Modulation of doxorubicin-induced cardiac dysfunction in dominant-negative p38α mitogen-activated protein kinase mice

Free Radical Biology & Medicine 49 (2010) 1422–1431

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Free Radical Biology & Medicine j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / f r e e r a d b i o m e d

Original Contribution

Modulation of doxorubicin-induced cardiac dysfunction in dominant-negative p38α mitogen-activated protein kinase mice Rajarajan A. Thandavarayan a,b, Kenichi Watanabe a,⁎, Flori R. Sari a, Meilei Ma a, Arun Prasath Lakshmanan a, Vijayasree V. Giridharan b, Narasimman Gurusamy c, Hiroshi Nishida b, Tetsuya Konishi b, Shaosong Zhang d, Anthony J. Muslin e, Makoto Kodama f, Yoshifusa Aizawa f a

Department of Clinical Pharmacology, Niigata University of Pharmacy and Applied Life Sciences, Niigata City 956-8603, Japan Department of Functional and Analytical Food Sciences, Niigata University of Pharmacy and Applied Life Sciences, Niigata City 956-8603, Japan c Department of Anesthesiology and Medicine, Harvard Medical School, Boston, MA 02115, USA d Lightlab Imaging, Inc., Westford, MA 01886, USA e Center for Cardiovascular Research, Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA f First Department of Medicine, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan b

a r t i c l e

i n f o

Article history: Received 23 February 2010 Revised 5 July 2010 Accepted 2 August 2010 Available online 10 August 2010 Keywords: Doxorubicin p38α MAPK Oxidative stress Apoptosis Free radicals

a b s t r a c t Doxorubicin (Dox) is a widely used antitumor drug, but its application is limited because of its cardiotoxic side effects. Increased expression of p38α mitogen-activated protein kinase (MAPK) promotes cardiomyocyte apoptosis and is associated with cardiac dysfunction induced by prolonged agonist stimulation. However, the role of p38α MAPK is not clear in Dox-induced cardiac injury. Cardiac dysfunction was induced by a single injection of Dox into wild-type (WT) mice and transgenic mice with cardiac-specific expression of a dominant-negative mutant form of p38α MAPK (TG). Left ventricular (LV) fractional shortening and ejection fraction were higher and the expression levels of phospho-p38 MAPK and phospho-MAPK-activated mitogen kinase 2 were significantly suppressed in TG mouse heart compared to WT mice after Dox injection. Production of LV proinflammatory cytokines, cardiomyocyte DNA damage, myocardial apoptosis, caspase-3-positive cells, and phospho-p53 expression were decreased in TG mice after Dox injection. Moreover, LV expression of NADPH oxidase subunits and reactive oxygen species was significantly less in TG mice compared to WT mice after Dox injection. These findings suggest that p38α MAPK may play a role in the regulation of cardiac function, oxidative stress, and inflammatory and apoptotic mediators in the heart after Dox administration. © 2010 Elsevier Inc. All rights reserved.

Introduction Doxorubicin (Dox), an anthracycline antibiotic, is a highly effective chemotherapeutic agent used in the treatment of solid and hematopoietic tumors; however, a major limiting factor for the clinical use of Dox is its cumulative, irreversible cardiac toxicity [1,2]. In fact, multiple intravenous Dox treatments over a period of several months result in the development of cardiomyopathy and congestive heart failure in humans [2]. The precise cellular mechanisms responsible for this chronic cardiotoxicity of Dox remain enigmatic, but the antitumor activity of Dox is likely to be distinct from the mechanism of its cardiotoxicity [3]. Doxinduced cardiomyopathy has been linked to apoptosis and DNA damage, free radical formation, and alterations in calcium metabolism [4]. Accumulating evidence indicates that Dox-induced cardiomyopathy is mainly caused by increased oxidant production, which eventually leads to the apoptotic loss of cardiomyocytes [2,5,6]. Therefore, we speculated

