In the present study, primary cultured cardiomyocytes from neonatal rats were used to investigate changes in TRAP1 expression after hypoxia treatment as well as the mechanism and effect
Trang 1receptor-associated protein 1 protects cardiomyocytes
from hypoxic injury by regulating mitochondrial
permeability transition pore opening
Fei Xiang, Yue-Sheng Huang, Xiao-Hua Shi and Qiong Zhang
Institute of Burn Research, State Key Laboratory of Trauma, Burns and Combined Injury, Southwest Hospital, Third Military Medical University, Chongqing, China
Introduction
Hypoxia is one of the main causes of myocardial
damage after the receipt of a burn In the early stages
after a severe burn, myocardial damage not only
causes cardiac insufficiency, but also induces or
aggravates burn shock, which can cause or aggravate
ischaemic⁄ hypoxic injury to other organs [1,2] Hence,
it is important to protect cardiomyocytes from hypoxic
damage Mitochondria are the primary target of hypoxic damage in cardiomyocytes Several inter-related factors, including calcium overload, an increase
in reactive oxygen species (ROS) and a decrease in adenine nucleotides, contribute to mitochondrial impairment during hypoxia and ischaemia [3] Mito-chondrial dysfunction in cardiomyocytes can also
Keywords
cardiomyocytes; cell damage; hypoxia;
mitochondrial permeability transition pore;
tumour necrosis factor receptor-associated
protein 1
Correspondence
Y.-S Huang, Institute of Burn Research,
State Key Laboratory of Trauma, Burns and
Combined Injury, Southwest Hospital, Third
Military Medical University, Chongqing
400038, China
Fax: +86 23 65461696
Tel: +86 23 65461696
E-mail: yshuang.tmmu@gmail.com
(Received 3 December 2009, revised 3
February 2010, accepted 11 February
2010)
doi:10.1111/j.1742-4658.2010.07615.x
Tumour necrosis factor receptor-associated protein 1 (TRAP1) is a mito-chondrial chaperone that plays a role in maintaining mitomito-chondrial func-tion and regulating cell apoptosis The opening of the mitochondrial permeability transition pore (MPTP) is a key step in cell death after hypoxia However, it is still unclear whether TRAP1 protects cardiomyo-cytes from hypoxic damage by regulating the opening of the pore In the present study, primary cultured cardiomyocytes from neonatal rats were used to investigate changes in TRAP1 expression after hypoxia treatment
as well as the mechanism and effect of TRAP1 on hypoxic damage The results obtained showed that TRAP1 expression increased after 1 h of hypoxia and continued to increase for up to 12 h of treatment Hypoxia caused an increase in cell death and decreased cell viability and mitochon-drial membrane potential; overexpressing TRAP1 prevented hypoxia-induced damage to cardiomyocytes The silencing of TRAP1 hypoxia-induced an increase in cell death and decreased both cell viability and mitochondrial membrane potential in cardiomyocytes under normoxic and hypoxic condi-tions Furthermore, cell damage induced by the silencing of TRAP1 was prevented by the mitochondrial permeability transition pore inhibitor, cyclosporin A These data demonstrate that hypoxia induces an increase in TRAP1 expression in cardiomyocytes, and that TRAP1 plays a protective role by regulating the opening of the mitochondrial permeability transition pore
Abbreviations
Ad-TRAP1, recombinant adenovirus vector for TRAP1 overexpression; CsA, cyclosporin A; CypD, cyclophilin D; GFP, green fluorescent protein; HSP, heat shock protein; MPTP, mitochondrial permeability transition pore; ROS, reactive oxygen species; siRNA, small interfering RNA; TRAP1, tumour necrosis factor receptor-associated protein 1.
