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Cardiovascular Research 2002 53(4):936-943; doi:10.1016/S0008-6363(01)00506-5
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Copyright © 2002, European Society of Cardiology

Bcl-xl reduces doxorubicin-induced myocardial damage but fails to control cardiac gene downregulation

Keita Kunisada*,1, Eiroh Tone1, Shinji Negoro, Yoshikazu Nakaoka, Yuichi Oshima, Tomoaki Osugi, Masanobu Funamoto, Masahiro Izumi, Yasushi Fujio, Hisao Hirota and Keiko Yamauchi-Takihara

Department of Molecular Medicine, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan

keita.kunisada{at}ma5.seikyou.ne.jp

* Corresponding author. Tel.: +81-6-6879-3835; fax: +81-6-6879-3839

Received 4 May 2001; accepted 10 October 2001


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: We recently reported that doxorubicin (Dox), an effective anti-cancer drug, induces apoptosis in cardiac myocytes in association with reduction of Bcl-xl expression. In the present study, we further examined whether overexpression of Bcl-xl ameliorates Dox-induced cardiac myocyte damage. Methods and results: Overexpression of the Bcl-xl gene by adenovirus vector resulted in an 11-fold increase in Bcl-xl protein in neonatal rat cardiac myocytes (BCL) compared to that in cells with β-galactosidase gene transfection (CTL). Although Dox treatment generated similar amounts of reactive oxygen species (ROS) in BCL and CTL, cell viability was maintained and the number of apoptotic cardiac myocytes was significantly decreased in BCL. Cytochrome c release and enhanced caspase-3 activity after Dox treatment were significantly suppressed and Bax expression level was decreased in BCL. Cardiac-specific gene expression is known to be inhibited by Dox. The expression of cardiac {alpha}-actin and sarcoplasmic reticulum Ca2+-ATPase 2a mRNA was equally inhibited in BCL and CTL after Dox treatment. Conclusions: Overexpression of Bcl-xl in cardiac myocytes failed to regulate Dox-induced ROS generation and cardiac-specific gene downregulation but inhibited apoptosis accompanied by reduction of Bax protein.

KEYWORDS Apoptosis; Free radicals; Gene therapy; Heart failure; Mitochondria


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Doxorubicin (Dox), a clinically effective anti-cancer drug, has beneficial effects in treating a variety of soft and solid human malignancies, but its use is restricted because of cardiac toxicity that finally leads to heart failure [1]. The mechanism of Dox-mediated cardiac toxicity involves the generation of reactive oxygen species (ROS), which damage cell membranes, and the selective inhibition of cardiac-specific gene expression, such as the cardiac {alpha}-actin, sarcoplasmic reticulum Ca2+-ATPase2a (SERCA2a) and cardiac troponin-I genes [2]. Siveski-Iliskovic et al. reported that probucol, a clinically available anti-oxidant drug, had a protective effect against Dox-induced cardiomyopathy [1]. In addition, mouse with cardiac-specific overexpression of the gene for metallothionein, a scavenger of free radicals, was resistant to the cardiac toxicity induced by Dox [3]. Transgenic mice with cardiac-specific overexpression of the signal transducer and activator of transcription 3 (STAT3) gene, a transcription factor downstream of several cytokines including the interleukin (IL)-6 cytokine family, was also tolerant against Dox, in association with inhibition of cardiac-contractile protein gene downregulation and production of cardiac-protective factors [4].

We recently reported that leukemia inhibitory factor, a member of the IL-6 cytokine family, protected cardiac myocytes from Dox-mediated apoptosis by activation of the phosphatidylinositol 3-kinase pathway and inhibition of Bcl-xl reduction [5]. The level of Bcl-xl expression strongly correlates with reduction of the response rate in myeloma cells against chemotherapeutic drugs including Dox [6]. On the other hand, overexpression of Bcl-2 in a human ovarian carcinoma cell line conferred a trend toward sensitivity but not resistance to anti-cancer drug [7]. In HL-60 cells overexpressing both Bcl-2 and Bcl-xl, downregulation of only Bcl-2 by antisense oligodeoxynucleotide (ODN) increased sensitivity to cytosine arabinoside despite Bcl-xl overexpression [8]. These results indicate that the anti-apoptotic effect of Bcl-2 family members is cell-specific. Therefore, in the present study, we examined whether overexpression of Bcl-xl protein protects cardiac myocytes from Dox-induced cell damage, and we attempted to clarify the molecular mechanism of Bcl-xl-mediated cardiac myocyte protection.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
All experiments were performed in accordance with Guidelines for Animal Experiments of Osaka University Graduate School of Medicine.

