Results Overexpression of Hsp70 protects cardiomyocytes from H2O2-induced apoptosis First, to overexpress Hsp70 we transiently transfected cardiomyocytes with a plasmid carrying human Hs
Trang 1shock protein 70 during oxidative stress
Bimei Jiang1, Bin Zhang1, Pengfei Liang2, Juan Song1, Hongbing Deng1, Zizhi Tu1, Gonghua Deng1 and Xianzhong Xiao1
1 Department of Pathophysiology, Xiangya School of Medicine, Central South University, Changsha, Hunan, China
2 Department of Burns and Plastic Surgery, Xiangya Hospital, Central South University, Changsha, Hunan, China
Introduction
Characterized by cellular and nuclear shrinkage,
cyto-plasmic blebbing, chromatin condensation and DNA
fragmentation [1,2], apoptosis can cause irreversible
loss of terminally differentiated cardiac myocytes and,
therefore, contributes significantly to the pathogenesis
of many cardiovascular diseases Apoptosis has been
identified in cardiac myocytes from patients suffering
from myocardial infarction, diabetic cardiomyopathy,
and end-stage congestive heart failure
Apoptosis is a highly regulated programme of cell
death and can be mediated by death receptors in the
plasma membrane, as well as in the mitochondria and
the endoplasmic reticulum [3] Studies on apoptotic
pathways in cardiomyocytes have revealed several
molecules as key regulators [4] Heat shock protein
70 (Hsp70) is a major stress-inducible heat shock pro-tein that has been shown to protect cells from apoptosis induced by heat shock, tumour necrosis factor, growth factor withdrawal, oxidative stress and radiation [5,6] Hsp70 is also a major self-preservation protein in the heart; its overexpression enhances myocardial tolerance to ischaemia⁄ reperfusion injury
in both transgenic animals [7] and cell cultures [8] Although substantial progress has been made in understanding the control and mechanisms of apopto-sis, how Hsp70 protects cardiomyocytes against apop-tosis induced by a variety of stresses remains to be investigated
Keywords
apoptosis; cardiomyocytes; heat shock
protein 70; hydrogen peroxide;
nucleolin ⁄ C23
Correspondence
Xianzhong Xiao, Department of
Pathophysiology, Xiangya School of
Medicine, Central South University,
Changsha, Hunan 410008, China
Fax: +86 731 82355019
Tel: +86 731 2355019
E-mail: xianzhongxiao@xysm.net
(Received 22 August 2009, revised 26
October 2009, accepted 23 November
2009)
doi:10.1111/j.1742-4658.2009.07510.x
Although heat shock protein 70 (Hsp70) has been shown to markedly inhi-bit H2O2-induced apoptosis in C2C12 cells, and nucleolin⁄ C23 has also been implicated in apoptosis, the relationship of these two molecules is still largely unknown The aim of the current study was to investigate the potential involvement of nucleolin⁄ C23 in the antiapoptotic mechanism of Hsp70 We found that primary cultures of neonatal rat cardiomyocytes underwent apoptosis upon H2O2 treatment, and in these cells nucleo-lin⁄ C23 protein was highly unstable and had a half-life of less than 4 h However, transfection with Hsp70 greatly stabilized nucleolin⁄ C23 and also protected the cells from H2O2-induced apoptosis When nucleolin⁄ C23 was knocked down with an antisense oligomer, H2O2-induced apoptosis became more severe, even in Hsp70-overexpressed cells, demonstrating an essential role of nucleolin⁄ C23 in the antiapoptotic effects of Hsp70 Similar results were obtained by both nuclear morphology observation and caspase-3 activity assay Therefore, these data provide evidence that nucleolin⁄ C23 is
an essential downstream effecter of Hsp70 in the protection of cardiomyo-cytes against oxidative stress-induced apoptosis
Abbreviations
DMEM, Dulbecco’s modified Eagle’s medium; FITC, fluorescein isothiocyanate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Hsp70, heat shock protein 70; PBS, phosphate-buffered saline; PI, pyridine iodination; SEM, standard error of the mean.
