the rat cardiomyoblast H9c2 cell line (Zordoky and El-Kadi, 2007).

P450 enzymes are considered one of the major metabolic pathways for the metabolism of arachidonic acid (AA) in addition to cyclooxygenases and lipoxygenases. In the presence of NADPH and oxygen, P450 metabolizes AA to epoxyeicosatrienoic acid (EET) and hydroxyeicosatetraenoic acid (HETE) metabolites. EETs can be either incorporated into membrane phospholipid pools or efficiently hydrolyzed by soluble epoxide hydrolase (sEH) to biologically less active dihydroxyeicosatrienoic acids (DHETs) or secreted into the extracellular space (Imig et al., 2002). P450 epoxygenases are known to metabolize AA to four regioisomeric metabolites, 5,6-, 8,9-, 11,12-, and 14,15-EET, and by P450 -hydroxylases to 20-HETE. The EETs and HETEs are regarded as important mediators in hypertension, cardiovascular disease, and inflammation (Roman, 2002).

The expression of several P450 genes identified in the heart has been reported to be altered during cardiac hypertrophy; however, there is a great discrepancy among various reports on P450 alterations during cardiac hypertrophy, likely because of differences in the etiology of hypertrophy, disease severity, species in question, and other underlying conditions (Zordoky and El-Kadi, 2008). In the present study, we investigated the expression of multiple P450 genes in the heart, lung, liver, and kidney of male SD rats during isoproterenol-induced cardiac hypertrophy. Furthermore, we investigated the effect of isoproterenol-induced cardiac hypertrophy on the formation of AA metabolites and to determine whether the changes in P450 and sEH gene expression have led to changes in AA metabolism. Our findings provide the first evidence for the cardiac-specific changes in P450 and sEH gene expression and AA metabolism during isoproterenol-induced cardiac hypertrophy.

	Materials and Methods

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Materials. TRIzol reagent was purchased from Invitrogen (Carlsbad, CA). High-Capacity cDNA Reverse Transcription Kit, SYBR Green SuperMix, and 96-well optical reaction plates with optical adhesive films were purchased from Applied Biosystems (Foster City, CA). Real-time PCR primers were synthesized by Integrated DNA Technologies, Inc. (Coralville, IA) according to previously published sequences. AA, isoproterenol, and 4-hydroxybenzophenone were purchased from Sigma-Aldrich (St. Louis, MO). AA metabolite standards 5,6-EET, 8,9-EET, 11,12-EET, 14,15-EET, 5,6-DHET, 8,9-DHET, 11,12-DHET, 14,15-DHET, and 20-HETE were obtained from Cayman Chemical (Ann Arbor, MI). Reagents used for liquid chromatography (LC)-electron spray ionization (ESI)-mass spectrometry (MS) were at HPLC grade. Acetonitrile and water (HPLC grade) were purchased from EM Scientific (Gibbstown, NJ). Other chemicals were purchased from Thermo Fisher Scientific (Waltham, MA).

Animals. All experimental procedures involving animals were approved by the University of Alberta Health Sciences Animal Policy and Welfare Committee. Male SD rats weighing 300 to 350 g were obtained from Charles River Canada (Montreal, QC, Canada). Animals were treated i.p. with 5 mg/kg isoproterenol (dissolved in normal saline) daily for 7 days (n = 6). Weight-matched controls received the same volume of normal saline daily for 7 days (n = 6). Animals were euthanized 24 h after the last injection under isoflurane anesthesia. All animals were allowed free access to food and water throughout the treatment period. Heart, lung, kidney, and liver tissues were excised, immediately frozen in liquid nitrogen, and stored at -80°C until analysis.

RNA Extraction and cDNA Synthesis. Total RNA from the frozen tissues was isolated using TRIzol reagent (Invitrogen) according to the manufacturer's instructions and quantified by measuring the absorbance at 260 nm. RNA quality was determined by measuring the 260/280 ratio. Thereafter, first strand cDNA synthesis was performed by using the High-Capacity cDNA reverse transcription kit (Applied Biosystems) according to the manufacturer's instructions. Briefly, 1.5 µg of total RNA from each sample was added to a mix of 2.0 µl of 10x reverse transcriptase buffer, 0.8 µl of 25x dNTP mix (100 mM), 2.0 µl of 10x reverse transcriptase random primers, 1.0 µl of MultiScribe reverse transcriptase, and 3.2 µl of nuclease-free water. The final reaction mix was kept at 25°C for 10 min, heated to 37°C for 120 min, heated for 85°C for 5 s, and finally cooled to 4°C.