Trang 2directly lead to cell death after hypoxia The
mitochon-drial permeability transition pore (MPTP) is a
nonspe-cific pore that opens during the time of calcium
overload, oxidative stress, adenine nucleotide depletion
and elevated phosphate levels Many studies have
dem-onstrated the role of MPTP opening during an
ischae-mia⁄ reperfusion injury to the heart and other organs
[4–6] We have also demonstrated that more MPTPs
open in cardiomyocytes after hypoxia compared to
normoxic conditions [7] Once the pore opens, the
membrane potential and pH gradient dissipate,
pre-venting ATP generation by oxidative phosphorylation
Ultimately, these changes lead to cell death through
the activation of phospholipases, nucleases and
prote-ases [8] Indeed, the irreversible mitochondrial injury
caused by MPTP opening is the key step in cell death
that occurs during hypoxia and other conditions [9]
Tumour necrosis factor receptor-associated protein 1
(TRAP1) localizes to the mitochondria and its targeting
sequence, which is found in the N-terminus of the
pro-tein, is for mitochondria matrix An analysis of the
cDNA sequences reveals that TRAP1 is identical to
heart shock protein (HSP) 75, which is a member of the
HSP90 family [10] HSP90 comprises an important
molecular chaperone that is involved in many cellular
processes After hypoxia treatment, HSP90 expression
increases, and this plays a protective role against
dam-age [11] However, the changes in TRAP1 in
cardiomyo-cytes under hypoxic conditions remain unclear TRAP1
comprises a mitochondrial chaperone that is critical for
importing proteins into the mitochondrial matrix [12] A
previous study showed that up-regulation of TRAP1
expression suppressed arsenite-induced apoptosis in
lung epithelium cells [13] Apoptogenic inducers, such as
the protein-tyrosine kinase inhibitor
b-hydroxyisovaler-ylshikonin or the topoisomerase II inhibitor VP16, can
decrease TRAP1 expression [14] At the same time,
TRAP1 antagonizes ROS production and protects
tumour cells from granzyme M-mediated apoptosis [15]
A recent study also demonstrated that TRAP1
over-expression preserves the mitochondrial membrane
potential (Dw) and maintains ATP levels and cell
viabil-ity during ischaemic-like injury in vivo [16] These data
suggest that TRAP1 may play an important role in
maintaining mitochondrial function As noted above,
MPTP is recognized as a key player in cell death
How-ever, whether TRAP1 can protect cells from hypoxic
damage by regulating MPTP opening in cardiomyocytes
has remained unclear until now
The present study aimed to observe changes in
TRAP1 expression after hypoxia treatment and to
investigate the effect of TRAP1 on cell death and
MPTP opening in primary cardiomyocytes
Results
Hypoxia increases TRAP1 expression in cardiomyocytes
Western blot analysis was used to investigate TRAP1 expression after hypoxia treatment in cardiomyocytes TRAP1 content increased after 1 h of hypoxia and continued to increase until for up to 12 h compared to the normoxic group At the same time, longer hypoxic treatments yielded higher TRPA1 expression (Fig 1A,B) We then examined TRAP1 immunoreac-tivity with an immunofluorescence assay After 1 h of hypoxia, TRAP1 fluorescence intensity was brighter in hypoxic cells than in normoxic cells, which meant that TRAP1 expression increased after 1 h of hypoxia (Fig 1C,D) Furthermore, increases in TRAP1 fluores-cence intensity became greater with an extension of hypoxic treatment time (Figs 1E–G and 2I) The results obtained were similar to those observed with the western blot
TRAP1 overexpression decreases hypoxic damage to cardiomyocytes
Because TRAP1 expression of cardiomyocytes was increased after hypoxia treatment, we performed exper-iments to determine whether the increase in TRAP1 expression plays a protective role in hypoxic cardio-myocytes We constructed a recombinant adenovirus vector for TRAP1 overexpression (Ad-TRAP1) and transfected the cardiomyocytes After 48 h of infection, infection efficiency was visualized by the expression of green fluorescent protein (GFP), and more than 90%
