Báo cáo khoa học: Expression of CYP2E1 increases oxidative stress and induces apoptosis of cardiomyocytes in transgenic mice pdf

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:
(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.
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