⁎ Corresponding author. Fax: + 81 250 25 5021. E-mail address: [email protected] (K. Watanabe). 0891-5849/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2010.08.005

that suppression of apoptosis may largely rescue Dox-triggered cardiotoxicity. p38 mitogen-activated protein kinase (p38 MAPK), a member of the MAPK family, is activated by physical and chemical stress factors resulting in growth promotion, apoptosis, oxidative stress, and vasoconstriction [7,8]. The p38 MAPK occurs in various isoforms such as α, β, γ, and δ, of which p38α MAPK is the major isoform in human heart [9]. Each protein in the cascade activates the subsequent kinase by phosphorylation of specific amino acid residues. Once activated, p38α MAPK phosphorylates a variety of intracellular targets, including transcription factors and protein kinases, and some of these targets may promote apoptosis [10]. In particular, transfection experiments using primary cultures of neonatal rat cardiomyocytes have shown that p38α is critically involved in myocyte apoptosis [11]. In any event, the common observation is that p38 MAPK activation is associated with accumulation of reactive oxygen species (ROS) generated under stress conditions. Therefore, in this study, we focused on the possible role of p38 MAPK in mediating Dox-induced apoptosis. Though previous studies [12,13] have indicated a role for p38 MAPK in Dox-induced cardiomyopathy, the specific role of p38α MAPK is not known.

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In this study, to clarify the role of p38α MAPK in Dox-induced cardiomyopathy, we attempted to use p38α MAPK knockout mice. The p38α MAPK knockout mice died during embryonic development [14]. Thus we used transgenic mice with cardiac-specific overexpression of a dominant-negative mutant (DN) of p38α MAPK to examine the role of p38α MAPK in the Dox myocardium. Materials and methods Generation of transgenic mice Transgenic DN p38α MAPK mice (TG) in the Swiss Black genetic background were generated as described previously [15] at the Neuroscience Transgenic Facility of the Washington University School of Medicine. Progeny were analyzed by polymerase chain reaction to detect transgene integration using mouse-tail DNA as template. TG mice were compared to wild-type (WT) littermates in every experiment. Mice were given free access to water and chow throughout the period of study, and the animals were treated in accordance with the Guidelines for Animal Experimentation of our institute. All animals were handled according to the approved protocols and animal welfare regulations of the Institutional Review Board at Niigata University of Pharmacy and Applied Life Sciences. Animal protocol The well-established protocol of Myer et al. [16] was used to produce subacute Dox injury in mice. Eight- to 10-week-old WT (n = 25) and TG mice (n = 20) were treated with a single dose of Dox (Kyowa Hakko Co. Ltd., Tokyo, Japan; 20 mg/kg ip, WT-Dox and TG-Dox, respectively). Untreated WT (n = 10) and TG (n = 10) mice were used as controls. The survival of the mice 10 days after doxorubicin treatment was analyzed using Kaplan–Meier methods and the log-rank test. Transthoracic echocardiography Cardiac function was evaluated using M-Mode echocardiography performed by an experienced echocardiographic analyst without knowledge of mouse genotype or previous treatment. Two-dimensional echocardiography studies were performed in anesthetized mice (pentobarbital, 50 mg/kg, ip) to evaluate cardiac function using an echocardiographic machine equipped with 7.5- and 12-MHz transducers linked to an ultrasound system (SSD-5500; Aloka, Tokyo, Japan). The short-axis view of the left ventricle was recorded to measure the left ventricular (LV) dimension in systole (LVDs) and diastole (LVDd) as well as the percentage fractional shortening (%FS) and percentage ejection fraction (%EF). Hearts were harvested for analysis from control and Dox mice. The left ventricle was quickly dissected and cut into two parts. One part was immediately transferred into liquid nitrogen and then stored at −80 °C for protein analysis. The other part was stored either in 10% formalin or at −80 °C after the addition of Tissue-Tek OCT compound (Sakura Co. Ltd., Tokyo, Japan) for histopathological and immunohistochemical analysis. Protein analysis Protein lysate was prepared from heart tissue as described previously [17]. The total protein concentration in samples was measured by the bicinchoninic acid method. For Western blotting experiments, 30 μg of total protein was loaded and proteins were separated by SDS–PAGE (200 V for 40 min) and electrophoretically transferred to nitrocellulose filters (semidry transfer at 10 V for 30 min). Filters were blocked with 5% nonfat dry milk in Tris-buffered saline (20 mM Tris, pH 7.6, 137 mM NaCl) with 0.1% Tween 20, washed, and then incubated with primary antibody. Primary antibody included rabbit polyclonal p38 MAPK; phosho-p38 MAPK; mitogen-activated protein kinase-activated protein