Trang 2Nucleolin⁄ C23 is a 110 kDa multifunctional
phos-phoprotein It is abundantly expressed in both normal
and cancerous cells [9], and ubiquitously distributed in
the nucleolus, nucleus and cytoplasm of the cell, as
well as at the cell surface [10] Nucleolin⁄ C23 regulates
many aspects of DNA and RNA metabolism, such as
RNA polymerase I-mediated transcription, the folding,
maturation, ribosome assembly and nucleocytoplasmic
transport of newly synthesized pre-rRNAs [11]
Because of its widespread distribution and broad range
of involvement, as well as the cross-talking among
molecules of different cellular processes, dissection of
nucleolin⁄ C23’s specific functions is particularly
chal-lenging
Recently, nucleolin⁄ C23 has also been implicated in
apoptosis All-trans retinoic acid-induced apoptosis
leads to nucleolin downregulation and bcl-2 mRNA
instability; overexpression of nucleolin⁄ C23 in chronic
lymphocytic leukaemia cells stabilizes bcl-2 mRNA
[12,13] Our previous study showed that oxidative
stress induces nucleolin⁄ C23 cleavage and apoptosis
[14], and that nucleolin downregulation induces
apop-tosis in C2C12 cells [15] These data suggest the roles
of nucleolin⁄ C23 in oxidative stress-induced apoptosis,
but whether nucleolin⁄ C23 participates in
Hsp70-medi-ated protection against H2O2-induced apoptosis is not
clear In the present investigation, we further studied
the effects of nucleolin⁄ C23 ablation on
Hsp70-medi-ated protection from apoptosis and examined the
expression and stability of nucleolin⁄ C23 with Hsp70
overexpression in response to H2O2-induced apoptosis
using primary culture cardiomyocytes
Results
Overexpression of Hsp70 protects
cardiomyocytes from H2O2-induced apoptosis
First, to overexpress Hsp70 we transiently transfected
cardiomyocytes with a plasmid carrying human Hsp70
cDNA, and examined the protein levels by western
blot analysis with Hsp70 antiserum after 36 h The
effi-ciency of transfection mediated by the Lipofectamine
Plus reagent ranged between 30 and 50% (data not
shown) As can be seen in Fig 1A, Hsp70
overexpres-sion was confirmed in the cells transfected with Hsp70
cDNA, but not in the cells transfected with the vector
alone
To determine the effect of Hsp70 overexpression on
H2O2-induced apoptosis, the above transfected cells
were exposed to H2O2 (0.5 mmolÆL)1) for the indicated
time periods and the percentages of apoptotic cells
examined by morphological observation (Fig 1B)
A
B
C
Fig 1 Overexpression of Hsp70 renders cardiomyocytes resistant
to H2O2 Cardiomyocytes were transiently transfected with full-length Hsp70 plasmid pcDNA ⁄ Hsp70 (Hsp70 ⁄ C) or pcDNA vector (pcDNA ⁄ C) (A) Immunoblot analysis of Hsp70 expression At 36 h post-transfection, cells were analysed by immunoblotting with anti-bodies against Hsp70 or GAPDH (as an internal control of protein loading) The results shown are representative of three indepen-dent experiments (B) Quantification of apoptotic cells by nuclear staining Untransfected or transfected cells were exposed to 0.5 mmolÆL)1 H 2 O 2 for the indicated durations and stained with Hoechst 33258; apoptotic cells showing condensed chromatin fragments were scored and expressed as the percentage of the total cell number counted The results from three indepen-dent experiments are presented as mean ± SEM *P < 0.05 versus control group; #P < 0.05 versus pcDNA3.1 + H2O2 group (n = 3) (C) Caspase-3 activity assay Thirty-six hours after transient transfection, cells were treated with or without 0.5 mmolÆL)1H2O2.
At different time points, cells were harvested for protease activity
of caspase-3 using the caspase colorimetric assay kit Data were obtained from four independent experiments *P < 0.05 versus control group; #P < 0.05 versus pcDNA3.1 + H2O2group.
Trang 3After 24 h of H2O2 exposure, 44% of untransfected
control cells and 45% of cells transfected with the
empty vector (pcDNA3.1) exhibited similar features of
apoptotic cells, such as highly condensed nuclei and
decreased cell size However, only 22% of the cells
transfected with Hsp70 underwent apoptosis (Fig 1B)
We also investigated the effects of Hsp70 on caspase-3
activation induced by oxidative stress Using an in vitro
substrate-cleavage reaction, H2O2 treatment
signi-ficantly induced the activation of caspase-3 in the
untransfected control cells as well as cells transfected
with the vector alone in a time-dependent manner, but
Hsp70 overexpression significantly reduced caspase-3
activation in response to H2O2treatment (Fig 1C)
Increased nucleolin/C23 protein levels in
Hsp70-transfected cells in response to
H2O2-induced apoptosis
We next examined the expression of nucleolin⁄ C23
protein in these cells in response to H2O2 exposure
Upon treatment with H2O2 for up to 8 h, all cells
showed a time-dependent decrease in the 110 kDa
nucleolin⁄ C23 fragment accompanied by the
appear-ance and an increase in the 80 kDa fragment (Fig 2)
Although both the untransfected cells and the cells
transfected with the vector alone started to show the
80 kDa fragment at time points as early as 2–4 h after
H2O2 treatment, this smaller protein fragment
appeared in the Hsp70-overexpressed cells at the 8 h
time point (Fig 2A,B) Overall, the change in
nucleo-lin⁄ C23 protein levels was least significant in cells
transfected with Hsp70 cDNA (Fig 2B), suggesting a
protective effect of Hsp70 on the degradation of
nucleolin⁄ C23 protein
Prolonged half-life of nucleolin/C23 in Hsp70-transfected cells in response to H2O2 exposure
To compare the stability of nucleolin⁄ C23 protein in cells transfected with the vector alone and cells trans-fected with Hsp70 cDNA during H2O2 exposure, the cells were treated with H2O2 for 2 h Protein synthesis was then inhibited by cycloheximide (5 lgÆmL)1) Western blot analysis revealed a time-dependent rapid decline in nucleolin⁄ C23 110 kDa bands in H2O2 -trea-ted cells transfec-trea-ted with the vector alone; the half-life