Quantification by Real-Time PCR. Quantitative analysis of specific mRNA expression was performed by real-time PCR by subjecting the resulting cDNA to PCR amplification using 96-well optical reaction plates in the ABI Prism 7500 System (Applied Biosystems). The 25-µl reaction mix contained 0.1 µl of 10 µM forward primer and 0.1 µl of 10 µM reverse primer (40 nM final concentration of each primer), 12.5 µl of SYBR Green Universal Mastermix, 11.05 µl of nuclease-free water, and 1.25 µl of cDNA sample. The primers used in the current study were chosen from previously published studies (Bleicher et al., 2001; Kalsotra et al., 2002; Wang et al., 2003; Hirasawa et al., 2005; Rollin et al., 2005; Baldwin et al., 2006) and are listed in Table 1. Assay controls were incorporated onto the same plate, namely, no-template controls to test for the contamination of any assay reagents. After sealing the plate with an optical adhesive cover, the thermocycling conditions were initiated at 95°C for 10 min, followed by 40 PCR cycles of denaturation at 95°C for 15 s and annealing/extension at 60°C for 1 min. Melting curve (dissociation stage) was performed by the end of each cycle to ascertain the specificity of the primers and the purity of the final PCR product.

Real-Time PCR Data Analysis. The real-time PCR data were analyzed using the relative gene expression (i.e., CT) method, as described in Applied Biosystems User Bulletin No. 2 and explained further by Livak and Schmittgen (2001). Briefly, the data are presented as the -fold change in gene expression normalized to the endogenous reference gene (glyceraldehyde-3-phosphate dehydrogenase) and relative to a calibrator. The untreated control was used as the calibrator when the change of gene expression by isoproterenol was being studied, whereas the P450 of lowest expression was used as a calibrator to compare the expression of different P450 genes.

Microsomal Preparation and Incubation. Microsomal protein was prepared from the heart tissue as described previously (Barakat et al., 2001). Briefly, hearts were washed in ice-cold potassium chloride [1.15% (w/v)], cut into pieces, and homogenized separately in cold sucrose solution (1 g of tissue in 5 ml of 0.25 M sucrose). Microsomal protein from homogenized tissues was separated by differential ultracentrifugation. The final pellet was reconstituted in cold sucrose and stored at -80°C. Heart microsomal protein concentration was determined by the Lowry method using bovine serum albumin as a standard (Lowry et al., 1951). Heart microsomes (1 mg protein/ml) were incubated in the incubation buffer (5 mM magnesium chloride hexahydrate dissolved in 0.5 M potassium phosphate buffer, pH 7.4) at 37°C in a shaking water bath (50 rpm). A pre-equilibration period of 5 min was performed. The reaction was initiated by the addition of 1 mM NADPH. AA was added to a final concentration of 50 µM and incubated for 30 min. The reaction was terminated by the addition of 600 µl of ice-cold acetonitrile followed by the internal standard, 4-hydroxybenzophenone. AA metabolites were extracted twice by 1 ml of ethyl acetate and dried using a speed vacuum (Thermo Fisher Scientific). The concentrations of these eicosanoids in the samples were calculated by comparing the ratios of peak heights with their corresponding standards.

View larger version (43K): [in this window] [in a new window]   FIG. 1. Constitutive expression of P450 genes and sEH in different tissues. Total RNA was isolated from different tissues, and the relative expression of P450 genes and sEH in the heart (A), lung (B), liver (C), and kidney (D) was determined by reverse transcription followed by real-time PCR. The data were analyzed using the relative gene expression method. The data were normalized to the endogenous reference gene (glyceraldehyde-3-phosphate dehydrogenase) and relative to a calibrator. The gene of lowest expression in each tissue was used as a calibrator as described under Materials and Methods. Results are presented as mean ± S.E.M. (n = 6).