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kinase 2 (MAPKAPK-2); phospho-MAPKAPK-2, a downstream effector of p38 MAPK; phospho-p53 and Bcl-XL (Cell Signaling Technology, Danvers, MA, USA); rabbit polyclonal p67-phox and p22-phox; goat polyclonal tumor necrosis factor-α (TNF-α); interleukin-1β (IL-1β); Nox4 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Santa Cruz Biotechnology, Santa Cruz, CA, USA); and mouse polyclonal p53 (Calbiochem, San Diego, CA, USA). After incubation with the primary antibody, the bound antibody was visualized with horseradish peroxidase-coupled secondary antibodies (Santa Cruz Biotechnology) and chemiluminescence developing agents (Amersham Biosciences, Buckinghamshire, UK). The level of expression of each protein in control WT mice was taken as 1 arbitrary unit. For Western blotting analysis, all primary and secondary antibodies were used at a dilution of 1:1000 and 1:5000, respectively. Films were scanned and band densities were quantified by densitometric analysis using Scion image software (Epson GT-X700; Tokyo, Japan). Cytochrome c reduction assay NADPH-dependent superoxide production was examined using superoxide dismutase (SOD)-inhibitable cytochrome c reduction [18]. Total protein from myocardial tissue (final concentration 1 mg/ml) was distributed in 96-well plates (final volume 200 μl/well). Cytochrome c (500 μmol/L) and NADPH (100 μmol/L) were added in the presence or absence of SOD (200 U/ml) and incubated at room temperature for 30 min. Cytochrome c reduction was measured by reading the absorbance at 550-nm wavelength on a microplate reader. Superoxide production was calculated from the difference between absorbance with and without SOD and extinction coefficient for the change of ferricytochrome c to ferrocytochrome c, i.e., 21.0 mM L− 1 cm− 1. Electron spin resonance (ESR) spectroscopy The formation of hydroxyl radical (•OH) was detected with 5,5′dimethyl-1-pyrroline-1-oxide (DMPO) as a spin trap [19,20]. The hearts were homogenized in cold phosphate-buffered saline (PBS) (100 mg hearts/ml), incubated with 200 μmol/L Dox (WT mice, n = 5; TG mice, n = 6) or without (WT mice and TG mice, n = 3 each) for 10 min, and then added to 0.05 ml of 9.0 M DMPO. The generation of hydroxyl radicals was observed as DMPO–•OH adducts on a (Jeol JES-TE 200) spectrometer. Quantification of the DMPO signal intensity was performed by comparing the observed signal to a standard Mn2+ marker; the hydroxyl radical signal relative to the internal standard of manganese ion was calculated. The spectrophotometer settings were as follows: microwave frequency, 9.43 GHz; microwave power, 8.0 mW; modulation amplitude, 0.1 mT; time constant, 0.03 s; sweep time, 30 s; and center fields, 342/332 mT. In situ detection of superoxide production in hearts To evaluate in situ superoxide production from hearts, unfixed frozen cross sections of the specimens were stained with dihydroethidium (DHE; Molecular Probes, Eugene, OR, USA) according to the previously validated method [21–23]. In the presence of superoxide, DHE is converted to the fluorescent molecule ethidium, which can then label nuclei by intercalating with DNA. Briefly, the unfixed frozen tissues were cut into 10-μm-thick sections and incubated with 10 μM DHE at 37 °C for 30 min in a light-protected humidified chamber. Fluorescence images were obtained using a fluorescence microscope equipped with a rhodamine filter. Single-cell gel electrophoresis assay Oxidative stress, radiation, and anthracyclines cause DNA damage that can be detected and quantified using the alkaline single-cell gel electrophoresis assay (comet assay [24]) During electrophoresis,