of the cardiomyocytes were infected (Fig 2A) Protein was then harvested and the results obtained by western blotting revealed that TRAP1 expression increased sig-nificantly in cardiomyocytes infected with Ad-TRAP1 compared to the expression in negative vector-trans-duced cardiomyocytes and to endogenous TRAP1 levels in normoxic cells (Fig 2B)
To evaluate the role of TRAP1 overexpression in cardiomyocytes under hypoxic conditions, we investi-gated cell viability, Dw and cell death After 6 h of hypoxia, cell viability and Dw were significantly lower
in the uninfected and vector-infected cardiomyocytes compared to normoxic cells By contrast, TRAP1 overexpression increased hypoxic cell viability (Fig 2C) and preserved Dw (Fig 2D) Additionally, propidium iodide staining was used to investigate the effect of TRAP1 overexpression on cell death As shown in Fig 3, hypoxia treatment resulted in increased cell death, which was reduced by TRAP1
Trang 3overexpression At the same time, infection with
the negative vector had no effect on hypoxia-induced
cell death
Silencing of TRAP1 expression induces cardiomyocyte damage
After demonstrating that TRAP1 overexpression can prevent hypoxic damage in cardiomyocytes, we next examined whether silencing TRAP1 expression induced damage in cardiomyocytes After infection with TRAP1-small interfering RNA (siRNA) or control vector adenovirus for 4 days, more than 90% of the cardiomyocytes were determined to be infected by observing GFP expression using a fluorescent micro-scope (Fig 4A) The effective silencing of endogenous TRAP1 by TRAP1-siRNA adenovirus infection was also confirmed by western blotting (Fig 4B)
After TRAP1-siRNA infection, the viability of the cardiomyocytes was significantly decreased compared
to that of normoxic cells and vector-infected cells (Fig 4C) Furthermore, silencing TRAP1 expression induced a decrease in Dw of cardiomyocytes under normoxic conditions and aggravated Dw loss induced
by hypoxia (Fig 4D) As shown in Fig 5, TRAP1 depletion also induced a significant increase in cardio-myocytes death, whereas there was very little cell death
in the normoxic cardiomyocytes and vector-infected cardiomyocytes
In addition, we also observed the effect of silencing TRAP1 expression on cardiomyocyte damage under hypoxic conditions It was found that hypoxia induced more injuries in cardiomyocytes in terms of both viability and cell death after TRAP1-siRNA infection (Fig 6A,B)
MPTP mediates the TRAP1 effect TRAP1 is a mitochondria chaperon and plays a role
in maintaining mitochondrial homeostasis, whereas MPTP opening is a key step in the process of cell death Therefore, we aimed to determine whether MPTP opening mediates TRAP1 behaviour After cardiomyocytes were infected with TRAP1-siRNA or negative vector for 2 days, cyclosporin A (CsA;
2 lm), a selective inhibitor of MPTP opening, was added to the cardiomyocytes Cells were then infected for an additional 2 days (4 days in total) Treatment with CsA prevented the decrease in cardiomyocyte viability and the increase in cell death induced by TRAP1-siRNA infection under normoxic conditions (Fig 7) However, there were no differences between vector-infected cells and vector-infected cells after CsA treatment (Fig 7)
Because silencing TRAP1 expression aggravated hypoxic damage of cardiomyocytes, we next investi-gated the effect of CsA on cell viability and cell death
A
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Fig 1 Effects of hypoxia on the TRAP1 levels in primary cultured
cardiomyocytes (A) Western blots show TRAP1 immunoreactivity
in normoxic or hypoxic cells at the indicated times b-actin was
used as an internal control (B) TRAP1 levels were normalized with
b-actin under normoxic or hypoxic conditions (C–G) TRAP1
expres-sion detected by immunofluorescence under normoxic conditions
(C) and hypoxic conditions for 1 h (D), 3 h (E), 6 h (F) and 12 h (G).
TRAP1 primary antibody was omitted as a negative control (H).
(I) Differences in fluorescence intensity of TRAP1 in normoxic or
hypoxic cells Data are the mean ± SEM Scale bar = 25 lm.
*P < 0.05 compared to the normoxic group The experiment was
repeated three times.