Statistical Analysis. Data are presented as mean ± S.E.M. Control and treatment measurements were compared using Student's t test. Comparative gene expression across tissues was analyzed using a one-way analysis of variance followed by a Student-Newman-Keuls post hoc comparison. A result was considered statistically significant where p < 0.05.

	Results

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Expression of P450 Genes and sEH in the Heart, Lung, Kidney, and Liver. To examine the constitutive expression of various P450 genes and sEH in the heart, lung, liver, and kidney tissues of male SD rats, total RNA was isolated from different tissues, and different genes were determined by real-time PCR. The gene expression profile of different P450 and sEH genes was found to be tissue specific. All of the examined genes except CYP4A2 are found to be constitutively expressed in the heart at different levels (Fig. 1A). CYP4A1 was the lowest expressed gene and was considered as a calibrator. CYP4F5, CYP2E1, CYP1B1, and sEH were the most highly expressed genes, about 10,000-fold higher than the calibrator (Fig. 1A). CYP1A1 and CYP2J3 were also highly expressed, about 1000-fold higher than the calibrator (Fig. 1A). CYP2B1, CYP2B2, CYP2C11, and CYP4F4 were moderately expressed genes, about 100-fold higher than the calibrator. CYP4A3 was constitutively expressed at low level, about 2-fold higher than the calibrator (Fig. 1A).

Similar to the heart, not all the examined genes were expressed in the lung; CYP4A1, CYP4A2, and CYP4A3 mRNA were not detected in the lung tissue. CYP2B2 was the lowest expressed gene and was considered as calibrator. CYP2B1 was the most highly expressed gene, about 120,000-fold higher than the calibrator (Fig. 1B). CYP1A1, CYP2E1, CYP4F5, and sEH were highly expressed, about 5000-, 4600-, 2200-, and 1400-fold higher than the calibrator, respectively (Fig. 1B). CYP2J3, CYP1B1, CYP4F4, and CYP2C11 were moderate to low expressed genes, about 290-, 65-, 17-, and 11-fold higher than the calibrator, respectively (Fig. 1B).

View larger version (15K): [in this window] [in a new window]   FIG. 2. Effect of isoproterenol treatment on the hypertrophic markers. A, hypertrophic markers gene expression was determined in the heart. Total RNA was isolated from control and isoproterenol-treated animals. ANP and BNP gene expressions were determined by real-time PCR. B, heart/body weight ratio (milligrams per gram) were determined for each animal after seven daily i.p. injections of isoproterenol or saline. Results are presented as mean ± S.E.M. (n = 6). *, p < 0.05 compared with control.

Similar to the liver, all the examined genes were found to be constitutively expressed at different levels in the kidney. CYP2B2 was the lowest expressed gene and was considered as the calibrator (Fig. 1D). CYP4A2 and CYP4A3 were the most highly expressed genes, about 50,000- and 24,000-fold higher than the calibrator, respectively. sEH and CYP2E1 were highly expressed genes, about 6000-fold higher than the calibrator (Fig. 1D). CYP2C11, CYP4A1, CYP4F5, CYP2J3, CYP1A1, and CYP1B1 were high to moderate expressed genes, about 750-, 360-, 210-, 200-, 150-, and 60-fold higher than the calibrator, respectively (Fig. 1D). The constitutive expression of CYP4F4 and CYP2B1 was very low, about 2- and 1.5-fold higher than the calibrator, respectively (Fig. 1D).

Effect of Isoproterenol Treatment on Hypertrophic Markers and the Heart/Body Weight Ratio. To investigate whether isoproterenol treatment caused cardiac hypertrophy in the treated SD rats, we measured the cardiac gene expression of the hypertrophic markers, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) relative to control rats. Our results showed that isoproterenol treatment caused statistically significant induction of both hypertrophic markers, ANP and BNP, by 10- and 3-fold, respectively (Fig. 2A). In addition, isoproterenol treatment caused a statistically significant increase in the heart/body weight ratio by about 30% (Fig. 2B).

Effect of Isoproterenol Treatment on P450 Gene Expression. To examine the effect of isoproterenol-induced cardiac hypertrophy on the expression of several P450 genes and sEH in different tissues, total RNA was extracted from the heart, lung, liver, and kidney tissues of both control and isoproterenol-treated rats. Thereafter, the expression of different genes was measured using reverse transcription followed by real-time PCR as described under Materials and Methods.