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undamaged DNA is largely confined to the nucleus, whereas damaged DNA migrates apart from the nucleus in the shape of a comet. The length and fluorescence intensity of the comet are proportional to the number of DNA strand breaks. The extent of DNA damage can be quantified by the use of this sensitive technique, with the most frequently reported measure being the tail length and tail moment, a product of tail length and percentage tail DNA [24]. Cardiomyocytes were isolated as described previously [25]. We performed the assay essentially as described by Chaubey et al. [24]. Cardiomyocytes were mixed with 0.8% low-melting agarose at 38 °C and spread on fully frosted slides. After solidification, slides were immersed in lysis buffer (2.5 M NaCl, 100 mM Na2-EDTA, 1% Triton X-100, and 10% dimethyl sulfoxide) for 1 h at 4 °C and electrophoresed in alkaline buffer (300 mM NaOH, 1 mM Na2-EDTA, pH 13) using 25 V, 400 mA for 30 min. After electrophoresis, slides were stained with SYBR Green II dye and observed under a fluorescence microscope at 200× magnification. Images of at least 50 cells per slide were captured (CIA-102; Olympus, Tokyo, Japan). The digital imaging Casp software (http://casp.sourceforge.net/) was used to measure the indexes of DNA damage. Tail length and tail moment were selected as the parameters to quantify DNA damage. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay (TUNEL) Frozen LV tissues embedded in OCT compound were cut into 4-μmthick sections and fixed in 4% paraformaldehyde (pH 7.4) at room temperature. TUNEL apoptosis analysis was performed as specified in the in situ apoptosis detection kit (Takara Bio, Shiga, Japan) and sections were examined under fluorescence microscopy at 200× magnification (CIA-102; Olympus). For each animal, five sections were scored for apoptotic nuclei. For each slide 10 fields were randomly chosen, and by using a defined rectangular field area, a total of 100 cells per field were counted. The percentage of total myocytes that were TUNEL positive (apoptotic index) was then calculated. This evaluation was performed by one person who was blinded to treatment group. Immunofluorescence For immunofluorescence, tissues were fixed in 10% buffered formaldehyde solution and embedded in paraffin. Sections underwent microwave antigen retrieval, were blocked with 10% goat serum in PBS and were incubated with polyclonal rabbit anti-caspase-3 antibody (Cell Signaling Technology). Binding sites of the primary antibody were revealed with fluorescein isothiocyanate-conjugated secondary antibody (Sigma–Aldrich, St. Louis, MO, USA). Samples were visualized with a fluorescence microscope at 400× magnification (CIA-102; Olympus) [18]. Statistical analysis Data are represented as means ± standard error (SE). Statistical analysis among groups was determined by t test or by one-way analysis of variance followed by Tukey's method. Differences were considered statistically significant at P b 0.05. Results