2.1. Reagents and cell culture
Neonatal rat cardiac myocytes were isolated from 1- or 2-day-old Wistar rats (Kiwa Jikken Dobutsu, Wakayama, Japan) as previously described [9]. Dox was obtained from Kyowa Hakko (Tokyo, Japan). Anti-Bax and Bcl-xl antibodies were purchased from Santa-Cruz Biotechnology (CA, USA) and anti-{alpha}-tubulin and {alpha}-actinin antibodies from Sigma (MO, USA). Human Bcl-xl cDNA was kindly provided by Dr. S. Nagata, SERCA2a cDNA by Dr. M. Tada [10] (Osaka University, Japan) and cardiac {alpha}-actin cDNA by Dr. H. Ito [2] (Tokyo Medical and Dental University, Japan). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA was purchased from Cayman (NY, USA).

2.2. Generation of recombinant adenovirus and adenovirus infection
Recombinant replication-defective adenovirus carrying the Bcl-xl gene or the β-galactosidase gene was prepared and purified as described previously [11]. Two days after plating, cardiac myocytes were infected with adenovirus diluted in D-MEM (Gibco BRL, NY, USA) with 5% fetal calf serum (FCS) at 1 to 30 multiplicity of infection (moi). Thereafter, viral suspension was removed and cells were cultured for an additional 2 days with medium 199 (M-199, Gibco BRL) containing 10% newborn calf serum (NCS).

2.3. TUNEL assay
In situ labeling of fragmented DNA was performed with the MEBSTAIN Apoptosis Kit Direct (MBL) as described previously [5]. In brief, cardiac myocytes were grown on fibronectin-coated glass slides, fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 20 min, and permeabilized with 0.2% Triton X-100 for 5 min. The cells were first incubated with anti-{alpha}-actinin antibody at 37°C for 1 h and subsequently with anti-mouse IgG TRITC-labeled antibody for 1 h. Thereafter, 50 µl terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) reaction mixture containing both TdT and FITC-dUTP was added to each sample for 1 h at 37°C. The cells were analyzed by fluorescence microscopy. Numbers of TUNEL-positive cells counted in {alpha}-actinin-positive cardiac myocytes (n=500) are expressed as percentages.

2.4. Measurement of cell viability (MTS assay)
Cardiac myocytes were cultured at a density of 1·104/100 µl in 96-well dishes, and were infected with adenovirus carrying the Bcl-xl gene (BCL) or the β-galactosidase gene (CTL) after 2 days. The cells were stimulated with Dox for 18 h and their viability was examined by MTS {3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt} cell respiratory assay (Promega, Madison, WI, USA) according to the manufacturer's instructions. Viability was expressed as the absorbance at 490 nm measured with a spectrophotometer.

2.5. Measurement of creatine phosphokinase (CPK)
Determination of CPK in cultured conditioned medium was performed using a commercially available kit (MERCK-1-TEST CK, Kanto Kagaku, Tokyo, Japan).

2.6. Measurement of ROS
Cardiac myocytes were incubated with 10 µM dichlorodihydrofluorescein diacetate (DCHF/DA, Nakarai, Osaka, Japan) for 30 min at 37°C in the dark, washed with PBS and detached with 0.25% trypsin and 0.05% EDTA. Then cardiac myocytes were rinsed three times with PBS containing 5% NCS to remove trypsin and filtered through 35-µm nylon mesh. They were analyzed immediately using a Becton-Dickinson FACScan with excitation at 488 nm and emission at 510 nm.