Figure 3 shows the effect of isoproterenol-induced cardiac hypertrophy on CYP1 family gene expression. Our results demonstrate that CYP1A1 is highly expressed in the lung, moderately expressed in the liver and kidney, and of low expression in the heart (Fig. 3A). Similarly, CYP1B1 was highly expressed in the lung, moderately expressed in the heart and kidney, and of low expression in the liver (Fig. 3B). Isoproterenol treatment caused a significant induction of CYP1A1 and CYP1B1 gene expression in the heart by about 2.8- and 4.2-fold, respectively (Fig. 3, C and D). In addition, isoproterenol treatment caused a significant induction of CYP1A1, but not CYP1B1, in the kidney by 2.8-fold (Fig. 3, C and D). However, there was no change in CYP1A1 and CYP1B1 gene expression in the other tissues (Fig. 3, C and D).

View larger version (27K): [in this window] [in a new window]   FIG. 3. Expression of CYP1A1 and CYP1B1 in different tissues and their modulation during isoproterenol-induced cardiac hypertrophy. Gene expression of CYP1A1 (A) and CYP1B1 (B) in the lung, liver, and kidney relative to the heart. Total RNA was isolated from different tissues, and the relative expression of CYP1A1 and CYP1B1 was determined by real-time PCR. *, p < 0.05 compared with the heart. Gene expression of CYP1A1 (C) and CYP1B1 (D) during isoproterenol-induced cardiac hypertrophy. Cardiac hypertrophy was induced by seven daily i.p. injections of isoproterenol. Twenty-four hours after the last injection, the heart, lung, liver, and kidney were collected. Total RNA was isolated, and gene expression of CYP1A1 and CYP1B1 was determined by real-time PCR in control and isoproterenol-treated rats. Results are presented as mean ± S.E.M. (n = 6). *, p < 0.05 compared with control.

View larger version (31K): [in this window] [in a new window]   FIG. 4. Expression of CYP2B1 and CYP2B2 in different tissues and their modulation during isoproterenol-induced cardiac hypertrophy. Gene expression of CYP2B1 (A) and CYP2B2 (B) in the lung, liver, and kidney relative to the heart. Total RNA was isolated from different tissues, and the relative expression of CYP2B1 and CYP2B2 was determined by real-time PCR. *, p < 0.05 compared with the heart. Gene expression of CYP2B1 (C) and CYP2B2 (D) during isoproterenol-induced cardiac hypertrophy. Cardiac hypertrophy was induced by seven daily i.p. injections of isoproterenol. Twenty-fours after the last injection, the heart, lung, liver, and kidney were collected. Total RNA was isolated, and gene expression of CYP2B1 and CYP2B2 was determined by real-time PCR in control and isoproterenol-treated rats. Results are presented as mean ± S.E.M. (n = 6).

View larger version (30K): [in this window] [in a new window]   FIG. 5. Expression of CYP2C11 and CYP2E1 in different tissues and their modulation during isoproterenol-induced cardiac hypertrophy. Gene expression of CYP2C11 (A) and CYP2E1 (B) in the lung, liver, and kidney relative to the heart. Total RNA was isolated from different tissues, and the relative expression of CYP2C11 and CYP2E1 was determined by real-time PCR. *, p < 0.05 compared with the heart. Gene expression of CYP2C11 (C) and CYP2E1 (D) during isoproterenol-induced cardiac hypertrophy. Cardiac hypertrophy was induced by seven daily i.p. injections of isoproterenol. Twenty-four hours after the last injection, the heart, lung, liver, and kidney (n = 6) were collected. Total RNA was isolated, and gene expression of CYP2C11 and CYP2E1 was determined by real-time PCR in control and isoproterenol-treated rats. Results are presented as mean ± S.E.M. (n = 6). *, p < 0.05 compared with control.