Myocardial expression of phospho-p38 MAPK and phospho-MAPKAPK-2 Mice with cardiac-specific expression of DN p38α MAPK have reduced p38α MAPK activity in the heart compared to the WT mice (Fig. 2A and B). Immunoblot analysis revealed that LV expression of phospho-p38 MAPK and phospho-MAPKAPK-2, a well-described substrate of p38 MAPK, was significantly increased in WT and TG mice at 5 days after Dox injection compared to their respective control mice (Fig. 2). However, TG mice had significantly less p38 MAPK and MAPKAPK-2 activation compared to WT mice (Fig. 2). Echocardiographic assessment of ventricular remodeling Intact chamber remodeling analysis by echocardiography revealed that cardiac dimensions did not differ between control TG and WT mice. The LVDd and LVDs were markedly increased in WT mice after Dox injection, as shown in Fig. 3. However, in TG mice, LV dilatation after Dox injection was significantly inhibited, and LVDd and LVDs were significantly smaller compared to WT mice 5 days after Dox injection (Figs. 3A and B). Dox caused a reduction in FS and EF in both WT and TG mice. However, the decreases in FS and EF were significantly less in TG mice compared with WT mice, as shown in Figs. 3C and D. Heart rate after Dox was similar between WT and TG mice (483± 6 vs 471 ±8 bpm). After echocardiography, mice were killed with a lethal ip injection of sodium pentobarbital (80 mg/kg), and LV weights after Dox were measured for WT and TG mice. Although body weight was the same, LV weight after Dox administration was less in TG mice than in WT mice (83 ±2 vs 95 ±6 mg, P b 0.01). DNA damage, TUNEL analysis, and caspase-3 activation Cardiomyocyte DNA damage was evaluated by performing the alkaline comet assay. The DNA damage measured in terms of tail moment and tail length was significantly increased in WT and TG mice at 5 days after Dox injection relative to their genetic controls (Figs. 4A–D, 5A, and B). However, the DNA damage was significantly less in TG mice relative to WT mice after Dox injection (Figs. 4B, D, 5A, and B). The number of TUNEL-positive cells in LV sections did not differ between control TG and WT mice (Fig. 5C), and expectedly, the number of TUNELpositive cells was markedly increased in LV sections of WT and TG mice after Dox injection relative to control mice (Fig. 5C). Interestingly, the number of TUNEL-positive cells after Dox injection was much less in TG WT-Control

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Survival of TG-Dox and WT-Dox mice was analyzed by the Kaplan– Meier approach (Fig. 1). Ten days after doxorubicin treatment, survival was significantly lower in WT-Dox compared with TG-Dox mice (35% vs 70%). The survival was also significantly lower in doxorubicin-treated mice compared with untreated controls (WT-Dox vs WT, 35% vs 100%, P b 0.01; TG-Dox vs TG, 70% vs 100%, P b 0.001).

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Days Fig. 1. Decreased survival rate in WT-Dox and TG-Dox mice, 10 days after Dox injection. Kaplan–Meier survival curves for Dox-treated and vehicle-treated WT and TG mice. *P b 0.05, **P b 0.01 vs WT-control mice; #P b 0.05, ##P b 0.01 vs TG-control mice; $$P b 0.01 vs WT-Dox mice on the same day.

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Fig. 2. Reduced p38 MAPK activity in TG mice at 5 days after Dox injection. Representative Western immunoblots and densitometry analysis using Scion image software for (A and B) p-p38 MAPK and (C and D) p-MAPKAPK-2 levels in controls (−) and 5 days after Dox injection (+). Blots were normalized against p38 MAPK and MAPKAPK-2. Each bar represents the mean ± SE (n = 4 or 5). **P b 0.01 vs WT-control mice; #P b 0.05, ##P b 0.01 vs TG-control mice; $P b 0.05 vs WT-Dox mice; $$P b 0.01 vs WT-Dox mice.

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Fig. 3. Echocardiographic characterization of left ventricular function 5 days after Dox injection. (A) LVDd and (B) LVDs are left ventricular end-diastolic and systolic dimensions, respectively. (C) %FS. (D) %EF. Each bar represents the mean ± SE (n = 8 to 10). *P b 0.05 vs WT-control mice; **P b 0.01 vs WT-control mice; #P b 0.05 vs TG-control mice; ##P b 0.01 vs TG-control mice; $P b 0.05 vs WT-Dox mice on the same day.