of the protein was estimated to be less than 2 h (Fig 3, left panel) At the same time, the 80 kDa degraded product was detected in an increasing amount in H2O2-treated cells transfected with the vector alone shortly after cycloheximide treatment (2–8 h) By contrast, nucleolin⁄ C23 in cells transfected with Hsp70 cDNA was more stable in response to
H2O2 exposure, reaching its half-life at 8 h after cyclo-heximide treatment (Fig 3, right panel)
Overexpression of nucleolin/C23 protects cardiomyocytes from H2O2-induced apoptosis
To determine the effect of nucleolin⁄ C23 overexpres-sion on H2O2-induced apoptosis in cardiomyocytes, we first transiently transfected cells with plasmids carrying human nucleolin⁄ C23 cDNA or the empty vector Using western blot analysis we confirmed nucleo-lin⁄ C23 overexpression only in cells transfected with nucleolin⁄ C23 cDNA but not the empty vector (pcDNA⁄ C) (Fig 4A) Then, transfected cells were exposed to H2O2 (0.5 mmolÆL)1) and apoptosis was assayed by both Hoechst nuclear staining (Fig 4B)
Ctrl 0 h
Nucleolin/C23
Nucleolin/C23 GAPDH
GAPDH PcDNA/C cells with H2O2 exposure Hsp70/C cells with H2O2 exposure
H2O2
A
B
Fig 2 Changes in nucleolin ⁄ C23 protein levels in cells transfected with pcDNA vector (pcDNA ⁄ C) or Hsp70 cDNA (Hsp70 ⁄ C) during H 2 O 2 exposure Cardiomyocytes were untransfected (A) or transiently transfected with pcDNA vector (pcDNA ⁄ C)
or pcDNA ⁄ Hsp70 (Hsp70 ⁄ C) for 36 h (B), and were treated with 0.5 mmolÆL)1H2O2 for 0–8 h Cells were harvested and protein lysates were prepared for western blot anal-ysis with monoclonal antibodies against nucleolin ⁄ C23 or GAPDH The results shown are the representatives of three independent experiments.
Trang 4and caspase-3 activity assays (Fig 4C) By counting
apoptotic nuclei, at 24 h H2O2 exposure, 45% of
cells transfected with the vector alone were apoptotic
However, only 22% of cells transfected with
nucleo-lin⁄ C23 cDNA underwent apoptosis (Fig 4B)
Consis-tently, H2O2 treatment significantly induced caspase-3
activation in cells transfected with the vector alone in
a time-dependent manner, and nucleolin⁄ C23
overex-pression significantly reduced caspase-3 activation
(Fig 4C) However, simultaneous Hsp70
overexpres-sion afforded the cells no significant protection by
nucleolin⁄ C23 against oxidant stress (Fig 4D)
In addition, H2O2 induced apoptosis in
cardiomyo-cytes with a marked downregulation of Bcl-2 protein,
and nucleolin overexpression could inhibit the
down-regulation of Bcl-2 protein (Fig 5A) Furthermore, we
compared the stability of Bcl-2 mRNA in cells
transfect-ed with nucleolin cDNA (pcDNA3.1-C23) or vector
control (pcDNA3.1) in the presence or absence of H2O2
by incubating the cells with actinomycin D (5 lgÆmL)1)
for up to 3 h Bcl-2 mRNA in H2O2-treated vector
con-trol cells was found to be highly unstable In contrast,
Bcl-2 in H2O2-treated nucleolin-overexpressed cells was
significantly more stable (Fig 5B)
Potentiation of apoptosis in Hsp70-transfected
cells by nucleolin/C23 antisense oligomer
Next, we used the gene knockdown approach with an
antisense oligonucleotide to examine whether
nucleo-lin⁄ C23 is necessary in the antiapoptotic function of
Hsp70 Fluorescein isothiocyanate (FITC)-labelled
phosphorothioate oligodeoxynucleotides were
success-fully transfected into primary cultured cardiomyocytes
with Lipofectamine 2000 and achieved a transfection
efficacy of 80% (Fig 6A) Transfection of
nucleo-lin⁄ C23 antisense oligonucleotide selectively reduced
the levels of endogenous nucleolin⁄ C23 protein by
more than 75% after 36 h, whereas transfection of the scrambled control oligonucleotide did not alter nucleo-lin⁄ C23 protein levels (Fig 6B)
Then, Hsp70 overexpressing cells were transfected with nonspecific control or nucleolin⁄ C23 antisense oligomers and the percentages of apoptotic cells in response to H2O2 treatment were examined by three methods First, by nuclear staining the apoptotic rates were 9–25% in cells transfected with the control oligo-mers and much lower at 13 and 44% in cells
transfect-ed with the nucleolin⁄ C23 antisense oligomer (Fig 6C) Second, the activity of caspase-3 was analy-sed as a marker of apoptotic cells using an in vitro substrate-cleaving reaction As shown in Fig 6D,
H2O2-induced apoptosis was increased in cells trans-fected with nucleolin⁄ C23 antisense oligonucleotide compared with those transfected with the scrambled control oligonucleotide, even in the presence of Hsp70 overexpression Finally, apoptosis was further assayed
by flow cytometry with annexin V–FITC⁄ pyridine iodination (PI) double staining, which identified apop-totic cells as annexin V positive, but PI negative This method obtained results consistent with those from the other two methods, showing that when nucleolin⁄ C23 was knocked down with an antisense oligomer, H2O2 -induced apoptosis was enhanced, even with the Hsp70-overexpressed cells (Fig 7A,B)
Discussion
Both our previous study [16] and the present study have provided evidence that oxidative stress induces apoptosis of cardiomyocytes, which can be character-ized by nuclear condensation and cell shrinkage Inter-estingly, upon transfection with an Hsp70-containing plasmid, these same cells become much less sensitive to
H2O2 exposure This observation was confirmed by both cell morphology and caspase-3 activity analyses
pcDNA/C cells with
H2O2 exposure 2 h
0 h
Nucleolin/C23
GAPDH
Hsp70/C cells with
H2O2 exposure 2 h
Fig 3 Nucleolin ⁄ C23 protein is stabilized in Hsp70-transfected cells during H 2 O 2 exposure Cardiomyocytes were transiently transfected with pcDNA vector (pcDNA ⁄ C) or pcDNA ⁄ Hsp70 (Hsp70 ⁄ C) After treatment with 0.5 mmolÆL)1 H2O2 for 2 h, cycloheximide (CHX;
5 lgÆmL)1) was added At different time points (2–8 h), cells were harvested and protein lysates were prepared for western blot analysis with monoclonal antibodies against nucleolin⁄ C23 or GAPDH The results shown are the representatives of three independent experiments.