Trang 4after TRAP1-siRNA infection under hypoxic
condi-tions After 6 h of hypoxia, treatment with CsA
abol-ished cardiomyocyte damage induced both by hypoxia
and silencing TRAP1 under hypoxic conditions
(Fig 6) On the basis of the results described above,
we conclude that silencing TRAP1 expression induces
MPTP opening in cardiomyocytes, resulting in cell
injury Furthermore, the up-regulation of TRAP1
expression may play a protective role in hypoxic
cardiomyocytes by reducing MPTP opening
Discussion
In the present study, we found that TRAP1 expression
of cardiomyocytes increases after hypoxia and that
TRAP1 overexpression protects cardiomyocytes from
hypoxic damage At the same time, silencing TRAP1
expression causes cell damage under normoxic and
hypoxic conditions Our data also indicate that
TRAP1 plays a role in cardiomyocytes by regulating MPTP opening
TRAP1 was initially identified by the yeast two-hybrid system as a novel protein that interacted with the intercellular domain of the type 1 tumour necrosis factor receptor [17] On the basis of the sequence of the homologue, TRAP1 was identified as a member of the HSP90 family The ATPase activity of TRAP1 is inhibited by geldanamycin, which is a specific inhibitor
of HSP90 Despite its ATP-binding activity, TRAP1 does not form a stable complex with the co-chaperones
of HSP90, such as Hop and p23 [18] Studies have shown that TRAP1 does not have a C-terminal EEVD sequence, which exists in HSP90 and is important for HSP90-Hop binding [19] Thus, it appears that TRAP1 has specific functions that are different from those of other well-characterized HSP90 homologues TRAP1
is up-regulated by glucose deprivation, oxidative stress and ultraviolet A irradiation, but cannot be induced
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Vector Control
Ad-TRAP1 Ad-TRAP1
75 kDa
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TRAP1
GAPDH
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Ad-TRAP1 Normoxia
D450
Vector Hypoxia Hypoxia
Hypoxia
#
*
*
#
Fig 2 TRAP1 overexpression prevented the hypoxia-induced reductions in cell viabil-ity and Dw in primary cultured cardiomyo-cytes (A) Cardiomyocytes were infected with negative vector or Ad-TRAP1 for 48 h and then observed under a fluorescence microscope to determine the infection efficiency by visualizing expression of the gene for GFP Scale bar = 200 lm (B) Expression of TRAP1 levels in the unin-fected control, negative vector-inunin-fected and Ad-TRAP1-infected cardiomyocytes as deter-mined by western blotting (C, D) Cardio-myocytes were infected with vector or Ad-TRAP1 for 48 h, starved, and then treated for 6 h under hypoxic conditions; cell viability was determined with a cell counting kit (C) and Dw was determined with tetram-ethylrhodamine ethylester; and then one hundred cells from each group were randomly chosen to measure fluorescence intensity (D) Data are the mean ± SEM.
*P < 0.05 compared to the normoxic group.
#P < 0.05 compared with the hypoxic and hypoxia + vector groups The experiment was repeated three times.
Trang 5by heat [16,20,21] Furthermore, deferoxamine, an iron
chelator, decreases TRAP1 levels in a dose- and
time-dependent manner and induces mitochondrial
dysfunc-tion in human hepatocytes [22] However, the changes
induced in TRAP1 expression in cardiomyocytes after
hypoxia treatment are still unclear In the present
study, we demonstrated that hypoxia treatment (for 1,
3, 6 and 12 h, respectively) induces a time-dependent
increase in the levels of TRAP1 protein
Hypoxia is a common pathophysiological process
in diseases such as shock, stroke and heart failure
Hypoxic damage of the myocardium is relevant not
only to coronary artery diseases, but also to
hyper-tensive and cardiomyopathic heart disease [23,24]
Mitochondria are the most susceptible organelles
to hypoxic damage in cardiomyocytes Although
hypoxia induced TRAP1 expression increases in
cardiomyocytes, the role of that TRAP1 increase remains unclear The question remains as to whether the hypoxia-induced TRAP1 increase is a protective reaction in cardiomyocytes Because TRAP1 is a mitochondrial chaperone, it has an important role in regulating cell apoptosis and maintaining mitochon-drial homeostasis and function Silencing TRAP1 enhances cytochrome c release from the mitochondria and apoptosis induced by b-hydroxyisovalerylshikonin and VP16 [14] TRAP1 depletion also sensitizes PC12 cells to oxidative stress-induced cytochrome c release and cell death, which means that TRAP1 play a role
in the modulation of the mitochondrial apoptotic cas-cade [25] Moreover, TRAP1 overexpression improves mitochondrial function after ischaemic injury in primary astrocytes in vitro [16] In the present study,
we found that TRAP1 overexpression abolishes the hypoxic damage in cardiomyocytes Silencing TRAP1 expression not only induces cell damage under normoxic conditions, but it also aggravates hypoxic damage of cardiomyocytes