2.7. Measurement of cytochrome c release
Cytochrome c (cyt-c) release into cytosol was examined as previously described [12]. In brief, cardiac myocytes were washed with ice-cold PBS and resuspended in 100 µl of extraction buffer containing 50 mM PIPES-KOH (pH 7.4), 220 mM mannitol, 68 mM sucrose, 5 mM EGTA, 2 mM MgCl2, 1 mM DTT and proteinase inhibitors for 30 min. Cells were homogenized by passage through a 25-gauge needle for 10 strokes, and the lysates were centrifuged at 750 g for 10 min to pellet out the nuclei. The remaining supernatants were centrifuged for 15 min at 10 000 g to remove mitochondria (mitochondrial fraction). This supernatant was considered a cytosolic fraction. Western blot analysis of control cardiac myocytes showed that the mitochondrial fraction contained much cyt-c and manganase-superoxide dismutase (Mn-SOD) protein but that the cytosolic fraction contained neither cyt-c nor Mn-SOD (data not shown).

2.8. Measurement of caspase-3 activity
The activity of caspase-3 was determined with a Caspase-3 Assay Kit (MBL, Nagoya, Japan). Cardiac myocytes were lysed in buffer containing 10 mM Tris–HCl (pH 8.0), 0.32 mM sucrose, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM sodium orthovanadate, 2 mM dithiothreitol (DTT), proteinase inhibitors and 1% Triton X-100. The concentration of total protein in the supernatant was measured with a protein assay system (Bio-Rad, CA, USA). Aliquots of the cytosolic extracts were mixed with equal volumes of 50 µM Ac-(Asp–Glu–Val–Asp) DEVD-p-nitroanilide (NA), in assay buffer containing 50 mM 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (HEPES) (pH 7.4), 100 mM NaCl, 10 mM DTT, 1 mM EDTA, 0.1% 3-[(cholamidopropyl)dimethylamino]-1-propanesulfonate (CHAPS) and 10% glycerol at 37°C for 1 h. The activity was assessed with a spectrophotometer at 405 nm.

2.9. Western blot analysis
The samples were separated in 15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gel and resolved proteins were electrophoretically transferred onto an Immobilon-P membrane (Millipore, MA, USA). Membranes were blocked with 5% skim milk and probed with rabbit anti-Bcl-xl, Bax or {alpha}-tubulin antibody at 1:1000 dilution for 1 h to detect each protein. They were then incubated with peroxidase-conjugated anti-rabbit IgG for an additional 1 h. The immune complexes were visualized with Kodak X-OMAT-AR film with an enhanced chemiluminescence system.

2.10. Northern blot analysis
Total RNA was isolated by ISOGEN (Nippon Gene, Tokyo, Japan) according to the manufacturer's instructions. Cardiac {alpha}-actin and SERCA2a cDNAs were labeled with an [{alpha}-32P]dCTP using PRIME IT labeling kit (Stratagene, TX, USA). Total RNA was separated on 1% formaldehyde-agarose gel and transferred to nylon membrane. Prehybridization was performed at 42°C for 6 h, followed by hybridization for 12 h. The membranes were washed three times with 2xSSC and 0.1% SDS at 60°C and the blots were exposed to X-ray film.

2.11. Statistical analysis
All values are expressed as mean±S.E.M. Statistical analysis was performed with Student's t-test (Statview). Values of p<0.05 were considered significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. Overexpression of Bcl-xl presents tolerance against Dox in cardiac myocytes
We first examined whether Bcl-xl overexpression suppresses Dox-induced apoptosis in cardiac myocytes. Fig. 1A shows typical results of TUNEL assay. TUNEL-positive cardiac myocytes were rarely detected among either BCL or CTL in control culture conditions (Dox: 0 µM). Dox treatment for 6 h increased the number of TUNEL-positive cardiac myocytes among CTL (a, b), while few TUNEL-positive cells were detected among BCL (c, d). As summarized in Fig. 1B, TUNEL-positive cardiac myocytes after Dox treatment accounted for 38% of CTL and 5% of BCL (p<0.05 vs. CTL).