Trang 5Hsp70 has been shown to regulate apoptotic
sig-nalling, effecter molecule activation, certain
down-stream events of caspase activation, the release of
cathepsins, cytochrome c, apoptosis-inducing factor
and even the death-associated lysosome permeabiliza-tion [17–21] Particularly, Hsp70 inhibits the release
of cytochrome c and apoptosis-inducing factor from mitochondria and prevents apoptosis [22,23] Our previous study showed that Hsp70 overexpression inhibited H2O2-mediated release of Smac (the second mitochondria-derived activator of caspases), activa-tion of caspase-3 and caspase-9, and apoptosis in C2C12 myogenic cells [24] Stankiewicz et al [25] found that Hsp70 overexpression stabilizes Mcl-1 protein in heat-shocked cells Hsp70 has also been suggested to play an important role in precondition-ing, a phenomenon of protection of a heart from strong ischaemic insult by prior exposure to mild ischaemia or other mild stresses In the present study, we have shown that Hsp70 renders nucleolin⁄ C23 enhanced stability; however, a decrease in nucle-olin⁄ C23 stability restrains such a protective effect of Hsp70 against the H2O2-induced apoptosis These data suggest that Hsp70 may inhibit apoptosis at multiple points and through a wide range of mechanisms
Nucleolin is a major nucleolar phosphoprotein that belongs to a large family of RNA-binding proteins [26] It plays a role in the pre-rRNA transcription
Ctrl pcDNA3.1 pcDNA3.1-C23
Nucleolin/C23
GAPDH
60
40
50
30
20
10
0
3
4
2.5
3.5
3.5
3
2.5
2
1.5
1
0.5
0
0.5 mmol·L –1 H2O2
2
1.5
1
0.5
0
Ctrl
Ctrl
pcDN
A3.1
pcDN A3.1-C23 pcDN A3.1-Hsp70
pcDN A3.1-C23 + pcDN
A3.1-Hsp70
pcDNA3.1 + H2O2
Ctrl pcDNA3.1 + H2O2
*
*
#
#
#
#
#
*
pcDNA3.1-C23 + H2O2
pcDNA3.1-C23 + H2O2
A
B
C
D
Fig 4 Overexpression of nucleolin ⁄ C23 protects cardiomyocytes from H2O2-induced apoptosis Cardiomyocytes were transiently transfected with full-length nucleolin plasmid (pcDNA3.1-C23) or pcDNA3.1 vector (A) Immunoblot analysis of nucleolin ⁄ C23 expres-sion At 36 h post-transfection, cells were analysed by immunoblot-ting with antibodies against nucleolin ⁄ C23 or GAPDH (as an internal control of protein loading) The results shown are represen-tatives of three independent experiments (B) Quantification of apoptotic cells by nuclear staining Untransfected or transfected cells were exposed to 0.5 mmolÆL)1H2O2 for the indicated dura-tions and stained with Hoechst 33258; apoptotic cells showing con-densed chromatin fragments were scored and expressed as a percentage of the total cell number counted The results from three independent experiments are presented as means ± SEM.
*P < 0.05 versus control group; #P < 0.05 versus pcDNA3.1 +
H2O2group (n = 3) (C) Caspase-3 activity assay Thirty-six hours after transient transfection, cells were treated with or without 0.5 mmolÆL)1H2O2for 12 h and harvested for the determination of protease activity of caspase-3 using the caspase colorimetric assay kit Data were obtained from four independent experiments.
*P < 0.05 versus control group; #P < 0.05 versus pcDNA3.1 +
H2O2 group (D) Caspase-3 activity assay Thirty-six hours after transfection with pcDNA3.1-C23 or pcDNA3.1-Hsp70 or transient cotransfection with pcDNA3.1-C23 and pcDNA3.1-Hsp70, cells were treated with or without 0.5 mmolÆL)1H2O2for 12 h and har-vested for protease activity of caspase-3 Data were obtained from four independent experiments *P < 0.05 versus control group;
#P < 0.05 versus pcDNA3.1 + H2O2group.
