Expression of CYP2E1 increases oxidative stress and induces apoptosis of cardiomyocytes in transgenic mice Wei Zhang 1 , Dan Lu 1 , Wei Dong 1 , Li Zhang 1 , Xiaojuan Zhang 1 , Xiongzhi Quan 1 , Chunmei Ma 2 , Hong Lian 1 and Lianfeng Zhang 1,2 1 Key Laboratory of Human Disease Comparative Medicine, Ministry of Health, Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences & Comparative Medical Center, Peking Union Medical College, Beijing, China 2 Key Laboratory of Human Diseases Animal Model, State Administration of Traditional Chinese Medicine, Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences & Comparative Medical Center, Peking Union Medical College, Beijing, China Introduction Cytochrome P450 2E1 (CYP2E1) is one of the cyto- chrome P450 (P450) isoforms. Overexpression of CYP2E1 is of direct importance to human health and has been associated with a range of diseases, including diabetes [1–3], alcoholic liver disease and cancer [4–8]. In addition, the reduction of molecular oxygen to water by NAD(P)H in the presence of CYP2E1 is widely considered to be substantially uncoupled under certain physiological situations and this may result in the generation of intracellular reactive oxygen species Keywords apoptosis; CYP2E1; dilated cardiomyopathy; oxidative stress; transgenic mice Correspondence L. Zhang, Building 5, Panjiayuan Nanli, Chaoyang District, Beijing 100021, China Fax: +86 010 67710812 Tel: +86 010 87778442 E-mail: zhanglf@cnilas.org (Received 14 November 2010, revised 11 February 2011, accepted 21 February 2011) doi:10.1111/j.1742-4658.2011.08063.x Cytochrome P450 2E1 (CYP2E1) is an effective generator of reactive oxy- gen species. Marked expression of CYP2E1 occurs in the heart and it is known to be regulated in the course of progression of myocardial ischemia and cardiomyopathy. We provide evidence that the expression of CYP2E1 is strongly up-regulated in cTnT R141W transgenic mice with dilated cardio- myopathy. Heart tissue-specific CYP2E1 transgenic mice were produced to study the effects of CYP2E1 overexpression on the heart. Increased morta- lity, chamber dilation and contractile dysfunction, as well as myocyte disar- ray, interstitial fibrosis, ultrastructural degeneration with myofibrillar disorganization and mitochondria damage, were observed in CYP2E1 transgenic mice and cTnT R141W transgenic mice. In addition, levels of H 2 O 2 and malondialdehyde were increased and levels of glutathione and total antioxidant capability were strongly reduced in CYP2E1 transgenic mice and cTnT R141W transgenic mice. Myocyte apoptosis was significantly increased by 19-fold in CYP2E1 transgenic mice and by 11-fold in cTnT R141W transgenic mice, respectively, compared to wild-type mice. Mitochondrial-dependent apoptotic signal transduction events, such as cytochrome c release from mitochondria into the cytosol and the expres- sion of cleaved (active) caspases 3 and 9, were significantly increased in CYP2E1 transgenic mice and cTnT R141W transgenic mice. These results demonstrate that CYP2E1 over-expression produces apoptosis and that the up-regulation of CYP2E1 in cTnT R141W transgenic mice also correlates with apoptosis in this model. Abbreviations Col3a1, collagen types III; CYP2E1, cytochrome P450 2E1; DCM, dilated cardiomyopathy; GSH, glutathione; MDA, malondialdehyde; a-MHC, a-myosin heavy chain; PNP, p-nitrophenol; ROS, reactive oxygen species; T-AOC, total antioxidant capability; TUNEL, terminal dUTP nick end-labeling; WT, wild-type. 1484 FEBS Journal 278 (2011) 1484–1492 ª 2011 The Authors Journal compilation ª 2011 FEBS (ROS) [9]. The overexpression of CYP2E1 both in vivo and in vitro is associated with several cellular markers of oxidative stress, including products of lipid peroxi- dation (e.g. 4-hydroxynonenal and malondialdehyde), as well as with decreased viability as a result of both necrosis and apoptosis [10,11]. CYP2E1 levels are increased in the human ischemic and dilated heart [12] and left ventricular tissue of the spontaneously hyper- tensive rats [13]. In the present study, we found that the expression of CYP2E1 was up-regulated in dilated cardiomyopathy (DCM) heart from cTnT R141W trans- genic mice. The effect of CYP2E1 in the heart was investigated using heat-specific CYP2E1 transgenic mice. Results Detection of CYP2E1 expression in the transgenic DCM mice and generation of CYP2E1 transgenic mice Heart tissues were sampled, respectively, from mice at embryonic age 16.5 days, and at 1, 2, 4 and 8 weeks of age, and protein translational levels of CYP2E1 were detected. CYP2E1 was not expressed in the heart of mice at embryonic age 16.5 days, although it was strongly up-regulated at 1–2 