View larger version (30K): [in this window] [in a new window]   FIG. 6. Expression of CYP2J3 and CYP4A1 in different tissues and their modulation during isoproterenol-induced cardiac hypertrophy. Gene expression of CYP2J3 (A) and CYP4A1 (B) in the lung, liver, and kidney relative to the heart. Total RNA was isolated from different tissues, and the relative expression of CYP2J3 and CYP4A1 was determined by real-time PCR. *, p < 0.05 compared with the heart. Gene expression of CYP2J3 (C) and CYP4A1 (D) during isoproterenol-induced cardiac hypertrophy. Cardiac hypertrophy was induced by seven daily i.p. injections of isoproterenol. Twenty-four hours after the last injection, the heart, lung, liver, and kidney (n = 6) were collected. Total RNA was isolated, and gene expression of CYP2J3 and CYP4A1 was determined by real-time PCR in control and isoproterenol-treated rats. Results are presented as mean ± S.E.M. (n = 6).

View larger version (27K): [in this window] [in a new window]   FIG. 7. Expression of CYP4A2 and CYP4A3 in different tissues and their modulation during isoproterenol-induced cardiac hypertrophy. Gene expression of CYP4A2 (A) and CYP4A3 (B) in the lung, liver, and kidney relative to the heart. Total RNA was isolated from different tissues, and the relative expression of CYP4A2 and CYP4A3 was determined by real-time PCR. *, p < 0.05 compared with the heart. Gene expression of CYP4A2 (C) and CYP4A3 (D) during isoproterenol-induced cardiac hypertrophy. Cardiac hypertrophy was induced by seven daily i.p. injections of isoproterenol. Twenty-four hours after the last injection, the heart, lung, liver, and kidney (n = 6) were collected. Total RNA was isolated, and gene expression of CYP4A2 and CYP4A3 was determined by real-time PCR in control and isoproterenol-treated rats. Results are presented as mean ± S.E.M. (n = 6). *, p < 0.05 compared with control.

With regard to CYP4F subfamily, CYP4F4 and CYP4F5 were found to be expressed in all the examined tissues. CYP4F4 was highly expressed in the liver compared with the other examined tissues (Fig. 8A). On the other hand, CYP4F5 was found to be highly expressed in the lung, moderately expressed in the heart and kidney, and of low expression in the liver (Fig. 8B). The gene expression of CYP4F4 and CYP4F5 was not altered during isoproterenol-induced cardiac hypertrophy in all the examined tissues (Fig. 8, C and D).

View larger version (29K): [in this window] [in a new window]   FIG. 8. Expression of CYP4F4 and CYP4F5 in different tissues and their modulation during isoproterenol-induced cardiac hypertrophy. Gene expression of CYP4F4 (A) and CYP4F5 (B) in the lung, liver, and kidney relative to the heart. Total RNA was isolated from different tissues, and the relative expression of CYP4F4 and CYP4F5 was determined by real-time PCR. *, p < 0.05 compared with the heart. Gene expression of CYP4F4 (C) and CYP4F5 (D) during isoproterenol-induced cardiac hypertrophy. Cardiac hypertrophy was induced by seven daily i.p. injections of isoproterenol. Twenty-four hours after the last injection, the heart, lung, liver, and kidney (n = 6) were collected. Total RNA was isolated, and gene expression of CYP4F4 and CYP4F5 was determined by real-time PCR in control and isoproterenol-treated rats. Results are presented as mean ± S.E.M. (n = 6).

View larger version (19K): [in this window] [in a new window]   FIG. 9. Expression of sEH in different tissues and its modulation during isoproterenol-induced cardiac hypertrophy. Gene expression of sEH in the lung, liver, and kidney relative to the heart (A). Total RNA was isolated from different tissues, and the relative expression of sEH was determined by real-time PCR. *, p < 0.05 compared with the heart. Gene expression of sEH during isoproterenol-induced cardiac hypertrophy (B). Cardiac hypertrophy was induced by seven daily i.p. injections of isoproterenol. Twenty-four hours after the last injection, the heart, lung, liver, and kidney (n = 6) were collected. Total RNA was isolated, and gene expression of sEH was determined by real-time PCR in control and isoproterenol-treated rats. Results are presented as mean ± S.E.M. (n = 6). *, p < 0.05 compared with control.