Trang 6and ribosome assembly that is implicated in the early
stage of preribosomal ribonucleoprotein assembly and
processing [27] Nucleolin has also been suggested to
regulate cell proliferation and growth, cytokinesis,
rep-lication, embryogenesis and nucleogenesis [28] by
forming large molecular complexes with other factors,
such as casein kinase II, c-Myb, midkine, histone H1,
nucleophosmin, p53 and protein phosphatase 1 A
number of studies, including our own, indicate that
nucleolin⁄ C23 may be one of the key components in
the regulation of apoptosis [12–14,29–31] In the
pres-ent study, we found that overexpression of
nucleo-lin⁄ C23 protects cardiomyocytes from H2O2-induced
apoptosis, and that the mechanism is probably
associ-ated with the increase in Bcl-2 mRNA stability (Fig 5) In addition, we have also established a strong relationship between nucleolin⁄ C23 and the antiapop-totic effect of Hsp70 Nucleolin⁄ C23 in Hsp70-trans-fected cells is significantly stabilized during H2O2 exposure, and simultaneous Hsp70 overexpression affords no additional protection against oxidant stress (Fig 4D) Therefore, Hsp70-transfected cells with a higher level of nucleolin⁄ C23 protein become more resistant to oxidative stress and less susceptible to apoptotic death More importantly, the role of nucleo-lin⁄ C23 in this scenario is indispensable, as the sup-pression of nucleolin⁄ C23 expression by an antisense oligomer potentiates apoptosis, even in the Hsp70-transfected cells These results also indicate that the nucleolin⁄ C23 gene has a functional role in the growth control of cardiomyocytes, and its regulation may be closely associated with the susceptibility of the cell to the induction of apoptosis
Caspases are important molecular mediators of apoptosis in the cell [32] Assays for caspase-3 activ-ity have also confirmed that H2O2 exposure induces cardiomyocytes to undergo apoptosis and Hsp70 overexpression prevents H2O2-induced caspase activa-tion; when nucleolin⁄ C23 protein is suppressed by the antisense oligonucleotide, the protection of Hsp70 is lost
Taken together, our results demonstrate an impor-tant relationship among the regulation of nucleo-lin⁄ C23, the activation of caspase-3 and the induction
of apoptosis under the setting of oxidative stress and Hsp70 overexpression More importantly, for the first time, we provide strong evidence that nucleolin⁄ C23 is
a downstream mediator of Hsp70’s antiapoptosis effects and it functions at the level of protein stability
in cardiomyocytes It is our hope that such studies on the mechanisms of apoptosis in cardiomyocytes will provide a molecular basis for new therapeutic strate-gies targeting specific pathways to treat human heart disease
Materials and methods
Animals
Neonatal Wistar rats (1-3 days) were purchased from the Animal Resource Center of Center South University The following procedures were approved by the Institutional Animal Care and Use Committee of the Center South Uni-versity, and were carried out in accordance with the National Institutes of Health Guide for the Care and Use
of Laboratory Animals All efforts were made to minimize the number of animals used and their suffering
H2O2 pcDNA3.1
100
10
Time (h)
pcDNA3.1 pcDNA3.1-C23
Bcl-2
GAPDH
A
B
Fig 5 Effect of nucleolin ⁄ C23 on Bcl-2 protein expression and
mRNA stability in the presence or absence of H 2 O 2 (A) Western
blot analyses showing the effect of nucleolin ⁄ C23 on Bcl-2 protein
expression Total cellular proteins were isolated and separated by
12% SDS ⁄ PAGE and analysed by immunoblotting with anti-Bcl-2.
Immunoblot analyses with GAPDH antiserum were used as the
protein loading control (B) Real-time PCR showing the effect of
nucleolin ⁄ C23 on Bcl-2 mRNA stability Cells transfected with
pcDNA3.1-C23 or the empty vector were cultured in the absence
or presence of H2O2(0.5 m M ) for 2 h Actinomycin D (5 lgÆmL)1)
was then added to the media and the cells were further incubated
for various times (0–3 h) Total RNAs were prepared and subjected
to real-time PCR analyses The amount of Bcl-2 mRNA was
normal-ized by that of GAPDH All experiments were performed in
tripli-cate and shown here by one representative *P < 0.05 versus
pcDNA3.1-transfected control group.