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Fig. 4. Representative photomicrographs of DNA damage and superoxide production 5 days after Dox injection. (A–D) DNA damage detected by single-cell gel electrophoresis assay (comet assay) in cardiomyocytes of (A) WT-control and (B) WT-Dox mice, as well as (C) TG-control and (D) TG-Dox, at 200× original magnification. (E–H) In situ superoxide production (bright area) using DHE-staining in hearts of (E) WT-control and (F) WT-Dox mice, as well as (G) TG-control and (H) TG-Dox mice, at 200× original magnification.

mice than in WT mice (Fig. 5C). As shown in Fig. 5D, WT and TG mice displayed significantly higher caspase-3-positive cells compared to control TG and WT mice at 5 days after Dox injection. But, the number of caspase-3-positive cells after Dox injection was significantly less in TG mice relative to WT mice (Fig. 5D).

mice compared to their genetic controls. However, these increases in TNF-α and IL-1β production after Dox treatment were significantly less in TG mice compared to WT mice (Figs. 6C–E).

Myocardial expression of phospho-p53 and Bcl-XL

ESR spectroscopic analyses showed that the formation of hydroxyl radical signals in heart homogenates of Dox TG mice was lower compared with Dox WT mice heart homogenates (Figs. 7A and B). The hydroxyl radical signals relative to the internal standard of manganese ion in Dox TG mice were lower compared with Dox WT mice (Figs. 7A and B). No signals were detected in the respective genetic control animals.

Immunoblot analysis revealed that LV expression of phospho-p53 was significantly increased in WT and TG mice at 5 days after Dox injection compared to their respective control mice (Figs. 6A and B). However, TG mice had significantly less phosphorylation on p53 compared to WT mice (Figs. 6A and B). Furthermore, there was no significant difference in the level of Bcl-XL expression in WT and TG mice at 5 days after Dox injection compared to their respective genetic controls (data not shown). Cytokine production in myocardium We examined proinflammatory cytokine production in myocardial tissue after Dox injection (Figs. 6C–E). Protein expression of TNF-α and IL-1β was markedly elevated after Dox treatment in WT and TG

ESR spectrometric analyses

Dox injection induces ROS and oxidative stress Dox cardiomyopathy has been reported to be associated with enhanced ROS generation and oxidative stress [26]. We used the fluorescent probe DHE, which has been used to detect intracellular superoxide formation [21–23]. Fig. 4F shows the intracellular red fluorescence due to the intercalation of ethidium into DNA in the heart of WT mice 5 days after Dox injection compared to their respective

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Fig. 5. Quantification of cardiomyocyte DNA damage, apoptosis, and caspase-3 expression 5 days after Dox injection. (A and B) DNA damage parameters (tail length and tail moment) analyzed by digital imaging Casp software (http://casp.sourceforge.net/). (C and D) Cardiomyocyte apoptosis and caspase-3-positive cells in control and Dox myocardium. Each bar represents the mean ± SE (n = 4 or 5). *P b 0.05, **P b 0.01 vs WT-control mice; #P b 0.05, ##P b 0.01 vs TG-control mice; $P b 0.05 vs WT-Dox mice; $$P b 0.01 vs WT-Dox mice.

genetic controls. Dox-induced enhancement of ethidium fluorescence was inhibited in TG mice compared to WT mice (Figs. 4F and H), indicating an overall reduced oxidative stress. Furthermore, NADPHdependent O− 2 production by LV homogenates assessed by cytochrome c reduction was significantly increased in the hearts of WT mice 5 days after Dox injection compared to their respective genetic controls (Fig. 7C). Interestingly, the ROS production was significantly lower in the hearts of TG mice compared to WT mice 5 days after Dox injection (Fig. 7C). Because NADPH oxidase is the main source of ROS in the cardiovascular tissues [27], we next measured the protein expression of p22-phox, p67-phox, and Nox4 in the hearts of control and Dox animals. Myocardial expression of p22-phox was significantly elevated in WT and TG mice at 5 days after Dox injection compared to their respective genetic controls (Figs. 8A and B). However, the increase was significantly less in TG mice compared to WT mice. In addition, the levels of expression of p67-phox and Nox4 were significantly increased in WT mice after Dox injection but not in TG mice relative to their genetic controls (Figs. 8A, C, and D). These data suggest that the increased cardiac lipid peroxidation level and NADPH oxidase subunits may contribute to the increased ROS, which caused the cardiac damage in Dox mice. Discussion In this study, TG mice showed less p53 phosphorylation; lower production of proinflammatory cytokines, ROS, and oxidative stress; less cardiomyocyte DNA damage; fewer TUNEL-positive nuclei in the myocardium; and fewer caspase-3-positive cells after Dox injection than WT mice. Consequently, cardiac function was preserved and survival rate was higher in TG mice compared with WT mice. This study provides direct evidence for the involvement of p38α MAPK-mediated signaling pathways in Dox-induced cardiotoxicity. Dox is one of the most important anticancer agents. However, clinical use of Dox is limited by its cardiotoxicity. In experimental

