Myocyte apoptosis was significantly increased by 19-fold in CYP2E1 transgenic mice and by 11-fold in cTnTR141W transgenic mice, respectively, compared to wild-type mice.. Results Detectio
Trang 1induces apoptosis of cardiomyocytes in transgenic mice Wei Zhang1, Dan Lu1, Wei Dong1, Li Zhang1, Xiaojuan Zhang1, Xiongzhi Quan1, Chunmei Ma2, Hong Lian1and Lianfeng Zhang1,2
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 cTnTR141Wtransgenic 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 cTnTR141W transgenic mice In addition, levels of
H2O2 and malondialdehyde were increased and levels of glutathione and total antioxidant capability were strongly reduced in CYP2E1 transgenic mice and cTnTR141Wtransgenic mice Myocyte apoptosis was significantly increased by 19-fold in CYP2E1 transgenic mice and by 11-fold in cTnTR141W 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 cTnTR141W transgenic mice These results demonstrate that CYP2E1 over-expression produces apoptosis and that the up-regulation of CYP2E1 in cTnTR141W 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.
Trang 2(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 cTnTR141W
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 cTnTR141W 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
GAPDH
Sal I Hind III
CYP2E1 GAPDH
WT
a-MHC-CYP2E1
9897 bp
Not I
CYP2E1 level (Relative units)
4 3 2 1 0 cTnT R141W
Not I
3 2 1 CYP2E1
Micr
WT CYP2E1 12
9 6 3
0
WT CYP2E1 CYP2E1 activity (nmol·min
WT #26 #36 CYP2E1 level (Relative units) 0
A
C
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 cTnTR141W 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)1Æmg)1total homogenate protein and are given as the mean ± SD (n = 4) (H).
Trang 3(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
cTnTR141W transgenic mice and CYP2E1 transgenic
mice beginning at 3 months of age, with respective
death rates of 17.5% in cTnTR141W 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, cTnTR141W, CYP2E1(#26)
and CYP2E1(#36) transgenic mice (Fig 3A and
Table 1) The cTnTR141Wtransgenic 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 cTnTR141W 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
cTnTR141W 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 H2O2 and malondialdehyde (MDA) were increased by 40% and 64%, respectively, in CYP2E1 transgenic mice, and by 28% and 36%, respectively, in cTnTR141W 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 cTnTR141W 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, cTnTR141W 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 disrupdeforma-tion were found in both CYP2E1 transgenic mice and cTnTR141W 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 cTnTR141W 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 cTnTR141W 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 cTnTR141Wtransgenic mice
WT 1.00
0.95
CYP2E1
*
1 2 3 4 5 6 7 8 9
0.85
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.
Trang 4CYP2EI 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
6
*
WT CYP2E1 cTnT R141W
(Relative units) WT CYP2E1 cTnT R141W
4 2 0
Col3a1 GAPDH
A
B
C
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 durdur-ing diastole (systole) All values are given as the mean ± SEM.
*P < 0.01 versus WT group.
Trang 5The 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 cTnTR141Wtransgenic 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 cTnTR141Wtransgenic 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 H2O2 and MDA were signifi-cantly increased, whereas GSH and T-AOC were strongly reduced, in both CYP2E1 transgenic mice and cTnTR141W 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 cTnTR141W 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 cTnTR141W transgenic mice, and the apoptosis of myocytes was increased in CYP2E1 transgenic mice and cTnTR141W 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
0.0
0.9
0.6
3.0
*
0.3
1.0 0.0
WT CYP2E1 cTnT R141W WT CYP2E1 cTnT R141W
WT CYP2E1 cTnT R141W WT CYP2E1 cTnT R141W
Fig 4 Measurements of H2O2, MDA, GSH and T-AOC in the
heart WT mice (n = 12), CYP2E1 (n = 12) and cTnTR141W(n = 12)
transgenic mice were sacrificed at 5 months of age and the total
lysates of heart tissues were collected Levels of H2O2(A), MDA
(B), GSH (C) and T-AOC (D) were determined by colorimetric
assays (*P < 0.01 versus WT group).
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).
Trang 6heart failure [30–37], and the results obtained in the
present study suggest that CYP2E1-mediated oxidative
stress and myocyte apoptosis are also evident in
cTnTR141W 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 cTNTR141Wtransgenic 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 cTNTR141W transgenic
mice In the present study, we found that the
overex-pression of CYP2E1 caused a DCM phenotype similar
to that in cTnTR141W transgenic mice, and the expres-sion level of CYP2E1 was correlated with myocyte apoptosis (r = 0.997) in CYP2E1 transgenic mice and cTnTR141Wtransgenic 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 cTnTR141Wor 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 cTnTR141W 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
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
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 O2,
as described previously [40], using 200 lg of homogenate in a
3 2 1
Cyto Mito
A
Cyt-c
in cyto
Cyt-c
B
(Relative units) 0 C
Cyt c
in mito
Cleaved
2
4
caspase 9 GAPDH
F
1 0
2 1 0
*
E
G
Cleaved caspase 3 GAPDH
6
*
*
4
2
0
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).
Trang 7100-lL reaction system containing 100 mmolÆL)1
potassium-phosphate buffer (pH 7.4), 0.2 molÆL)1PNP and 1 mmolÆL)1
NADPH Duplicate reaction mixtures were initiated with
NAD(P)H, incubated at 37C, 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 molÆL)1 sodium
deter-mined using: PNP activity (nmolÆmin)1Æmg)1 protein) =
D546⁄ 9.53 ⁄ 0.2 ⁄ 60 ⁄ 7.1 · 103
Echocardiography
Mice were lightly anesthetized by intraperitoneal injection of
tribromo-ethanol at a dose of 180 mLÆkg)1body 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)1phosphate
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 H2O2, 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)1KCl, 1.0 mmolÆL)1 EDTA) to obtain 1 : 10 (w⁄ v) homogenates
30 min to collect the supernatant for assay Levels of MDA were evaluated by the thiobarbituric acid reactive sub-stances method [41] Levels of H2O2 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
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
Trang 8addition, hearts tissues from mice at 5 months of age were
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
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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