weeks of age. Expression was found to be down-regulated after 4 weeks (Fig. 1A). CYP2E1 protein levels in DCM affected hearts from cTnT R141W transgenic mice at 1, 3 and 5 months of age were up-regulated by one- to four- fold compared to those measured in wild-type (WT) mice (Fig. 1B, C). To study the effect of CYP2E1 on the heart, C57BL ⁄ 6J mice carrying the CYP2E1 trans- gene were established. The transgenic plasmids were individually constructed by inserting the mouse CYP2E1 cDNA downstream of the a-myosin heavy chain (a-MHC) promoter (Fig. 1D). Two lines of CYP2E1 transgenic mice (founders #26 and #36) with a seven- to nine-fold increase in CYP2E1 levels compared to WT mice were selected from among 12 founders (Fig. 1E, F). Transgene copy numbers (mean ± SE) were determined in mice from F2 and F3 generations separately to be 16.5 ± 2.5 for founder #26 (n = 4) and 8.2 ± 3.1 for founder #36 (n = 4). The transgene was stable during the propagation of the transgenic mice. Transgenic expressed CYP2E1 was mainly localized in microsomal compartments CYP2E1 E16.5 1 week 2 week 4 week 8 week CYP2E1 WT DCM WT DCM WT DCM 1 m 3 m 5 m GAPDH Sal I Hind III CYP2E1 GAPDH WT a -MHC-CYP2E1 9897 bp Not I CYP2E1 level (Relative units) 1 m 3 m 5 m 4 3 2 1 0 cTnT R141W Not I 3 2 1 CYP2E1 WT #26 #36 Micr WT CYP2E1 12 9 6 3 0 WT CYP2E1 CYP2E1 activity (nmol·min –1 ·mg –1 ) GAPDH Cyto WT #26 #36 CYP2E1 level (Relative units) 0 A C EFGH D B Fig. 1. Expression of CYP2E1 in mouse heart tissue and the generation of transgenic mice. Protein translational levels of CYP2E1 in heart tissue from mice at embryonic age 16.5 days and at 1, 2, 4 and 8 weeks of age (A) and in the DCM-affected hearts from cTnT R141W trans- genic mice (DCM) at 1, 3 and 5 months of age (B) were detected by western blotting, using GADPH as a normalization standard; bars repre- sent the relative levels quantified by densitometry using QUANTITY ONE software (C; n = 3). The CYP2E1 transgenic construct was generated by inserting the target gene under the control of the a-MHC cardiac-specific promoter (D) and transgenic mice were created following micro- injection. Mouse lines (#26 and #36) with overexpression of CYP2E1 were selected by western blotting using GADPH as a normalization standard (E); bars represent the relative levels (F; n = 3). Transgenic expression levels of CYP2E1 in the microsome (micr) or cytosol (cyto) compartment were detected by western blotting (G). CYP2E1 enzyme activity in heart homogenate from CYP2E1 transgenic mice and WT mice was measured using the rate of oxidation of PNP to p-nitrocatechol; the results are expressed as nmolÆmin )1 Æmg )1 total homogenate protein and are given as the mean ± SD (n = 4) (H). W. Zhang et al. CYP2E1 causes apoptosis of cardiomyocytes in vivo FEBS Journal 278 (2011) 1484–1492 ª 2011 The Authors Journal compilation ª 2011 FEBS 1485 (Fig. 1G). CYP2E1 activity, analyzed by the oxidation of p-nitrophenol (PNP) in heart homogenates from CYP2E1 transgenic mice, was 8.1-fold higher than the activity in homogenates from WT mice (Fig. 1H). Overexpression of CPY2E1 in the heart leads to death of mice Zero mortality was observed in the WT group between 1 and 9 months of age. Death occurred in the cTnT R141W transgenic mice and CYP2E1 transgenic mice beginning at 3 months of age, with respective death rates of 17.5% in cTnT R141W transgenic mice and 13.75% in CYP2E1 transgenic mice being detected between 3 and 9 months of age (Fig. 2). Overexpression of CPY2E1 in the heart leads to the DCM phenotype and increases myocyte disarray and interstitial fibrosis Left ventricular dimensions and functions were deter- mined using hearts from WT, cTnT R141W , CYP2E1(#26) and CYP2E1(#36) transgenic mice (Fig. 3A and Table 1). The cTnT R141W transgenic mice and the two lines of CYP2E1 transgenic mice exhibited the DCM phenotype (i.e. chamber dilation and dysfunction). On light microscopy, both myocyte disarray and fibrosis were observed in the cTnT R141W transgenic mice and the two lines of CYP2E1 transgenic mice, in contrast to WT mice (Fig. 3B, C). mRNA expression levels of collagen types III (Col3a1) were increased by two-fold in CYP2E1 transgenic mice and by three-fold in cTnT R141W transgenic mice compared to WT mice (Fig. 3D, E). These results suggest that the overexpres- sion of CYP2E1 enhances the remodeling of the myo- cardial arrangement. Overexpression of CPY2E1 in the heart increases oxidative stress Levels of H 2 O 2 and malondialdehyde (MDA) were increased by 40% and 64%, respectively, in CYP2E1 transgenic mice, and by 28% and 36%, respectively, in cTnT R141W transgenic mice (Fig. 4A, B), whereas glutathione (GSH) and total antioxidant