MPTP is a channel consisting of several proteins that is usually in a low permeability or closed state Some models have proposed the presence of other molecular components of the pore, although there is still no consensus regarding the exact components However, cyclophilin D (CypD) is generally accepted
as a critical regulatory component of MPTP and plays an important role in regulating MPTP opening [8,26] CsA, a selective MPTP inhibitor, prevents MPTP opening by inhibiting the activity of the pept-idyl-prolyl cis-trans isomerase of CypD [27,28] The consequences of MPTP opening are cell necrosis and apoptosis and, even if MPTP opening is insufficient
to cause necrosis, apoptosis can occur After the MPTP opens, apoptogenic substrates (i.e cytochrome c) are released into the cytoplasm and activate cas-pase-dependent apoptotic pathways Because MPTP plays a critical role in cell necrosis and apoptosis, it
is also involved in protecting cell against hypoxic and ischaemic damages [29,30] MPTP not only contrib-utes to the early and delayed protective effects of ischaemic preconditioning in rat or rabbit heart, but
it is also relevant to ischaemic post-conditioning [31]
We had also previously demonstrated that adenosine A1 receptor activation reduces hypoxic damage by preventing MPTP opening in rat cardiomyocytes [7] Many studies have demonstrated that Dw loss is accompanied by an increase in MPTP opening [32– 34] It is considered that Dw reflects the state of MPTP opening indirectly In the present study, we found that silencing TRAP1 induces Dw loss in cardiomyocytes, and that overexpression of TRAP1
Fig 3 TRAP1 overexpression decreased hypoxia-induced cell
death in primary cultured cardiomyocytes Cell death was
deter-mined by incubating normoxic cells, hypoxic cells, vector-infected
hypoxic cells and Ad-TRAP1-infected cells after 6 h of hypoxia with
Hoechst 33342 (10 lgÆmL)1, blue) and propidium iodide (PI)
(10 lgÆmL)1, red) Scale bar = 50 lm Graphs show the
quantifica-tion of cell death (mean ± SEM) and 200–300 cells were counted
for each group *P < 0.05 compared to the normoxic group.
#P < 0.05 compared to the hypoxic and hypoxic + vector groups.
The experiment was repeated three times.
Trang 6suppresses Dw loss caused by hypoxia Furthermore,
our present data also show that CsA prevents the cell
damage induced by TRAP1 depletion under normoxic
and hypoxic conditions, which means that silencing
TRAP1 expression can cause MPTP opening and lead
to damage Because the opening of MPTP increases
after hypoxia treatment, and TRAP1 overexpression
abolishes hypoxic damage, we therefore assume that
TRAP1 overexpression may prevent MPTP opening
and having a protective effect under hypoxic
condi-tions in cardiomyocytes In tumour cells, TRAP1
interacts with CypD, and the association of TRAP1
with CypD is prevented by CsA and not
geldanamy-cin, suggesting that this association may be necessary
for CypD activity [35]
Many factors are involved in inducing MPTP
open-ing, especially calcium overload and oxidative stress
[36,37] ROS increases could lead to the MPTP
open-ing persistently However, TRAP1 also shows an
important role in regulating ROS generation ROS
production is decreased by TRAP1 overexpression and promoted by silencing TRAP1 expression [15,16,38] Because TRAP1 plays a role against cell damage by MPTP, further studies are needed to determine whether ROS are mediators between TRAP1 and MPTP in cardiomyocytes
In summary, hypoxia increases the level of TRAP1 in cardiomyocytes, which may protect cells from hypoxic damage by regulating MPTP opening These results provide us with a deeper understanding of the protective role of TRAP1 in cardiomyocytes and offer new consid-erations for myocardial protection after burn shock
Materials and methods
Cardiomyocyte culture and hypoxia treatment Primary cardiomyocyte cultures were prepared from the ventricles of neonatal Sprague-Dawley rats (days 1–3) and trypsinized as described previously [39] in accordance with
A
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TRAP1-siRNA TRAP1-siRNA
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GAPDH
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Normoxia
D450
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Hypoxia Hypoxia
TRAP1-siRNA TRAP1-siRNA
*
*
#
Fig 4 Silencing TRAP1 expression induced cell viability and Dw in primary cultured cardiomyocytes (A) Cardiomyocytes were infected with negative vector or
TRAP1-siRNA for 4 days, and then a fluorescence microscope was used to observe the infection efficiency by visualizing expression of the gene for GFP Scale bar = 200 lm (B) Expression of TRAP1 levels in uninfected control, vector-infected and TRAP1-siRNA-infected cardiomyocytes as determined by western blotting (C) Cardiomyocytes were infected with vector or TRAP1-siRNA for 4 days, starved, and then cell viability was determined under normoxic conditions (D) Cardiomyocytes were infected with vector
or TRAP1-siRNA for 4 days, starved, and then Dw was determined under normoxic conditions or after 6 h of hypoxia The results are shown as the mean ± SEM.