Trang 7Cell culture and treatment
Primary cultures of neonatal rat cardiomyocytes were
culti-vated as previously described [33,34] Briefly, hearts from
neonatal Wistar rats (1–3 days after birth) were removed, minced and trypsinized at 37C with gentle stirring in D-Hanks buffer containing 0.1% trypsin (Gibco, Rockville,
MD, USA) The cells were then centrifuged and resus-pended in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) containing 15% fetal bovine serum (Hyclone, Logan, UT, USA) After incubation at 37C for 120 min, the suspended cardiomyocytes were seeded at a density of
5· 105cellsÆmL)1 All cells were cultured in DMEM con-taining 15% fetal bovine serum for 24 h before the initia-tion of experiments 5-Bromo-2¢-deoxyuridine (Sigma, St Louis, MO, USA) (0.1 mmolÆL)1) was added to the culture for 36 h to inhibit the proliferation of nonmyocytes H2O2
was first diluted in phosphate-buffered saline (PBS, pH 7.4) and further diluted in culture medium to a final concentra-tion of 0.5 mmolÆL)1 for all treatments Cycloheximide (Sigma) was diluted in the medium to a final concentration
of 5 lgÆmL)1
Quantification of apoptotic cells
At the predetermined time points after H2O2exposure, cells were detached from tissue culture plates with tryp-sin⁄ EDTA or cell scrapers, and collected together with the nonadherent cells After centrifugation at 500 g for 5 min, the cells were fixed with 4% paraformaldehyde for 30 min
at room temperature and then washed once with PBS The fixed cells were then incubated with Hoechst 33258 (50 ngÆmL)1) for 30 min at room temperature, and washed
60
Hsp70/C + NCL/C23-AsODNs Hsp70/C + NCL/C23-ScrODNs
Time (h) of H2O2 exposure
*
* 50
40
30
20
10
0
Hsp70/C + NCL/C23-AsODNs
Hsp70/C + NCL/C23-ScrODNs
Time (h) of H2O2 exposure
0
0.5
1
1.5
2
3
2.5
Hsp70/C cells
Nucleolin/C23
Hsp70
GAPDH
A
B
C
D
Fig 6 Potentiation of apoptosis in Hsp70-overexpressed cells by nucleolin ⁄ C23 antisense oligomer (A) Transfection efficiency of phosphorothioate oligodeoxynucleotides in primary cultures of car-diomyocytes FITC-labelled phosphorothioate oligodeoxynucleotides were transfected into cells with Lipofectamine 2000 and cells were stained with Hoechst 33258 Right, the image under blue fluores-cence channel showing all cells with nuclear Hoechst staining; left, image under green fluorescence channel showing only cells posi-tive for FITC-labelled oligomers (B) Knocking-down of nucleo-lin ⁄ C23 in cardiomyocytes The Hsp70-transfected cardiomyocytes (Hsp70 ⁄ C) were cotransfected with nucleolin ⁄ C23 scrambled (NCL ⁄ C23-ScrODNs) or antisense (NCL ⁄ C23-AsODNs) oligomers After 36 h, cells were harvested for western blot analysis with monoclonal antibody against nucleolin ⁄ C23 (NCL ⁄ C23) or GAPDH (C) Determination of the percentage of apoptotic cells by Hoechst staining Hsp70 ⁄ C cells were cotransfected with NCL ⁄ C23-Scr-ODNs or NCL ⁄ C23-AsODNs and then treated with H 2 O 2 for 6–36 h The results from three independent experiments are shown as mean ± SEM *P < 0.05 versus NCL ⁄ C23-ScrODNs group (D) Determination of percentage of apoptotic cells by cas-pase-3 activity assay Hsp70 ⁄ C cells were transfected and treated
as above, and assayed for caspase-3 activity Data were obtained from four independent experiments *P < 0.05 versus NCL ⁄ C23-ScrODNs group.
Trang 8again with PBS Cells were mounted on to glass slides and
examined by fluorescence microscopy Apoptotic cells were
identified by the condensation and fragmentation of their
nuclei The percentage of apoptotic cells was calculated as
the number of apoptotic cells divided by the total number
of cells counted A minimum of 500 cells were counted for
each slide
Flow cytometry
Both adherent and floating cells were collected after treatment, washed with ice-cold PBS and stained with FITC-conju-gated annexin V (BD Biosciences, Franklin Lakes, NJ, USA) and PI for 20 min at room temperature in the dark The stained cells were then analysed by a flow cytometer
A
B
Fig 7 Flow cytometry analysis of apoptosis
with annexin V–FITC ⁄ PI double staining.
Hsp70-transfected cardiomyocytes
(Hsp70 ⁄ C) were cotransfected with the
nucleolin ⁄ C23 scrambled (NCL ⁄
C23-ScrO-DNs) or antisense (NCL⁄ C23-AsOC23-ScrO-DNs)
oligomers Twenty-four hours later, they
were treated with 0.5 mmolÆL)1H2O2for 6
or 12 h Cells were harvested and
pro-cessed for annexin V–FITC and PI costaining
and analysed by flow cytometry (A) Q3
cells were the control cells, Q4 cells were
cells at the early apoptosis stage, Q2 cells
were cells at the late apoptosis stage, and
Q1 cells were necrotic cells The percentage
of apoptotic cells was also calculated (B).
The results are representative of three
independent experiments Data are the
mean ± SEM of triplicate samples.
*Significant difference (P < 0.05) versus the
pcDNA3.1 control group; #significant
difference (P < 0.05) versus the control
group.
