Mitochondrial chaperone tumour necrosis factor
receptor-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

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.
FEBS Journal 277 (2010) 1929–1938 ª 2010 The Authors Journal compilation ª 2010 FEBS 1929
directly 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

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

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

13 6
H
yp
oxia treatment (h)
12
0.4
TRAP1
β-actin
0.3
0.2
0.1
TRAP1/β-actin
0
Normoxia
*
*
*
*
136
Hypoxia treatment (h)
Hypoxia treatment (h)
12
Normoxia
13 612
75 kD
a
43 kD
a
Fig. 1. Effects of hypoxia on the TRAP1 levels in primary cultured
cardiomyocytes. (A) Western blots show TRAP1 immunoreactivity

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
A
B
C
D
Vector
Vector
Control
Ad-TRAP1
Ad-TRAP1
75 kD
a

oxia
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-infected 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

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

The experiment was repeated three times.
F. Xiang et al. TRAP1 protects cells from hypoxic injury by MPTP
FEBS Journal 277 (2010) 1929–1938 ª 2010 The Authors Journal compilation ª 2010 FEBS 1933
suppresses 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

30
20
10
0
Normoxia
Normoxia
Vector
TRAP1-siRNA
Normoxia
Fluorescence intensity
(arbitrary units)
D
450
Normoxia
Vector Vector
Hypoxia
HypoxiaTRAP1-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.

cin. Cells were maintained in a 5% CO
2
incubator at
37 °C. Before hypoxia treatment, the cardiomyocytes were
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 con-
taining 94% nitrogen, 5% CO
2
and 1% O
2
overnight and
allowed to equilibrate with the hypoxic atmosphere.
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 pro-
duced by Shanghai GeneChem, Co. Ltd (Shanghai,
China). The vectors encoded the GFP sequence, which
served as a marker gene. A high titre adenovirus stock
was made after several rounds of amplification in
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

hypoxic + TRAP1-siRNA group (data are the mean ± SEM). The
experiment was repeated three times.
F. Xiang et al. TRAP1 protects cells from hypoxic injury by MPTP
FEBS Journal 277 (2010) 1929–1938 ª 2010 The Authors Journal compilation ª 2010 FEBS 1935
48 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
Cells were washed three times with ice-cold NaCl ⁄ P
i
at the
appropriate time after treatment, and lysed in radioimmuno-
precipitation assay (Sigma-Aldrich) lysis buffer that
contained 2 lgÆmL
)1
aprotinin, 2 lgÆmL

Immunofluorescence assay
Cardiomyocytes were grown on coverslips. After hypoxia
treatment, the cells were fixed with 4% (w ⁄ v) formaldehyde
in NaCl ⁄ P
i
for 10 min and permeabilized with 0.2% (v ⁄ v)
Triton X-100 for 15 min at room temperature. Nonspecific
binding sites were blocked by incubating the coverslips with
10% (v ⁄ v) goat serum in NaCl ⁄ P
i
for 1 h. Cells were probed
with primary anti-TRAP1 serum at a 1 : 100 dilution over-
night at 4 °C, washed with NaCl ⁄ P
i
, and incubated in the
dark at 37 °C for 1 h with fluorescein isothiocyanate-conju-
gated IgG. The cells were then washed again with NaCl ⁄ P
i
and stained with 0.4 mgÆmL
)1
4¢,6-diamidino-2-phenylindole
(Sigma-Aldrich) for 10 min at room temperature. Micro-
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

)1
; 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
incubated in the dark with 200 nmolÆL
)1
tetramethylrhod-
amine ethylester at 37 °C for 15 min. Cells were then washed
with NaCl ⁄ P
i
and observed using a laser scanning confocal
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

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