Separation of AA Metabolites Using the LC-ESI-MS Selected Ion Chromatogram. LC-ESI-MS has been used in this study for the separation and quantification of P450-derived metabolites of AA. The mass spectrometer was operated in negative ionization mode with single-ion recorder acquisition, where the most abundant ion corresponds to the m/z = [M-1]^-. All four regioisomeric epoxyeicosatrienoic acids (5,6-, 8,9-, 11,12-, and 14,15-EET) exhibited the most abundant ions, corresponding to m/z = 319 ion, whereas their corresponding DHET has m/z = 337 ion, and 20-HETE has m/z = 319 ion. The fact of having functional groups at different positions in the eicosanoid structure allowed successful separation that was achieved through the use of the reverse phase C18 HPLC column and linear gradient mobile phase. Using authentic standards, 14,15-, 11,12-, 8,9-, and 5,6-EET were found to be eluted at 26.33, 28.56, 29.38, and 30.09 min, respectively, whereas that of 20-HETE was eluted at 15.07 min. The elution patterns of 14,15-, 11,12-, 8,9-, and 5,6-DHET were at 11.35, 12.58, 13.59 and 14.73 min, respectively.

Effect of Isoproterenol Treatment on AA Metabolism. To investigate the effect of isoproterenol treatment on the formation of AA metabolites, heart microsomes of either control or isoproterenol-treated rats were incubated with 50 µM AA for 30 min. Thereafter, AA metabolites were determined using LC-ESI-MS. Our results clearly show that 5,6-EET was the major metabolite produced in both control and isoproterenol-treated rats, followed by 8,9-, 14,15-, and 11,12-EET, respectively. In comparison with control animals, in microsomes of hypertrophied hearts, the formation of 5,6-, 8,9-, 11,12-, and 14,15-EET was significantly lower, by 34.20, 44.34, 50.84, and 44.17%, respectively (Fig. 10A).

View larger version (17K): [in this window] [in a new window]   FIG. 10. Effect of isoproterenol treatment on EETs (A), DHETs (B), and 20-HETE formation (C). Heart microsomes of control or isoproterenol-treated rats were incubated with 50 µM arachidonic acid. The reaction was started by the addition of 1 mM NADPH and lasted for 30 min. The reaction was terminated by the addition of ice-cold acetonitrile. EETs, DHETs, and 20-HETE were extracted twice by 1 ml of ethyl acetate and dried using speed vacuum. Reconstituted metabolites were injected into LC-ESI-MS for metabolite determination. Results are presented as mean ± S.E.M. (n = 5). *, p < 0.05 compared with control.

To determine the effect of isoproterenol treatment on P450 -hydroxylase activity, we determined the formation of 20-HETE in microsomes from control and hypertrophied rats. Isoproterenol treatment significantly increased the 20-HETE formation by 192% in comparison with the control group (Fig. 10C).

	Discussion

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Although cardiac hypertrophy is an important condition that usually precedes heart failure (Carreno et al., 2006), there are limited reports of altered expression of P450 genes during cardiac hypertrophy. The expression of P450 has been studied in SHRs as compared with SD rats (Thum and Borlak, 2002) and the expression of sEH has been studied in spontaneously hypertensive heart failure (SHHF) rats as compared with normal strains (Monti et al., 2008). Nevertheless, there are no data about the effect of experimentally induced cardiac hypertrophy on the expression of P450 and sEH genes and their associated P450-derived metabolites of AA. To the best of our knowledge, this work demonstrates for the first time the effect of experimentally induced cardiac hypertrophy on the expression of several P450 genes and P450-dependent AA metabolism in rats. In addition, the current study demonstrates the tissue-specific expression of many P450 genes and sEH in male SD rats.

Tissue-specific expression of P450 genes in the rat has been a subject of discrepancies (Zordoky and El-Kadi, 2007; Zordoky and El-Kadi, 2008). Several factors may have led to these discrepancies. The first factor is the use of a conventional PCR technique in most of these studies, which may be insensitive because of the low level of P450 expression, especially in extrahepatic tissues. Secondly, most of the previous studies focused on only one tissue without giving comparative information regarding the other tissues. Therefore, it was necessary to examine the expression of multiple P450 genes simultaneously in different organs by a sensitive technique, such as the real-time PCR technique. In the current study, we showed that CYP1A1, CYP1B1, CYP2B1, CYP2B2, CYP2C11, CYP2E1, CYP2J3, CYP4F4, CYP4F5, and sEH genes are constitutively expressed in all the examined tissues, whereas CYP4A1 and CYP4A3 genes are expressed in the heart, kidney, and liver but not in the lung, and the CYP4A2 gene was expressed in the liver and kidney only. The lung has the highest constitutive expression of CYP1A1, CYP1B1, CYP2B1, and CYP4F5 genes, and the liver and kidney have the highest expression of CYP4A2 and CYP4A3, whereas the liver has the highest expression level of all other P450 genes and sEH. Generally, the heart has the lowest level of constitutive P450 expression except for CYP1B1 and CYP4F5, which are expressed in the heart at higher levels than those in the liver.