Figure 1
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  		Fig. 1 Overexpression of Bcl-xl protects cardiac myocytes from Dox-induced toxicity. (A) In situ labeling of DNA fragmentation (TUNEL assay). Cultured cardiac myocytes were treated with 1.0 µM of Dox for 6 h. The cells were stained with anti-{alpha}-actinin antibody (a, c) and the nuclei were labeled with TdT (b, d). Cardiac myocytes were transfected with adenovirus carrying the β-galactosidase gene (CTL; a, b), or the Bcl-xl gene (BCL; c, d). (B) Numbers of TUNEL-positive cells counted in five hundred {alpha}-actinin-positive cardiac myocytes are expressed as percentages. (C) Cardiac myocytes were treated with indicated dose of Dox for 18 h and cell viability was evaluated by MTS assay and expressed as absorbance at 490 nm measured with a spectrophotometer. (D) Cardiac myocytes were treated as described in (C). Open bars represent CTL and closed bars represent BCL in (C) and (D). Data are means±S.E.M. from five independent experiments. *p<0.05 vs. CTL, #p<0.05 vs. 0 µM Dox.

 
The effect of Bcl-xl overexpression on Dox-induced cardiac myocyte damage was also evaluated by MTS assay and CPK release. Viability of CTL decreased after 18 h of Dox treatment in a dose-dependent manner, but BCL significantly maintained viability after 0.5 and 1.0 µM of Dox (p<0.05 vs. CTL) (Fig. 1C). CPK concentration in cultured medium increased significantly after 0.5 and 1.0 µM Dox treatment in CTL, but significantly suppressed in BCL (p<0.05 vs. CTL) (Fig. 1D). These findings indicate that overexpression of Bcl-xl protects cardiac myocytes from Dox-induced cell injury.

3.2. Dox-induced ROS generation is not suppressed in cardiac myocytes with Bcl-xl overexpression
Dox is known to induce ROS in several kinds of cells, and ROS induction is one of the important mechanisms of cell injury [13]. We next examined whether Bcl-xl suppresses ROS production induced by Dox. ROS was equally produced in BCL and CTL after 10 µM Dox treatment (Fig. 2a, c). Augmented ROS generation was also detected in these cells cultured without serum (Fig. 2b, d). These results indicate that Bcl-xl failed to scavenge ROS induced by 10 µM of Dox or serum deprivation in cardiac myocytes.


Figure 2
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  		Fig. 2 ROS generation in cardiac myocytes. Cardiac myocytes were cultured with or without 10 µM Dox for 1 h in the medium containing 10% serum (a, c), or in the medium with or without 10% serum (b, d). Cardiac myocytes were transfected with adenovirus carrying the β-galactosidase gene (CTL; a, b), or the Bcl-xl gene (BCL; c, d).

 
3.3. Cyt-c release and caspase-3 activation after Dox treatment are significantly suppressed in cardiac myocytes with Bcl-xl overexpression
Release of cyt-c from mitochondria to cytosol is a key step in the execution of the cell death pathway [14]. Cyt-c release was observed with 0.5 and 1.0 µM Dox treatment in CTL, but was inhibited in BCL (Fig. 3A). Caspase-3 also functions as an executor of apoptosis [15], and inhibition of this enzyme suppresses induction of apoptosis. Caspase-3 activity increases in CTL after 3 h of Dox treatment and this increased activity continued up to 12 h (Fig. 3B). On the other hand, caspase-3 activity in BCL increased after 6 h of Dox treatment but returned to control level after 12 h. Caspase-3 activity after Dox treatment was significantly lower in BCL than CTL at all time points examined (p<0.05). Overexpression of Bcl-xl significantly suppressed cyt-c release and subsequent activation of caspase-3 in cardiac myocytes after Dox treatment.


Figure 3
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  		Fig. 3 Cytochrome c release in cytosol and caspase-3 activation after Dox treatment. (A) Cultured cardiac myocytes were treated with indicated doses of Dox for 2 h and prepared for the detection of cyt-c in cytosolic fraction as described in Methods. A 20-µg amount of protein was loaded for Western blot analysis and probed with anti-cyt-c antibody. (B) Cultured cardiac myocytes were treated with 1.0 µM Dox for indicated periods and harvested for the detection of caspase-3 activity as described in Methods. Open bars represent CTL and closed bars represent BCL. Data are means±S.E.M. from three independent experiments. *p<0.05 vs. CTL, #p<0.05 vs. 0 h.