capability (T- AOC) levels were reduced by 28% and 46%, respec- tively, in CYP2E1 transgenic mice, and by 18% and 30%, respectively, in cTnT R141W transgenic mice (Fig. 4C, D) compared to levels in WT mice. These results indicate that the overexpression of CYP2E1 increased oxidative stress in heart tissues. Overexpression of CPY2E1 in the heart causes ultrastructural damage Transmission electron microscopy was used to exam- ine the myocardial ultrastructure of hearts from WT, cTnT R141W and CYP2E1 transgenic mice at 5 months of age (Fig. 5). Mitochondria were present as clus- ters between the parallel arrays of myofibrils in the WT mice. Lysed and disorganized myofibrils and interspersed clusters of mitochondria with deforma- tion and cristae disruption were found in both CYP2E1 transgenic mice and cTnT R141W transgenic mice. Overexpression of CYP2E1 in the heart increases the release of mitochondrial cytochrome c, activates caspases 3 and 9, and causes cardiomyocyte apoptosis Mitochondrial lesions may induce the release of mito- chondrial factors, such as cytochrome c, triggering cell death pathways. The release of cytochrome c may activate caspases 3 and 9 and execute the apoptotic system [14]. By western blotting, we found that up-regulation of CYP2E1 increased the release of cytochrome c from mitochondria into the cytosol, as well as the levels of cleaved active caspases 3 and 9 in both CYP2E1 transgenic mice and cTnT R141W transgenic mice compared to these parameters in WT mice (Fig. 6A–F). A terminal dUTP nick end-labeling (TUNEL) assay (Fig. 6G, H) indicated that the up-regulation of CYP2E1 caused myocyte apoptosis that was 19-fold higher in CYP2E1 transgenic mice and 11-fold higher in the cTnT R141W transgenic mice compared to WT mice (n =3, P < 0.05). The expression level of CYP2E1 was correlated with myo- cyte apoptosis (r = 0.997) in CYP2E1 transgenic mice and cTnT R141W transgenic mice. WT 1.00 0.95 CYP2E1 cTnT R141W Survival rate (%) * 123456 789 0.85 0.80 * Time (months) 0.90 Fig. 2. Kaplan–Meier survival analysis. Kaplan–Meier survival data for WT mice (n = 80), CYP2E1 transgenic mice (n = 80) and cTnT R141W transgenic mice (n = 40) were recorded at 1–9 months of age. *P < 0.01 versus WT group. CYP2E1 causes apoptosis of cardiomyocytes in vivo W. Zhang et al. 1486 FEBS Journal 278 (2011) 1484–1492 ª 2011 The Authors Journal compilation ª 2011 FEBS Discussion CYP2EI is a cytochrome P450 enzyme that catalyzes the oxidation of numerous exogenous compounds, includ- ing acetaminophen, benzene, carbon tetrachloride, ethanol and N-nitrosodimethylamine. CYP2E1 is also involved in the metabolism of endogenous aldehydes and ketones and plays a key role in gluconeogenesis in ketone bodies released as a result of energy deprivation [15–17], and also is markedly induced in fasting [18]. WT CYP2E1#26 CYP2E1#36 cTnT R141W WT CYP2E1#26 CYP2E1#36 cTnT R141W WT CYP2E1#26 CYP2E1#36 cTnT R141W 6 * WT CYP2E1 cTnT R141W Col3a1 (Relative units) WT CYP2E1 cTnT R141W 4 2 0 * * Col3a1 GAPDH A B C DE Fig. 3. Histopathological observations in transgenic mice. Heart tissue from WT, CYP2E1#26, CYP2E1#36 and cTnT R141W transgenic mice at 5 months of age was sampled and treated using standard pathological protocols. Whole-heart longitudinal sections are shown in (A) (magnifi- cation ·20; scale bars = 1 mm). The hemotoxylin and eosin-stained sections of the left ventricle are shown in (B) and Masson’s trichrome stained sections of the left ventricle are shown in (C) (myocytes stained red, collagenous tissue stained green; magnification ·400; scale bars = 100 lm). (D) Expression of mRNA for Col3a1 in hearts from WT mice, CYP2E1 and cTnT R141W transgenic mice at 5 months of age was measured by semi-quantitative RT-PCR and cTnT R141W . Sample loading was normalized using GAPDH. (E) Band intensities were quanti- fied by densitometry using QUANTITY ONE software (n =3;*P < 0.01 versus WT group). Table 1. M-mode echocardiographic analysis of mice at 5 months of age. LVIDd (LVIDs), left ventricular internal diameter at end-diastole (end-systole); LVEDV (LVESV), left ventricular end-diastolic volume (end-systolic volume); LVPWd (LVPWs), left ventricular posterior wall dur- ing diastole (systole); LVAWd (LVAWs), left ventricular anterior wall during diastole (systole). All values are given as the mean ± SEM. *P < 0.01 versus WT group. Parameter WT (n = 32) CYP2E1#26 (n = 28) CYP2E1#36 (n = 28) cTnT R141W (n = 30) Body weight (g) 26.0 ± 4.2 26.6 ± 4.6 26.6 ± 4.6 26.1 ± 3.2 Heart rate (beatsÆmin )1 ) 456 ± 42 479 ± 21 490 ± 23 480 ± 27 LVIDd (mm) 3.81 ± 0.23 4.08 ± 0.20* 4.16 ± 0.20* 4.27 ± 0.33* LVIDs (mm) 2.55 ± 0.26 2.91 ± 0.22* 2.96 ± 0.23* 3.40 ± 0.40* LVEDV (lL) 62.8 ± 9.08 71.6 ± 8.84* 74.3 ± 8.53* 83.0 ± 15.18* LVESV (lL) 23.7 ± 5.73 32.0 ± 6.43* 32.6 ± 6.70* 45.6 ± 13.12* LVPWd (mm) 0.83 ± 0.16 0.69 ± 0.13* 0.71 ± 0.11* 0.78 ± 0.11* LVPWs (mm) 1.21 ± 0.17 1.02 ± 0.18* 1.07 ± 0.15* 0.98 ± 0.13* LVAWd (mm) 0.82 ± 0.11 0.75 ± 0.09* 0.72 ± 0.12* 0.79 ± 0.12* LVAWs (mm) 1.27 ± 0.14 1.16 ± 0.13* 1.11 ± 0.14* 1.07 ± 0.12* Ejection fraction (%) 62.5 ± 7.08 55.4 ± 6.56* 56.3 ± 6.58* 45.8 ± 7.82* Fractional shortening (%) 33.5 ± 5.23 28.6 ± 4.34* 29.2 ± 4.38* 22.8 ± 4.59* W. Zhang et al. CYP2E1 causes apoptosis of cardiomyocytes in vivo FEBS Journal 278 (2011) 1484–1492 ª 2011 The Authors Journal compilation ª 2011 FEBS 1487 The expression of this enzyme is marked in the liver, heart, lungs, pancreas, brain and intestine [19,20]. We found that CYP2E1 was expressed immediately after birth and was maximally transcribed within the first week (Fig. 1A), similar to previous observations made in rat liver [21]. The expression of CYP2E1 is increased in the human ischemic and dilated heart [12] and in left ventricular tissue of the spontaneously hypertensive rats [13]. In the present study, we observed that the CYP2E1 expression level was significantly increased in the heart of cTnT R141W transgenic mice (Fig. 1B, C), which was a model of DCM [22]. Because CYP2E1 is also involved in the metabolism of endogenous aldehydes and ketones, and plays a key role in gluconeogenesis associ- ated with energy deprivation [15–18], it is possible that the up-regulation of CYP2E1 in the dilated heart might meet the energy demand for enhancing systolic function, which is dysfunctional in DCM mice. However, CYP2E1-catalyzed metabolism may also cause toxicity or cell damage through the production of toxic meta- bolites, oxygen radicals and lipid peroxidation. To understand the effects of CYP2E1 on the heart, we produced heart tissue-specific CYP2E1 transgenic mice. The overexpression of CYP2E1 lead to a DCM pheno- type similar to that in cTnT R141W transgenic mice (e.g. increased mortality, chamber dilation and contractile dysfunction, as well as myocyte disarray, interstitial fibrosis, ultrastructural degeneration with myofibrillar disorganization and mitochondria damage) (Figs 3 and 5 and Table 1). A number of studies have demonstrated that CYP2E1 is a loosely coupled enzyme, and can generate ROS and promote oxidative stress in cells during its catalytic cycle [9,23]. The formation of ROS can occur even in the absence of added exogenous substrates in the CYP2E1-expressing cells [24–26]. In present study, we found that levels of H 2 O 2 and MDA were signifi- cantly increased, whereas GSH and T-AOC were strongly reduced, in both CYP2E1 transgenic mice and cTnT R141W transgenic mice compared to WT mice (Fig. 4). Damage to mitochondria by CYP2E1 is an impor- tant mechanism for CYP2E1-dependent cytotoxicity [9,23,27–29]. In the present study, interspersed clusters of mitochondria with deformation and cristae disrup- tion were found in CYP2E1 transgenic mice and cTnT R141W transgenic mice (Fig. 5). In addition, the mitochondrial-dependent apoptotic pathway, initiated by cytochrome c release and followed by caspase 9-dependent caspase 3 activation, was also triggered in CYP2E1 transgenic mice and cTnT R141W transgenic mice, and the apoptosis of myocytes was increased in CYP2E1 transgenic mice and cTnT R141W transgenic mice (Fig. 6), suggesting that CYP2E1 increases the apoptosis of myocytes through the mitochondrial- dependent pathway. Clinical and experimental studies support the hypothesis that oxidative stress and myocyte apoptosis play an important role in the pathogenesis of cardiovascular diseases such as ische- mic heart disease, atherosclerosis, cardiomyopathy and 10.0 8.0 6.0 5.0 * * * * 6.0 4.0 2.0 4.0 3.0 2.0 1.0 H 2 O 2 (mmol·g –1 protein) 0.0 MDA (nmol·g –1 protein) 0.0 1.2 4.0 0.9 0.6 3.0 2.0 * * * * GSH (mg·g –1 protein) 0.3 0.0 T-AOC (U·mg –1 protein) 1.0 0.0 WT CYP2E1 cTnT R141W WT CYP2E1 cTnT R141W WT CYP2E1 cTnT R141W WT CYP2E1 cTnT R141W AB CD Fig. 4. Measurements of H 2 O 2 , MDA, GSH and T-AOC in the heart. WT mice (n = 12), CYP2E1 (n = 12) and cTnT R141W (n = 12) transgenic mice were sacrificed at 5 months of age and the total lysates of heart tissues were collected. Levels of H 2 O 2 (A), MDA (B), GSH (C) and T-AOC (D) were determined by colorimetric assays (*P < 0.01 versus WT group). WT C ccTnT R141W CYP2E1 Fig. 5. Ultrastructural observations in transgenic mice. Heart tissue from WT mice, CYP2E1 and cTnT R141W transgenic mice at 5 months of age were sampled for observation by transmission electron microscopy after treatment with standard pathological protocols (magnification ·30000; scale bars = 1 lm). CYP2E1 causes apoptosis of cardiomyocytes in vivo W. Zhang et al. 1488 FEBS Journal 278 (2011) 1484–1492 ª 2011 The Authors Journal compilation ª 2011 FEBS heart failure [30–37], and the results obtained in the present study suggest that CYP2E1-mediated oxidative stress and myocyte apoptosis are also evident in cTnT R141W transgenic mice. These results support a possible correlation between CYP2E1 expression and apoptosis in this DCM model. The pathogenesis of DCM is complex. On the basis of microarray data, we found that many genes, includ- ing CYP2E1, were expressed differently in the heart tissues of WT mice and cTNT R141W transgenic mice at 1 and 3 months of age, representing the early stage of DCM (data not shown); therefore, we propose that the up-regulated expression of CYP2E1 was involved in the pathogenesis of DCM in cTNT R141W transgenic mice. In the present study, we found that the overex- pression of CYP2E1 caused a DCM phenotype similar to that in cTnT R141W transgenic mice, and the expres- sion level of CYP2E1 was correlated with myocyte apoptosis (r = 0.997) in CYP2E1 transgenic mice and cTnT R141W transgenic mice. In addition, the up-regula- tion of CYP2E1 was also found in the heart of a DCM patient [12]. These results suggest that CYP2E1 may be a modulator of DCM subsequent to mutation of cTnT R141W or other factors. The CYP2E1-mediated myocyte apoptosis may be associated with the progress of DCM, such that the inhibition of CYP2E1 may mitigate the progression of DCM. In summary, we report that the CYP2E1 expression level is strongly increased in the DCM-affected heart of cTnT R141W transgenic mice. The present study provides the first evidence indicating that the over- expression of CYP2E1 causes cardiac oxidative stress, myocyte apoptosis and the DCM phenotype. Materials and methods Animals The a-MHC-cTnT R141W transgenic mice generated in Key Laboratory of Human Disease Comparative Medicine (Ministry of Health, Peking Union Medical College, Beij- ing, China) exhibited DCM phenotypic characteristics con- sistent with those reported previously [22]. The cDNA encoding mouse CYP2E1 (GenBank accession number: NM_021282.2) was cloned into an expression plasmid with the a-MHC promoter. Transgenic mice were generated by the microinjection method [38]. Genotyping of CYP2E1 transgenic mice was facilitated by the PCR using primers 5¢-CCAAGTTGGCAAAGCGCT-3¢ and 5¢-AAAAGAC CAAAGGCCAGCC-3¢. The expression of the target gene was analyzed by western blot analysis using antibodies to CYP2E1. Polyclonal rabbit anti-CYP2E1 was obtained from Abcam (Cambridge, MA, USA) (ab28146, dilution 1 : 2000) and horseradish peroxidase-conjugated IgG was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The copy number of the transgene was detected using a protocol modified from the method reported previ- ously by Randy et al. [39]. All the mice were bred in an Association for Assessment and Accreditation of Labora- tory Animal Care accredited facility and the use of animals was approved by the Animal Care and Use Committees of the Institute of Laboratory Animal Science of Peking Union Medical College (GC08-2027). CYP2E1 activity CYP2E1 activity was measured by the rate of oxidation of PNP to p-nitrocatechol in the presence of NADPH and O 2 , as described previously [40], using 200 lg of homogenate in a 3 2 1 Cyto Mito * * * * A Cyt-c in cyto Cyt-c B Cytochrom c (Relative units) 0 C Cyt c in mito Cleaved D 3 2 * * 4 3 * caspase 9 GAPDH F Cleaved Caspase 9 (Relative units) Cleaved Caspase 3 (Relative units) 1 0 2 1 0 * E G Cleaved caspase 3 GAPDH WT CYP2E1 cTnT R141W H 8 6 * * TUNEL-positive cell (%) 4 2 0 WT CYP2E1 cTnT R141W WT CYP2E1 cTnT R141W WT CYP2E1 cTnT R141W WT CYP2E1 cTnT R141W WT CYP2E1 cTnT R141W WT CYP2E1 cTnT R141W WT CYP2E1 cTnT R141W Fig. 6. Determination of the levels of cytochrome c release, cleaved caspases 3 and 9, as well as apoptotic cells in heart tis- sues. Protein levels of cytochrome c (cyt-c) in mitochondrial (mito) or cytosolic (cyto) fractions (A), cleaved caspase 9 (C) and cleaved caspase 3 (E) extracted from heart tissues of WT mice, and CYP2E1 and cTnT R141W transgenic mice at 5 months were mea- sured by western blotting. Bars represent the released cyto- chrome c (B), cleaved caspase-9 (D) and cleaved caspase-3 (F) levels quantified by densitometry using QUANTITY ONE software (n =3; *P < 0.01 versus WT group). Apoptotic cardiomyocytes were detected by the TUNEL assay in heart tissues sections (G) (magnification ·200; scale bars = 50 lm) and the apoptotic cardio- myocytes (H) were enumerated (%) in eight microscopic fields (n =3;*P < 0.01 versus WT group). W. Zhang