Trang 9(Beckman Coulter, Fullerton, CA, USA) FITC-conjugated
annexin V binds to phosphatidylserine molecules only
pres-ent at the surface of apoptotic cells where they were
trans-located from the internal side of the plasma membrane
during apoptosis Cells were simultaneously stained with PI
to discriminate membrane-permeable necrotic cells from
FITC-labelled apoptotic cells Apoptotic cells were
identi-fied as those positive for annexin V–FITC staining but
negative for PI staining, and the percentage of apoptotic
cells in the total number of cells was calculated
Western blot analyses
Cells were washed with PBS and collected by
centrifuga-tion Cell pellets were resuspended with 5 volumes of cold
lysis buffer [50 mmolÆL)1 Tris⁄ HCl (pH 7.5), 250
mmo-lÆL)1 NaCl, 5 mmolÆL)1 EDTA, 50 mmolÆL)1 NaF, 0.5%
Nonidet P-40] containing a protease inhibitor mixture
(Roche Applied Science, Burgess Hill, UK) The cell lysate
was incubated on ice for 30 min and centrifuged at
10 000 g for 10 min at 4C The protein concentration of
the supernatant was determined using the Bradford assay
(Bio-Rad, Hercules, CA, USA) Equal amounts of protein
(5–10 lg) were loaded on to and separated by SDS⁄ PAGE
and transferred to a nitrocellulose membrane The blot
was blocked with 2% albumin in 20 mmolÆL)1 Tris⁄ HCl,
pH 8.0, 150 mmolÆL)1 NaCl, 0.1% Tween 20 (TBST)
overnight at 4C and then incubated with the rabbit
anti-C23 (Sigma) or anti-(glyceraldehyde-3-phosphate
dehydro-genase) (GAPDH; Santa Cruz Biotechnology, Santa Cruz,
CA, USA) for 2 h After washing with TBST buffer three
times for 15 min each, the blot was incubated with
horse-radish peroxidase-conjugated goat anti-mouse IgG (diluted
1 : 2000 in TBST buffer) at room temperature for 1 h,
and finally washed three times with TBST for 15 min
each Immunoreactivity was visualized using the enhanced
chemiluminescence reaction (Amersham, Piscataway, NJ,
USA)
Caspase activity assay
The activity of caspase-3 was measured by in vitro
sub-strate-cleavage reactions using a commercial kit according
to the manufacturer’s protocol (R&D Systems,
Minneapo-lis, MN, USA) Briefly, cells cultured in 60 mm dishes were
treated with 0.5 mmolÆL)1H2O2for the indicated time
peri-ods The cells (5· 106
) were lysed with 250 lL chilled cell lysis buffer on ice for 10 min After centrifugation
(10 000 g, 1 min, 4C), the protein concentration in the
supernatant was determined using the BioRad protein
assay Supernatants containing equal amounts of protein
(corresponding to 5 · 105
cells) were used for caspase-3 colorimetric assays After protein samples were incubated
with substrates at 37C for 1.5 h, the absorbance at
405 nm was measured using a microtitre plate reader
(Molecular Devices, Sunnyvale, CA, USA) Fold increases
in caspase-3 activity over that before treatment were deter-mined
Lipofectamine-mediated gene transfection
Transfection of cells was carried out following the manu-facturer’s instructions (Lipofectamine 2000, Invitrogen, Carlsbad, CA, USA) Briefly, 5 · 105
cells in 5 mL appropriate complete growth medium per flask were grown
at 37C in a CO2incubator until the cells reached 70–80% confluence (24 h) After rinsing with serum-free and antibi-otic-free medium, the cells were transfected with pcDNA3.1-Hsp70 (HspA1A was a gift from I Benjamin, University of Utah Health Sciences Center) (experimental group) or pcDNA3.1 (vector control), at 8 lg DNA per
20 lL Lipofectamine, followed by incubation at 37C in a
CO2incubator for 6 h The medium was then replaced with DMEM culture medium containing 20% fetal bovine serum
Nucleolin/C23 antisense oligodeoxynucleotide
The phosphorothioate oligodeoxynucleotides used in this study were manufactured by Bioasia Biotech (Shanghai, China) The sequence of nucleolin⁄ C23 antisense oli-godeoxynucleotide (NUL⁄ C23-AsODNs), corresponding to the rat nucleolin⁄ C23 translation initiation site, was 5¢-TG CGAGTTTCACCATGATGGC-3¢ A scrambled nucleolin ⁄ C23 oligodeoxynucleotide (NUL⁄ C23-ScrODNs; 5¢-CTGA TGTCACGTCCATGTGAG-3¢) was used as the control The oligodeoxynucleotides were diluted in 10 mmolÆL)1 Tris (pH 7.4) and 1 mmolÆL)1EDTA and kept at)20 C
RT-PCR
Total RNAs were prepared with the Rneasy kit (Qiagen, Cambridge, MA, USA) according to the manufacturer’s instructions and 2 lg samples were reverse transcribed using oligo(dT) primers The levels of gene expression were quanti-fied by real-time PCR, using a QuantiTect SYBR Green PCR Kit (Qiagen) and the 7500 Fast Real-Time PCR system (Applied Biosystems, Foster City, CA, USA) under the following conditions: 40 sequential cycles, each including
95C for 10 s and 60 C for 10 s Sequences of primers spe-cific to each target cDNA were: nucleolin, forward, 5-CA ATCAGGCTGGAGTTGCAAG-3; and reverse, 5-TGGC CCAGTCCAAGGTAACTT-3 (amplicon size: 282 bp);
and reverse, 5-TCCACCACCCTGTTGCTGTA-3 (size:
440 bp) The specificity of PCR products was verified by melt-ing curve analysis and electrophoresis on agarose gels The comparative threshold cycle method and an internal control (GAPDH) were used to normalize target gene expression
Trang 10Statistical analyses
Data are presented as mean ± standard error of the mean
(SEM) of the values obtained from the indicated number of
independent experiments Differences between two groups
were analysed by unpaired Student’s t-test Differences
between three or more groups were analysed by one-way
ANOVA followed by Student–Newman–Keuls posthoc test
P< 0.05 was considered statistically significant
Acknowledgements
This study was supported by grants from the National
Basic Research Program of China (2007CB512007),
the National Natural Science Foundation of China
(30700290) and Special Funds for PhD Training from
The Ministry of Education of China (20060533009)
References
1 Kerr JFR, Wyllie AH & Currie AR (1972) Apoptosis:
a basic biological phenomenon with wide-ranging
impli-cations in tissue kinetics Br J Cancer 26, 239–257
2 Jacobsen MD, Weil M & Raff MC (1997) Programmed
cell death in animal development Cell 88, 347–354
3 Lee Y & Gustafsson AB (2009) Role of apoptosis in
cardiovascular disease Apoptosis 14, 536–548
4 Mani K (2008) Programmed cell death in cardiac
myocytes: strategies to maximize post-ischemic salvage
Heart Fail Rev 13, 193–209
5 Arya R, Mallik M & Lakhotia SC (2007) Heat shock
genes – integrating cell survival and death J Biosci 32,
595–610
6 Zhao Y, Wang W & Qian L (2007) Hsp70 may protect
cardiomyocytes from stress-induced injury by inhibiting
Fas-mediated apoptosis Cell Stress Chaperones 12,
83–95
7 Marber MS, Mestril R, Chi RS, Sayen MR, Yellon
DM & Dillmann WH (1995) Overexpression of the rat
inducible 70-kD heat stress protein in a transgenic
mouse increases the resistance of the heart to ischemic
injury J Clin Invest 95, 1446–1456
8 Jayakumar J, Suzuki K, Khan M, Smolenski RT,
Farrell A, Latif N, Raisky O, Abunasra H, Sammut
IA, Murtuza B et al (2000) Gene therapy for
myocardial protection: transfection of donor hearts
with heat shock protein 70 gene protects cardiac
func-tion against ischemia-reperfusion injury Circulafunc-tion
102, 302–306
9 Derenzini M, Sirri V, Trere D & Ochs RL (1995) The
quantity of nucleolar proteins nucleolin and protein
B23 is related to cell doubling time in human cancer
cells Lab Invest 73, 497–502
10 Hovanessian AG, Puvion-Dutilleul F, Nisole S, Svab J,
Perret E, Deng JS & Krust B (2000) The
cell-surface-expressed nucleolin is associated with the actin cytoskel-eton Exp Cell Res 261, 312–328
11 Ginisty H, Amalric F & Bouvet P (1998) Nucleolin functions in the first step of ribosomal RNA processing EMBO J 17, 1476–1486
12 Otake Y, Sengupta TK, Bandyopadhyay S, Spicer EK
& Fernandes DJ (2005) Retinoid-induced apoptosis in HL-60 cells is associated with nucleolin down-regulation and destabilization of Bcl-2 mRNA Mol Pharmacol 67, 319–326
13 Otake Y, Soundararajan S, Sengupta TK, et al (2007) Overexpression of nucleolin in chronic lymphocytic leu-kemia cells induces stabilization of bcl2 mRNA Blood,
109, 3069–3075
14 Wang KK, Jiang L, Yi YX, Liu K, E SM, Tang DL, Wang JS, Shi YZ, Wang QP & Xiao XZ (2004) Effect
of heat shock response on the cleavage of nucleolin induced by oxidative stress Zhong Nan Da Xue Xue Bao Yi Xue Ban 29, 504–508
15 Wang KK, Jiang L, E SM, Liu K, Zhang LL, Liu MD
& Xiao XZ (2005) Effect of nucleolin down-regulation
on the proliferation and apoptosis in C2C12 cells Zhong Nan Da Xue Xue Bao Yi Xue Ban 30, 125–129
16 Jiang B, Xiao W, Shi Y, Liu M & Xiao X (2005) Heat shock pretreatment inhibited the release of Smac from mitochondria and apoptosis induced by hydrogen per-oxide in cardiomyocytes and C2C12 myogenic cells Cell Stress Chaperones 10, 252–262
17 Feng X, Bonni S & Riabowol K (2006) HSP70 induc-tion by ING proteins sensitizes cells to tumor necrosis factor alpha receptor-mediated apoptosis Mol Cell Biol
26, 9244–9255
18 Lui JC & Kong SK (2007) Heat shock protein 70 inhib-its the nuclear import of apoptosis-inducing factor to avoid DNA fragmentation in TF-1 cells during erythro-poiesis FEBS Lett 581, 109–117
19 Bivik C, Rosdahl I & Ollinger K (2007) HSP70 protects against UVB induced apoptosis by preventing release of cathepsins and cytochrome c in human melanocytes Carcinogenesis 28, 537–544
20 Matsumori Y, Hong SM, Aoyama K, Fan Y, Kayama
T, Sheldon RA, Vexler ZS, Ferriero DM, Weinstein PR
& Liu J (2005) HSP70 overexpression sequesters AIF and reduces neonatal hypoxic⁄ ischemic brain injury
J Cereb Blood Flow Metab 25, 899–910
21 Nylandsted J, Gyrd-Hansen M, Danielewicz A, Fehrenbacher N, Lademann U, Høyer-Hansen M, Weber E, Multhoff G, Rohde M & Ja¨a¨ttela¨ M (2004) Heat shock protein 70 promotes cell survival by inhibit-ing lysosomal membrane permeabilization J Exp Med
200, 425–435
22 Didelot C, Schmitt E, Brunet M, Maingret L, Parcellier
A & Garrido C (2006) Heat shock proteins: endogenous modulators of apoptotic cell death Handb Exp Pharma-col 26, 171–198