 
3.4. Bax expression is decreased in cardiac myocytes with Bcl-xl overexpression
To examine the molecular mechanism of Bcl-xl-mediated tolerance to Dox, we examined expression level of Bcl-2 protein family members in cardiac myocytes. Surprisingly, prior to exposure to Dox, cardiac myocytes overexpressing Bcl-xl demonstrated reduced expression of Bax protein, and the level of this expression was inversely correlated with that of Bcl-xl (Fig. 4). The level of Bax protein expression in cardiac myocytes with Bcl-xl overexpression continued to be reduced even after Dox treatment (data not shown). The expression levels of Bcl-2 protein and Bad protein were not changed in these cells with either Dox treatment or normal conditions (data not shown).


Figure 4
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  		Fig. 4 Bcl-xl overexpression reduces Bax protein level in cardiac myocyte. Cultured cardiac myocytes were transfected with indicated doses of adenovirus carrying the Bcl-xl gene. The whole cell lysates were prepared and Western blot analysis was performed by using anti-Bax, Bcl-xl or {alpha}-tubulin antibody.

 
3.5. Downregulation of cardiac-specific gene by Dox treatment
The expressions of cardiac {alpha}-actin and SERCA2a mRNAs after Dox treatment were examined in CTL and BCL by Northern blot analysis (Fig. 5). Expressions of cardiac {alpha}-actin and SERCA2a mRNAs were equally decreased in BCL after 1.0 µM Dox treatment, without affecting the expression of a housekeeping gene; GAPDH. Overexpression of Bcl-xl in cardiac myocytes failed to inhibit Dox-induced reduction of cardiac-specific gene expression.


Figure 5
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  		Fig. 5 Down regulation of cardiac-specific gene after Dox treatment. Cultured cardiac myocytes were treated with 1.0 µM Dox for 18 h. Northern blot analysis was performed by using cardiac {alpha}-actin, SERCA2a or GAPDH cDNA as a probe.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
Several studies have demonstrated that apoptosis occurs in murine hearts treated with Dox. Apoptosis is observed in rat heart exposed to 2.5 mg/kg intraperitoneal administration of Dox, with a peak at 24–48 h and return to baseline level within a week [16]. Kang et al. reported that apoptosis occurred in the heart 4 days after Dox injection at a single dose of 15 mg/kg [17]. These findings do not show that prolonged toxicity to myofibrils is responsible for the late cardiomyopathy observed in patients with Dox treatment. On the other hands, Nakamura et al. reported that apoptosis increased in the heart as early as 9 weeks with once a week intravenous administration of 2 mg/kg of Dox for 8 weeks [18]. They demonstrated that neutralization of Fas ligand inhibited Dox-induced apoptosis and that apoptosis increased with deterioration of morphological findings and cardiac function. The period during which apoptosis occurs in Dox-treated hearts may depend on several conditions, such as dose or mode of administration. Moreover, it has been reported that patients with chronic heart failure exhibit ongoing cardiac myocyte apoptosis for years [19]. These results suggest that apoptosis may be an important underlying mechanism of cardiac myocyte loss and cardiac dysfunction in patients undergoing Dox treatment.

Cardiac myocytes with overexpression of Bcl-xl were evaluated by expression of Bcl-2 family proteins, TUNEL assay, and for caspase-3 activity and phosphorylation of kinases, such as Akt, mitogen activated protein kinase (MAPK), p38-MAPK and JNK, by Western blot analysis. Regarding expression of Bcl-2 family proteins, Bcl-2 and Bad expressions were not altered but Bax expression was significantly decreased in BCL than in CTL. Other results were the same for BCL and CTL (data not shown).