et al. CYP2E1 causes apoptosis of cardiomyocytes in vivo FEBS Journal 278 (2011) 1484–1492 ª 2011 The Authors Journal compilation ª 2011 FEBS 1489 100-lL reaction system containing 100 mmolÆL )1 potassium- phosphate buffer (pH 7.4), 0.2 molÆ L )1 PNP and 1 mmolÆL )1 NADPH. Duplicate reaction mixtures were initiated with NAD(P)H, incubated at 37 °C, and terminated after 60 min by the addition of 30 lL of 20% trichloro-acetic acid. The supernatant was treated with 10 lL of 10 molÆ L )1 sodium hydroxide and D 546 was determined. Activity was deter- mined using: PNP activity (nmolÆmin )1 Æmg )1 protein) = D 546 ⁄ 9.53 ⁄ 0.2⁄ 60 ⁄ 7.1 · 10 3 . Echocardiography Mice were lightly anesthetized by intraperitoneal injection of tribromo-ethanol at a dose of 180 mLÆkg )1 body weight. M- mode echocardiography of the left ventricle was recorded at the tip of the mitral valve apparatus with a 30 MHz trans- ducer (Vevo770; VisualSonics, Toronto, Canada) [22]. Histological analysis Cardiac tissue from mice at the age of 5 months was fixed in 4% formaldehyde and mounted in paraffin blocks. Sec- tions were stained with hemotoxylin and eosin or Masson’s trichrome. RT-PCR Total RNA was isolated from heart tissue from each trans- genic mouse at the age of 5 months using TRIzol Reagent (In- vitrogen, Carlsbad, CA, USA). First-strand cDNA was synthesized from 2 lg of total RNA using random hexamer primers in accordance with the Superscript b reverse trans- criptase manufacturer’s protocol (Invitrogen). Detection of mRNA for Col3a1 was carried out by the RT-PCR, using GAPDH for normalization. Primers were: 5¢-GGCAGTGA TGGGCAACCT-3¢ and 5¢-TCCCTTCGCACCGTTCTT-3¢ for Col3a1; 5¢-CAAGGTCATCCATGACAACTTTG-3¢ and 5¢-GTCCACCACCCTGTTGCTGTAG-3¢ for GADPH. Transmission electron microscopy Myocardial samples of mice at 5 months of age were rou- tinely fixed in 2.5% glutaraldehyde in 0.1 molÆL )1 phosphate buffer (pH 7.4) and post-fixed in buffered 1% osmium tetrox- ide for 1 h. Samples were then dehydrated using several changes of ethanol and embedded in Epon 812. Thin sections were stained with uranyl acetate and lead citrate and exam- ined under a JEM-1230 Transmission Electronic Microscope (JEOL Ltd, Tokyo, Japan) equipped with a digital camera. Survival analysis Cumulative percentage mortality of the transgenic mice was calculated each month and the data from 1 to 6 months of age were summarized. Upon the death of each mouse, the body was autopsied by a pathologist and morphological and pathological changes of the heart were recorded. Kaplan–Meier curves for survival analysis were compared by the log-rank test using SPSS, version 10.0 (SPSS Inc., Chicago, IL, USA). Measurements of H 2 O 2 , MDA, GSH and T-AOC in heart tissue Hearts tissue from the mice at 5 months of age was homog- enized rapidly in nine volumes of buffer (0.15 molÆL )1 KCl, 1.0 mmolÆL )1 EDTA) to obtain 1 : 10 (w ⁄ v) homogenates. Homogenates were centrifuged at 13 000 g (4 °C) for 30 min to collect the supernatant for assay. Levels of MDA were evaluated by the thiobarbituric acid reactive sub- stances method [41]. Levels of H 2 O 2 were measured using an assay kit (DE3700; R&D Systems, Minneapolis, MN, USA). Levels of GSH were measured using the GSH-400 colorimetric assay kit (Promega, Madison, WI, USA). Levels of T-AOC were measured using assay kit ab65329 (Abcam). The protein concentration in heart homogenates was determined by the Bradford method using BSA as a standard [42]. TUNEL assay The in situ TUNEL assay was performed in sections of heart tissues using the In Site Cell Death Detection Kit (Roche Diagnostics GmbH, Mannheim, Germany) in accordance with the manufacturer’s instructions. Eight images per heart (three hearts per genotype group) were acquired and positive cells were counted individually. The results were expressed as the percentage of apoptotic cells among the total cell population. Separation of cytosolic and mitochondrial fractions To detect cytochrome c release, cytosolic and mitochon- drial fractions were isolated using a method described pre- viously [33]. Heart tissue from mice at 5 months of age was homogenized in buffer (50 mmolÆL )1 Tris, pH 7.5, 0.5 molÆL )1 NaCl, 1.0 mmolÆL )1 EDTA, 10% glycerol and proteinase inhibitor cocktail) using 40 strokes in a Dounce homogenizer. After centrifugation at 13 000 g for 15 min, the cytosol fraction was obtained as a supernatant, and the pelleted mitochondrial fraction was resuspended in lysis buffer. Western blot Cytosol and mitochondrial fractions were resolved by 15% SDS ⁄ PAGE to enable detection of cytochrome c release. In CYP2E1 causes apoptosis of cardiomyocytes in vivo W. Zhang et al. 1490 FEBS Journal 278 (2011) 