To induce cardiac hypertrophy experimentally, we used a regimen of seven daily i.p. injections of 5 mg/kg isoproterenol, which is known to induce cardiac hypertrophy without heart failure or blood pressure elevation (Kralova et al., 2008). In the current study, isoproterenol treatment caused cardiac hypertrophy in SD rats as manifested by significant induction of the hypertrophic markers, ANP and BNP, and the increase of the heart/body weight ratio. In agreement with our results, isoproterenol infusion of 4 mg/kg/day for 7 days caused a significant increase of the heart to body weight and a significant induction of ANP (Masson et al., 1998). Similarly, BNP is known to be increased in various models of cardiac hypertrophy (Magga et al., 1998); however, there is limited information about the effect of isoproterenol-induced cardiac hypertrophy on BNP gene expression.

CYP1A1 and CYP1B1 are found to be highly expressed in the heart at the constitutive level; therefore, their role in AA metabolism in the heart cannot be ignored. CYP1A1 has been shown to be involved in -terminal HETE synthesis, whereas CYP1B1 can metabolize AA to both midchain HETEs and EETs (Choudhary et al., 2004). In accordance, it was very important to investigate the effect of isoproterenol-induced cardiac hypertrophy on the gene expression of CYP1A1 and CYP1B1 in the heart and in other tissues. In the current study, we found that CYP1A1 gene expression is induced in the heart and kidney in isoproterenol-treated rats but not in the lung or liver, whereas CYP1B1 gene expression was induced only in the hypertrophied heart. In agreement with our results, expression of CYP1A1 and CYP1B1 was significantly increased in left ventricular tissues of SHRs as compared with normotensive SD rats (Thum and Borlak, 2002).

On the contrary to CYP1A1 and CYP1B1, CYP2C11 gene expression was significantly lower in the hypertrophied heart than the control hearts; however, its gene expression was not altered in the other tissues. CYP2C11 is an important epoxygenase enzyme that is involved in AA metabolism and EET synthesis (Ng et al., 2007). Unlike CYP2C11, CYP2E1 metabolizes AA to 18- and 19-HETEs (Laethem et al., 1993). Isoproterenol-induced cardiac hypertrophy caused a significant inhibition of CYP2E1 gene expression in the heart and a significant induction of its expression in kidney. On the contrary to this finding, CYP2E1 has been reported to be higher in SHR than in normotensive SD rats (Thum and Borlak, 2002).

Despite previous reports that showed increased expression of CYP2B1/2 and CYP2J3 in SHR as compared with normotensive SD rats (Thum and Borlak, 2002), our results showed that CYP2B1, CYP2B2, and CYP2J3 gene expression were not altered during isoproterenol-induced cardiac hypertrophy. The discrepancy between our results and that of Thum and Borlak is likely because of the strain differences. CYP4A3 was significantly induced in the heart, liver, and kidney in isoproterenol-treated rats as compared with the control. The premise of this observation emerges from the fact that CYP4A3 is an important enzyme involved in AA metabolism to 20-HETE (Wang et al., 1996, 1999; Roman, 2002). Isoproterenol treatment did not significantly alter the gene expression of CYP4A1, CYP4F4, and CYP4F5 in any of the examined tissues.

sEH enzyme is a crucial determinant of EETs level because it catalyzes the conversion of EETs to DHETs, thus abolishing their biological activity (Imig et al., 2002). Therefore, any change in AA metabolism because of alteration of P450 can be augmented or opposed by an altered level of sEH. Moreover, the gene encoding sEH was found to be a susceptibility factor for heart failure in SHHF rats (Monti et al., 2008). Therefore, it was necessary to investigate the effect of cardiac hypertrophy on sEH expression. In the current study, sEH gene expression in the heart was found to be increased during isoproterenol-induced cardiac hypertrophy. In agreement with our results, it has been shown that sEH activity is higher in SHHF rats (Monti et al., 2008); however, this is the first report to show an induction of sEH gene expression in experimentally induced cardiac hypertrophy.