*P < 0.05 compared to the normoxic and normoxic + vector groups #P < 0.05 compared to the hypoxic and hypoxic + vector groups The experiment was repeated three times.
Trang 7a protocol approved by the Animal Care and Use
Commit-tee of the Third Military Medical University The cultures
0.1 mm bromodeoxyuridine (Sigma-Aldrich, St Louis, MO,
deprived of serum for 12 h
Hypoxic conditions were prepared by using an anaerobic
jar (Mitsubishi, Tokyo, Japan) and a vacuum glove box
(Chunlong, Lianyungang, China) Serum-free medium was
placed in the vacuum glove box filled with a mixed gas
Cardiomyocytes were then subjected to hypoxic conditions
by replacing the normoxic medium with hypoxic medium and placing the cultures in an anaerobic jar All procedures were performed in vacuum glove box
Recombinant adenovirus vector for TRAP1 overexpression
Ad-TRAP1 and a negative adenovirus vector were
China) The vectors encoded the GFP sequence, which served as a marker gene A high titre adenovirus stock
HEK293A (American Type Culture Collection, Manassas,
VA, USA) All recombinant adenoviruses were tested for transgene expression in cardiomyocytes by western blot-ting Cardiomyocytes were infected with Ad-TRAP1 or a negative vector at a multiplicity of infection of 10 for
Fig 5 Silencing TRAP1 expression induced cell death in primary
cultured cardiomyocytes under normoxic conditions Cell death was
determined by incubating uninfected, vector-infected and
TRAP1-siRNA-infected cardiomyocytes under normoxic conditions with
Hoechst 33342 (10 lgÆmL)1, blue) and propidium iodide (PI)
(10 lgÆmL)1, red) Scale bar = 50 lm Graphs show the
quantifica-tion of cell death (mean ± SEM) and 200–300 cells were counted
for each group *P < 0.05 compared to the normoxic and
normoxic + vector groups The experiment was repeated three
times.
A
B
Fig 6 CsA prevented hypoxic damage after TRAP1-siRNA infec-tion in primary cardiomyocytes CsA (2 lM) was added into vector-infected and TRAP1-siRNA-vector-infected cardiomyocytes after 2 days of infection The cells were then starved, and subjected to hypoxia for
6 h after 4 days of infection (A) Effects of CsA on cell death in uninfected, vector-infected and TRAP1-siRNA-infected cells under hypoxic conditions In each group, 200–300 cells were counted (B) Effects of CsA on cell viability in uninfected, vector-infected and TRAP1-siRNA-infected cells under hypoxic conditions *P < 0.05 compared to the normoxic group #P < 0.05 compared to the oxic and hypoxic + vector groups **P < 0.05 compared to the hyp-oxic and hyphyp-oxic + vector groups ##P < 0.05 compared to the hypoxic + TRAP1-siRNA group (data are the mean ± SEM) The experiment was repeated three times.