1484–1492 ª 2011 The Authors Journal compilation ª 2011 FEBS addition, hearts tissues from mice at 5 months of age were homogenized and subjected to 15% SDS ⁄ PAGE to enable detection of cleaved caspases 3 and 9. After the transfer step, the membranes were incubated with antibodies specific for cytochrome c and cleaved (active) caspases 3 and 9 (Cell Signaling Technology, Beverly, MA, USA; dilution 1 : 1000). Primary antibodies were visualized with horse- radish peroxidase conjugated to goat anti-rabbit IgG as the second antibody (Santa Cruz Biotechnology; dilution 1 : 20 000) using a chemiluminescent detection system (Western Blotting Luminal Reagent, Santa Cruz Biotech- nology). Variations in sample loading were normalized rela- tive to the GAPDH signal. Bands were quantified by the densitometry function of quantity one software (Bio-Rad, Hercules, CA, USA). Statistical analysis All measurement data are expressed as the mean ± SEM. Statistical significance of differences among groups was analyzed by one-way analysis of variance. P < 0.05 was considered statistically significant. Acknowledgements The present work was supported in part by the Ministry of Health Foundation (200802036) and the National Science and Technology Major Projects (2009ZX09501-026). The authors declare that there are no conflicts of interest. References 1 Woodcroft KJ, Hafner MS & Novak RF (2002) Insulin signaling in the transcriptional and post-transcriptional regulation of CYP2E1 expression. Hepatology 35, 263–273. 2 Raza H, Ahmed I, John A & Sharma AK (2000) Modulation of xenobiotic metabolism and oxidative stress in chronic streptozotocin-induced diabetic rats fed with Momordica charantia fruit extract. J Biochem Mol Toxicol 14, 131–139. 3 Ohkuwa T, Sato Y & Naoi M (1995) Hydroxyl radical formation in diabetic rats induced by streptozotocin. Life Sci 56, 1789–1798. 4 Hodges NJ, Green RM, Chipman JK & Graham M (2007) Induction of DNA strand breaks and oxidative stress in HeLa cells by ethanol is dependent on CYP2E1 expression. Mutagenesis 22, 189–194. 5 McKillop IH & Schrum LW (2005) Alcohol and liver cancer. Alcohol 35, 195–203. 6 Stickel F, Schuppan D, Hahn EG & Seitz HK (2002) Cocarcinogenic effects of alcohol in hepatocarcinogene- sis. Gut 51, 132–139. 7 Seitz HK, Matsuzaki S, Yokoyama A, Homann N, Va ¨ keva ¨ inen S & Wang XD (2001) Alcohol and cancer. Alcohol Clin Exp Res 25, 137S–143S. 8 Ekstrom G & Ingelman-Sundberg M (1989) Rat liver microsomal NADPH-supported oxidase activity and lipid peroxidation dependent on ethanol-inducible cyto- chrome P-450 (P-450IIE1). Biochem Pharmacol 38, 1313–1319. 9 Cederbaum AI, Wu D, Mari M & Bai J (2001) CYP2E1-dependent toxicity and oxidative stress in HepG2 cells. Free Radic Biol Med 31, 1539–1543. 10 Schattenberg JM, Wang Y, Rigoli RM, Koop DR & Czaja MJ (2004) CYP2E1 over-expression alters hepatocyte death from menadione and fatty acids by activation of ERK1 ⁄ 2 signaling. Hepatology 39, 444– 455. 11 Wu D & Cederbaum AI (2001) Removal of glutathione produces apoptosis and necrosis in HepG2 cells overex- pressing CYP2E1. Alcohol Clin Exp Res 25, 619–628. 12 Sidorik L, Kyyamova R, Bobyk V, Kapustian L, Rozhko O, Vigontina O, Ryabenko D, Danko I, Maksymchuk O, Kovalenko VN et al. (2005) Molecular chaperone, HSP60, and cytochrome P450 2E1 co- expression in dilated cardiomyopathy. Cell Biol Int 29, 51–55. 13 Thum T & Borlak J (2002) Testosterone, cytochrome P450, and cardiac hypertrophy. FASEB J 16, 1537– 1549. 14 Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M, Alnemri ES & Wang X (1997) Cytochrome c and dATP-dependent formation of Apaf-1 ⁄ caspase-9 complex initiates an apoptotic protease cascade. Cell 91, 479–489. 15 Casazza JP, Felver ME & Veech RL (1984) The metab- olism of acetone in rat. J Biol Chem 259, 231–236. 16 Ekstrom G, von Bahr C & Ingelman-Sundberg M (1989) Human liver microsomal cytochrome P-450IIE1. Immunological evaluation of its contribution to micro- somal ethanol oxidation, carbon tetrachloride reduction and NADPH oxidase activity. Biochem Pharmacol 38, 689–693. 17 Koop DR & Casazza JP (1985) Identification of ethanol-inducible P-450 isozyme 3a as the acetone and acetol monooxygenase of rabbit microsomes. J Biol Chem. 260, 13607–13612. 18 Johansson I, Lindros KO, Eriksson H & Ingelman- Sundberg M (1990) Transcriptional control of CYP2E1 in the perivenous liver region and during starvation. Biochem Biophys Res Commun 173, 331–338. 19 Thum T & Borlak J (2000) Gene expression in distinct regions of the heart. Lancet 355, 979–983. 