Increase in Bcl-xl protein had a protective effect against Dox-induced cell damage associated with Bcl-2 expression. The mechanism of Bcl-xl-mediated suppression of cell damage has been described in several reports [20–22]. Bcl-xl and Bax change mitochondrial membrane permeability, causing cyt-c release mediated by outer membrane transition pores including voltage-dependent anion channels (VDAC) [23]. Shimizu et al. reported that Bax and Bak accelerate opening of VDAC, whereas Bcl-xl closes this channel by direct binding [24]. Bax and Bak thus allow cyt-c to pass through VDAC, but this passage is prevented by Bcl-xl. Activated caspase-9 subsequently cleaves downstream caspase-3 [25]. Our results showed that Bcl-xl inhibited cyt-c release from mitochondria, subsequent activation of caspase-3 and DNA fragmentation in cardiac myocytes.

Dox is known to produce ROS including oxygen radicals and hydrogen peroxide, which is a partially reduced form of oxygen, and to cause lipid peroxidation [26]. ROS induces contractile failure and apoptosis in cardiac myocytes [27]. Pretreatment with catalase, ebselen, a glutathione peroxidase mimetic, or nitrone spin traps is reported to dramatically inhibit Dox-induced apoptosis [28]. In addition to these direct antioxidants, the relationship between the generation of ROS and the expression of Bcl-2 or Bcl-xl has been examined. In HL60 cells overexpressing Bcl-2 protein, glutathione depletion-induced ROS generation was significantly suppressed in association with reduction of apoptotic cells [29]. On the other hand, Gottlieb et al. reported that overexpression of Bcl-xl prevented TNF-{alpha}-induced initial decrease in mitochondrial membrane potential and subsequent ROS production but did not prevent that induced by hydrogen peroxide [30]. They suggested that Bcl-xl controls membrane potential and subsequent ROS production in mitochondria rather than acting as a direct antioxidant.

The ROS analyzed by FACScan includes not only hydrogen peroxide but also lipid peroxide, and generation of lipid peroxide was also not suppressed in BCL (data not shown). The ROS generations caused by 10 µM Dox or serum-deprivation were similar in CTL and BCL in our study. Thus, the mechanism of inhibition of Dox-induced apoptosis in BCL is not suppression of ROS generation but inhibition of the events downstream from ROS, which involve cyt-c release from mitochondria and caspase-3 activation.

Dibbert et al. reported that Bax-deficient neutrophils exhibited delayed apoptosis, which is also observed when cells are treated with granulocyte macrophage-colony stimulating factor [31]. This finding suggests that Bax may be a possible target for an antisense strategy for protection against Dox stimulation in cardiac myocytes. Impaired myocardial relaxation is improved by overexpression of SERCA2a in isolated cardiac myocytes [32], and improvement of decreased SERCA2a activity in a mouse model of congestive heart failure has been reported to rescue cardiac function [33]. The reduced expression of cardiac-specific genes observed after Dox treatment is be one of the causes of Dox-induced heart failure.

In summary, overexpression of Bcl-xl suppressed Dox-induced cell death by downregulation of Bax and suppression of cyt-c release in cytosol. Bcl-xl regulates neither the generation of ROS nor the reduction in transcription of cardiac-specific genes caused by Dox treatment. Cardiac-specific overexpression of the Bcl-xl gene may be a therapeutic strategy useful against Dox-induced cardiac toxicity, but not sufficient to maintain cardiac function. We therefore conclude that additional combined therapy is necessary to protect cardiac myocytes from Dox-induced downregulation of cardiac-specific genes.

Time for primary review 32 days.


   Acknowledgements
 
We are grateful to J. Hironaka for her secretarial assistance. This study was supported by a Grant-in-Aid for Scientific Research on Priority Areas from The Ministry of Education, Science, Sports and Culture of Japan and a grant from the Ministry of Health and Welfare of Japan.