Trang 848 h and then subjected to experiments after being
deprived of serum for 12 h
Recombinant adenovirus vector for silencing of
TRAP1 expression
The recombinant adenovirus vector for silencing of TRAP1
expression (TRAP1-siRNA) was purchased from Shanghai
GeneChem, Co Ltd The targeting sequence of the siRNA
against rat TRAP1 was 5¢-CAACAGAGATTGATCAA
AT-3¢ A negative control adenovirus vector containing
nonspecific siRNA was constructed in the same way
(non-specific vector, 5¢-TTCTCCGAACGTGTCACGT-3¢) All
vectors contained the gene for GFP, which served as a
mar-ker Cardiomyocytes were infected with TRAP1-siRNA or
control vector by the addition of adenovirus to the cell
cul-ture at a multiplicity of infection of 10 After 4 days of
infection, the cells were serum starved for 12 h and then
treated
Preparation of cell lysates
appropriate time after treatment, and lysed in
phenylmethanesulfo-nyl fluoride Cells were then scraped, and the resulting lysate was ultrasonicated and centrifuged at 12 000 g for 20 min at
Western blot analysis Protein concentrations were determined by the RC DC assay (Bio-Rad, Hercules, CA, USA) Thirty micrograms of proteins were fractionated by 10% SDS-PAGE and then transferred to a poly(vinylidene difluoride) membrane
2 h at room temperature Next, the membrane was probed with a 1 : 500 dilution of primary anti-TRAP1 serum (BD
overnight The membrane was washed four times with TBST and incubated with a horseradish peroxidase-conju-gated antibody against mouse IgG for 1 h at room temper-ature The membrane was then rinsed with TBST, and the protein bands were visualized with ECL Western Blotting
USA) The images were analysed with quantity one 4.1 software (Bio-Rad) The experiment was repeated three times, and the same results were obtained
Immunofluorescence assay Cardiomyocytes were grown on coverslips After hypoxia
Triton X-100 for 15 min at room temperature Nonspecific binding sites were blocked by incubating the coverslips with
with primary anti-TRAP1 serum at a 1 : 100 dilution
(Sigma-Aldrich) for 10 min at room temperature scopic images were acquired using a Leica Confocal Micro-scope (Leica Microsystems, Wetzlar, Germany) In the negative control, the primary antibody was omitted
Detection of cardiomyocyte viability Cardiomyocyte viability was determined with a cell counting kit (CCK-8, Dojindo Molecular Technologies, Kumamoto,
A
B
Fig 7 CsA prevented the cell damage induced by silencing TRAP1
in primary cardiomyocytes under normoxic conditions CsA (2 lM)
was added to vector-infected and TRAP1-siRNA-infected
cardio-myocytes after 2 days of infection The cells were then subjected
to cell viability and cell death assay after 4 days of infection (A)
Effects of CsA on cell death in uninfected, vector-infected and
TRAP1-siRNA-infected cells In each group, 200–300 cells were
counted (B) Effects of CsA on cell viability in uninfected,
vector-infected and TRAP1-siRNA-vector-infected cells *P < 0.05 compared to
the normoxic and normoxic + vector groups #P < 0.05 compared
to the normoxic + TRAP1-siRNA group (data are the mean ± SEM).
The experiment was repeated three times.
Trang 9Japan) Cells were cultured in 96-well plates (10 000 cells
per well) and the original medium was removed after 6 h of
hypoxia Then, 10 lL of CCK-8 solution and 100 lL of
determined (n = 3) and the experiment was repeated three
times
Cell death assays
Sigma-Aldrich)-labelled cells Propidium iodide readily penetrates
cells with compromised plasma membranes (dead cells) but
does not cross intact plasma membranes Hoechst is a
cell-permeable nucleic acid stain that labels both live and dead
nuclei
Mitochondrial membrane potential
Dw was monitored by tetramethylrhodamine ethylester
(Sigma-Aldrich) Cells cultured in a serum-free medium were
microscope The experiment was repeated three times
Statistical analysis
All values were expressed as the mean ± SEM spss,
version 11.0 (SPSS Inc., Chicago, IL, USA) was used to
conduct analyses of variance and Tukey’s tests P < 0.05
was considered statistically significant
Acknowledgements
This work was supported by the Key Project of China
National Programs for Basic Research and
Develop-ment (2005CB522601), the Key Program of National
Natural Science Foundation of China (30430680), the
Program for Changjiang Scholars, and the Innovative
Research Team in University (IRT0712) We thank Sun
Wei and Wang Li-ting (Central Library of The Third
Military Medical University) for their technical
assis-tance with the laser scanning confocal microscope The
authors declare that there are no conflicts of interest
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