To investigate the effect of altered P450 gene expression on AA metabolism, we performed in vitro incubation of heart microsomes with AA. We found a significant decrease in 5,6-, 8,9-, 11,12-, and 14,15-EET formation in microsomes of hypertrophied hearts in comparison with the control. The decrease in EETs formation was accompanied by a significant increase in 8,9- and 14,15-DHET formation, whereas 5,6- and 11,12-DHET were not significantly altered. The decreased formation of EETs during isoproterenol-induced cardiac hypertrophy may be attributed to lower expression of CYP2C11 and higher expression of sEH. The significantly higher formation of 8,9- and 14,15-DHET is consistent with the higher expression of sEH because 14,15-EET is the best substrate for sEH followed by 8,9-EET (Karara et al., 1991).

Moreover, our results demonstrate that 20-HETE formation is significantly higher in microsomes of hypertrophied hearts in comparison with untreated rats. The increased formation of 20-HETE is suggestive of its role in cardiac hypertrophy. 20-HETE formation is mainly catalyzed by P450 -hydroxylases (Kroetz and Xu, 2005). P450 -hydroxylases involved in the formation of HETEs are CYP1A, CYP1B1, CYP4A, and CYP4F (Wang et al., 1996, 1999; Roman, 2002; Choudhary et al., 2004; Elbekai and El-Kadi, 2006). Therefore, the increase in 20-HETE formation in the present work could be attributed to the increased expression of CYP1A1, CYP1B1, and CYP4A3.

In conclusion, isoproterenol-induced cardiac hypertrophy caused significant changes of several P450 and sEH gene expression, which is mostly specific to the heart. The overall balance of these changes has led to higher production of 20-HETE and lower production of EETs in the hypertrophied hearts. 20-HETE is known to be involved in many cardiovascular diseases; however, there is indirect evidence about its role in the development of cardiac hypertrophy (Lee et al., 2004; Chabova et al., 2007). On the other hand, EETs are reported to have cardioprotective effects mainly by inhibiting the activation of nuclear factor B (Campbell, 2000; Hirotani et al., 2002; Xu et al., 2006). Increased expression of CYP1A1, CYP2E1, and CYP4A3 in the kidney during isoproterenol-induced cardiac hypertrophy could also lead to altered P450-mediated AA metabolites, which play a critical role in the kidney function (Elbekai and El-Kadi, 2006). Therefore, the cardiac and renal P450 may play an important role in the development and/or progression of cardiac hypertrophy. However, more studies are needed to explore the mechanisms by which cardiac hypertrophy modulates P450 gene expression.

	Acknowledgments

 
We thank Dr. Vishwa Somayaji for excellent technical assistance with liquid chromatography-electron spray ionization-mass spectrometry.

	Footnotes

 
This study was supported by a grant from the Heart and Stroke Foundation of Alberta, NWT, and Nunavut (to A.O.S.E.-K.). B.N.M.Z. and M.E.A. are the recipients of Egyptian Government Scholarships.

B.N.M.Z. and M.E.A. contributed equally to this work.

doi:10.1124/dmd.108.023077.

ABBREVIATIONS: P450, cytochrome P450; SD, Sprague Dawley; SHR, spontaneously hypertensive rat; AA, arachidonic acid; EET, epoxyeicosatrienoic acid; HETE, hydroxyeicosatetraenoic acid; sEH, soluble epoxide hydrolase; DHET, dihydroxyeicosatrienoic acid; PCR, polymerase chain reaction; LC, liquid chromatography; ESI, electron spray ionization; MS, mass spectrometry; HPLC, high-performance liquid chromatography; ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; SHHF, spontaneously hypertensive heart failure.

Address correspondence to: Dr. Ayman O. S. El-Kadi, Faculty of Pharmacy and Pharmaceutical Sciences, 3126 Dentistry/Pharmacy Centre, University of Alberta, Edmonton, AB, Canada T6G 2N8. E-mail: aelkadi{at}pharmacy.ualberta.ca

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