   Notes
 
1 These authors contributed equally to this study. Back


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

  1. Siveski-Iliskovic N., Kaul N., Singal P.K. Probucol promotes endogenous antioxidants and provides protection against adriamycin-induced cardiomyopathy in rats. Circulation (1994) 89:2829–2835.[Abstract/Free Full Text]
  2. Ito H., Miller S.C., Billingham M.E., et al. Doxorubicin selectively inhibits muscle gene expression in cardiac muscle cells in vivo and in vitro. Proc Natl Acad Sci USA (1990) 87:4275–4279.[Abstract/Free Full Text]
  3. Kang Y.J., Chen Y., Yu A., Voss-McCowan M., Epstein P.N. Overexpression of metallothionein in the heart of transgenic mice suppresses doxorubicin cardiotoxicity. J Clin Invest (1997) 100:1501–1506.[Web of Science][Medline]
  4. Kunisada K., Negoro S., Tone E., et al. Signal transducer and activator of transcription 3 in the heart transduces not only a hypertrophic signal but a protective signal against doxorubicin-induced cardiomyopathy. Proc Natl Acad Sci USA (2000) 97:315–319.[Abstract/Free Full Text]
  5. Negoro S., Oh H., Tone E., et al. Glycoprotein 130 regulates cardiac myocyte survival in doxorubicin-induced apoptosis through phosphatidylinositol 3-kinase/Akt phosphorylation and bcl-xL/caspase-3 interaction. Circulation (2001) 103:555–561.[Abstract/Free Full Text]
  6. Tu Y., Renner S., Xu F., et al. BCL-X expression in multiple myeloma: possible indicator of chemoresistance. Cancer Res (1998) 58:256–262.[Abstract/Free Full Text]
  7. Beale P.J., Rogers P., Boxall F., Sharp S.Y., Kelland L.R. BCL-2 family protein expression and platinum drug resistance in ovarian carcinoma. Br J Cancer (2000) 82:436–440.[CrossRef][Web of Science][Medline]
  8. Konopleva M., Tari A.M., Estrov Z., et al. Liposomal Bcl-2 antisense oligonucleotides enhance proliferation, sensitize acute myeloid leukemia to cytosine-arabinoside, and induce apoptosis independent of other antiapoptotic proteins. Blood (2000) 95:3929–3938.[Abstract/Free Full Text]
  9. Kunisada K., Hirota H., Fujio Y., et al. Activation of JAK-STAT and M.A.P. kinases by leukemia inhibitory factor through gp130 in cardiac myocytes. Circulation (1996) 94:2626–2632.[Abstract/Free Full Text]
  10. Kimura Y., Otsu K., Nishida K., Kuzuya T., Tada M. Thyroid hormone enhances Ca2+ pumping activity of the cardiac sarcoplasmic reticulum by increasing Ca2+ ATPase and decreasing phospholamban expression. J Mol Cell Cardiol (1994) 26:1145–1154.[CrossRef][Web of Science][Medline]
  11. Fujio Y., Nguyen T., Wencker D., Kitsis R.N., Walsh K. Akt promotes survival of cardiomyocytes in vitro and protects against ischemia-reperfusion injury in mouse heart. Circulation (2000) 101:660–667.[Abstract/Free Full Text]
  12. Cook S.A., Sugden P.H., Clerk A. Regulation of bcl-2 family proteins during development and in response to oxidative stress in cardiac myocytes: association with changes in mitochondrial membrane potential. Circ Res (1999) 85:940–949.[Abstract/Free Full Text]
  13. DeAtley S.M., Aksenov M.Y., Aksenova M.V., et al. Antioxidants protect against reactive oxygen species associated with adriamycin-treated cardiomyocytes. Cancer Lett (1999) 136:41–46.[CrossRef][Web of Science][Medline]
  14. Skulachev V.P. Cytochrome c in the apoptotic and antioxidant cascades. FEBS Lett (1998) 423:275–280.[CrossRef][Web of Science][Medline]
  15. Casciola-Rosen L., Nicholson D.W., Chong T., et al. Apopain/CPP32 cleaves proteins that are essential for cellular repair: a fundamental principle of apoptotic death. J Exp Med (1996) 183:1957–1964.[Abstract/Free Full Text]
  16. Arola O.J., Saraste A., Pulkki K., Kallajoki M., Parvinen M., Voipio-Pulkki L.M. Acute doxorubicin cardiotoxicity involves cardiomyocyte apoptosis. Cancer Res (2000) 60:1789–1792.[Abstract/Free Full Text]