20 Ingelman-Sundberg M, Johansson I, Yin H, Terelius Y, Eliasson E, Clot P & Albano E (1993) Ethanol-induc- ible cytochrome P4502E1, genetic polymorphism, W. Zhang et al. CYP2E1 causes apoptosis of cardiomyocytes in vivo FEBS Journal 278 (2011) 1484–1492 ª 2011 The Authors Journal compilation ª 2011 FEBS 1491 regulation, and possible role in the etiology of alcohol- induced liver disease. Alcohol 10, 447–452. 21 Song BJ, Gelboin HV, Park SS, Yang CS & Gonzalez FJ (1986) Complementary DNA and protein sequences of ethanol-inducible rat and human cytochrome P-450s. Transcriptional and post-transcrip- tional regulation of the rat enzyme. J Biol Chem 261, 16689–16697. 22 Juan F, Wei D, Xiongzhi Q, Ran D, Chunmei M, Lan H, Chuan Q & Lianfeng Z (2008) The changes of the cardiac structure and function in cTnTR141W transgenic mice. Int J Cardiol 128, 83–90. 23 Dey A & Cederbaum AI (2006) Geldanamycin, an inhibitor of Hsp90, potentiates cytochrome P4502E1- mediated toxicity in HepG2 cells. J Pharmacol Exp Ther 317, 1391–1399. 24 Nieto N, Friedman SL & Cederbaum AI (2002) Stimulation and proliferation of primary rat hepatic stellate cells by cytochrome P450 2E1-derived reactive oxygen species. Hepatology 35, 62–73. 25 Kim HR, Lee GH, Cho EY, Chae SW, Ahn T & Chae HJ (2009) Bax inhibitor 1 regulates ER-stress- induced ROS accumulation through the regulation of cytochrome P450 2E1. J Cell Sci 122, 1126–1133. 26 Dai Y, Rashba-Step J & Cederbaum AI (1993) Stable expression of human cytochrome P4502E1 in HepG2 cells, characterization of catalytic activities and produc- tion of reactive oxygen intermediates. Biochemistry 32, 6928–6937. 27 Wu D & Cederbaum AI (2002) Cyclosporine A protects against arachidonic acid toxicity in rat hepatocytes, role of CYP2E1 and mitochondria. Hepatology 35, 1420– 1430. 28 Cederbaum AI (2006) Cytochrome P450 2E1-dependent oxidant stress and upregulation of anti-oxidant defense in liver cells. J Gastroenterol Hepatol 21(Suppl 3), S22–S25. 29 Nieto N, Friedman SL & Cederbaum AI (2002) Cytochrome P450 2E1-derived reactive oxygen species mediate paracrine stimulation of collagen I protein synthesis by hepatic stellate cells. J Biol Chem 277, 9853–9864. 30 Giordano FJ (2005) Oxygen, oxidative stress, hypoxia, and heart failure. J Clin Invest 115, 500–508. 31 Lo ´ pez Farre ´ A & Casado S (2001) Heart failure, redox alterations, and endothelial dysfunction. Hypertension 38, 1400–1405. 32 Ungvari Z, Gupte SA, Recchia FA, Batkai S & Pacher P (2005) Role of oxidative-nitrosative stress and downstream pathways in various forms of cardiomyop- athy and heart failure. Curr Vasc Pharmacol 3, 221–229. 33 Narula J, Pandey P, Arbustini E, Haider N, Narula N, Kolodgie FD, Dal Bello B, Semigran MJ, Bielsa-Mas- deu A, Dec GW et al. (1999) Apoptosis in heart failure, release of cytochrome c from mitochondria and activa- tion of caspase-3 in human cardiomyopathy. Proc Natl Acad Sci USA 96, 8144–8149. 34 Toko H, Takahashi H, Kayama Y, Oka T, Minamino T, Okada S, Morimoto S, Zhan DY, Terasaki F, Anderson ME et al. (2010) Ca 2+ ⁄ calmodulin-dependent kinase IIdelta causes heart failure by accumulation of p53 in dilated cardiomyopathy. Circulation 122, 891– 899. 35 Narula J, Kolodgie FD & Virmani R (2000) Apoptosis and cardiomyopathy. Curr Opin Cardiol 15, 183–188. 36 Di Napoli P, Taccardi AA, Grilli A, Felaco M, Balbone A, Angelucci D, Gallina S, Calafiore AM, De Caterina R & Barsotti A (2003) Left ventricular wall stress as a direct correlate of cardiomyocyte apoptosis in patients with severe dilated cardiomyopathy. Am Heart J 146, 1105–1111. 37 Latif N, Khan MA, Birks E, O’Farrell A, Westbrook J, Dunn MJ & Yacoub MH (2000) Upregulation of the Bcl-2 family of proteins in end stage heart failure. JAm Coll Cardiol 35, 1769–1777. 38 Gordon JW & Ruddle FH (1981) Integration and stable germ line transmission of genes injected into mouse pronuclei. Science 214, 1244–1246. 39 Randy R, Tom M, Mark H & Carl W (1998) Quantita- tive PCR by continuous fluorescence monitoring of a double strand DNA specific binding dye. Biochemistry 2, 8–11. 40 Chen Q & Cederbaum AI (1998) Cytotoxicity and apoptosis produced by cytochrome P450 2E1 in Hep G2 cells. Mol Pharmacol 53, 638–648. 41 Ohkawa H, Ohishi N & Yagi K (1979) Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 95, 351–358. 42 Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248–254. CYP2E1 causes apoptosis of cardiomyocytes in vivo W. Zhang et al. 1492 FEBS Journal 278 (2011) 1484–1492 ª 2011 The Authors Journal compilation ª 2011 FEBS . triggered in CYP2E1 transgenic mice and cTnT R141W transgenic mice, and the apoptosis of myocytes was increased in CYP2E1 transgenic mice and cTnT R141W transgenic mice. Expression of CYP2E1 increases oxidative stress and induces apoptosis of cardiomyocytes in transgenic mice Wei Zhang 1 , Dan Lu 1 ,