  17. Kang Y.J., Zhou Z.X., Wang G.W., Buridi A., Klein J.B. Suppression by methallothionein of doxorubicin-induced cardiomyocyte apoptosis through inhibition of p38 mitogen-activated protein kinases. J Biol Chem (2000) 275:13690–13698.[Abstract/Free Full Text]
  18. Nakamura T., Ueda Y., Juan Y., et al. Fas-mediated apoptosis in adriamycin-induced cardiomyopathy in rats: in vivo study. Circulation (2000) 102:572–578.[Abstract/Free Full Text]
  19. Olivetti G., Abbi R., Quiani F., et al. Apoptosis in the failing human heart. New Engl J Med (1997) 336:1131–1141.[Abstract/Free Full Text]
  20. Narita M., Shimizu S., Ito T., et al. Bax interacts with the permeability transition pore to induce permeability transition and cytochrome c release in isolated mitochondria. Proc Natl Acad Sci USA (1998) 95:14681–14686.[Abstract/Free Full Text]
  21. Zoratti M., Szabo I. The mitochondrial permeability transition. Biochim Biophys Acta (1995) 1241:139–176.[Medline]
  22. Jurgensmeier J.M., Xie Z., Deveraux Q., et al. Bax directly induces release of cytochrome c from isolated mitochondria. Proc Natl Acad Sci USA (1998) 95:4997–5002.[Abstract/Free Full Text]
  23. Crompton M. The mitochondrial permeability transition pore and its role in cell death. Biochem J (1999) 341:233–249.[CrossRef][Web of Science][Medline]
  24. Shimizu S., Narita M., Tsujimoto Y. Bcl-2 family proteins regulate the release of apoptogenic cytochrome c by the mitochondrial channel VDAC. Nature (1999) 399:483–487.[CrossRef][Medline]
  25. Li P., Nijhawan D., Budihardjo I., et al. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell (1997) 91:479–489.[CrossRef][Web of Science][Medline]
  26. Kitada M., Horie T., Awazu S. Chemiluminescence associated with doxorubicin-induced lipid peroxidation in rat heart mitochondria. Biochem Pharmacol (1994) 48:93–99.[CrossRef][Web of Science][Medline]
  27. Matsubara T., Dhalla N.S. Relationship between mechanical dysfunction and depression of sarcolemmal Ca(2+)-pump activity in hearts perfused with oxygen free radicals. Mol Cell Biochem (1996) 160–161:179–185.
  28. Kotamraju S., Konorev E.A., Joseph J., Kalyanaraman B. Doxorubicin-induced apoptosis in endothelial cells and cardiomyocytes is ameliorated by nitrone spin trap and ebselen. Role of reactive oxygen and nitrogen species. J Biol Chem (2000) 275:33585–33592.[Abstract/Free Full Text]
  29. Kane D.J., Sarafian T.A., Anton R. Bcl-2 inhibition of neural death: decreased generation of reactive oxygen species. Science (1993) 262:1274–1277.[Abstract/Free Full Text]
  30. Gottlieb E., Vander Heiden M.G., Thompson C.B. Bcl-x(L) prevents the initial decrease in mitochondrial membrane potential and subsequent reactive oxygen species production during tumor necrosis factor alpha-induced apoptosis. Mol Cell Biol (2000) 20:5680–5689.[Abstract/Free Full Text]
  31. Dibbert B., Weber M., Nikolaizik W.H., et al. Cytokine-mediated Bax deficiency and consequent delayed neutrophil apoptosis: a general mechanism to accumulate effector cells in inflammation. Proc Natl Acad Sci USA (1999) 96:13330–13335.[Abstract/Free Full Text]
  32. del Monte F., Harding S.E., Schmidt U., et al. Restoration of contractile function in isolated cardiomyocytes from failing human hearts by gene transfer of SERCA2a. Circulation (1999) 100:2308–2311.[Abstract/Free Full Text]
  33. Minamisawa S., Hoshijima M., Chu G., et al. Chronic phospholamban-sarcoplasmic reticulum calcium ATPase interaction is the critical calcium cycling defect in dilated cardiomyopathy. Cell (1999) 99:313–322.[CrossRef][Web of Science][Medline]

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