studies, electron microscopy revealed extensive cardiac damage characterized by mitochondrial degeneration and swelling, intracytoplasmic vacuolization, and focal myofilament disarray, although histopathologic changes by light microscopy were not observed at 5 days after 10 to 25 mg/kg Dox injection [28]. Olson et al. [29] have also shown that mice treated with 20 mg/kg Dox developed cardiac failure. Although the precise mechanisms whereby Dox induces myocardial injury have not been fully documented, it is widely accepted that the cardiac toxicity of Dox is mediated by ROS [30,31]. On the other hand, a previous study suggested that ROS generated from the mitochondria during ischemia and reperfusion activate p38α MAPK, and inhibition of p38α significantly prevented cell death arising from ischemia reperfusion [32]. Recently, we have reported that p38α MAPK is involved in the oxidative stress-induced activation of cardiomyocyte apoptosis [18]. On the basis of these studies, we injected a single dose of Dox (20 mg/kg) and focused our attention on changes in cardiac function, cytokine production, ROS production, oxidative stress, DNA damage, and apoptosis 5 days after Dox treatment in this study. Administration of a single dose of Dox to mice has been validated extensively in the study of Dox-induced cardiac dysfunction in vivo [33,34]. In agreement with others, WT-DOX mice were found to display severe systolic and diastolic LV dysfunction, resulting in impaired cardiac dysfunction [34]. In TG-Dox mice the LV function was improved relative to WT-Dox mice as a result of enhanced systolic and diastolic performance measured by echocardiography. Thus, we conclude that p38α MAPK plays a pivotal role in LV dysfunction due to Dox-induced cardiomyopathy. To further analyze the mechanisms involved, we characterized cardiac oxidative stress, inflammatory response, DNA damage, and apoptosis, which are all known to be relevant in this disease. In this study, survival rates 10 days after 20 mg/kg Dox injection were 35% in WT mice and 70% in TG mice. The survival rate in WT mice after 20 mg/kg Dox is consistent with that found in previous studies, which used the same dose of Dox [35,36]. Although we did not

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Fig. 6. LV p53 and cytokine expression 5 days after Dox injection. Representative Western immunoblots and densitometry analysis using Scion image software for (A, B) p-p53 normalized against p53, (C, D) TNF-α, and (C, E) IL-1β, normalized against GAPDH. Each bar represents the mean ± SE (n = 4 or 5). *P b 0.05, **P b 0.01 vs WT-control mice; #P b 0.05, ## P b 0.01 vs TG-control mice; $P b 0.05 vs WT-Dox mice.

investigate pathologic changes 10 days after Dox treatment, recent studies have demonstrated that surviving mice show extensive necrosis, and mineralization of cardiomyocytes combined with a mild degree of cardiomyocyte vacuolation has been seen in mouse hearts 18 days after 20 mg/kg Dox treatment [29,35]. Our observations suggest that significant pathophysiologic changes in WT mice 5 days after Dox injection may contribute to the 10-day survival rate. The p38 MAPK family is activated by physical and chemical stress factors, resulting in growth promotion, apoptosis, oxidative stress, and vasoconstriction [7,8,37,38]. p38 MAPK is present as four isoforms, α, β, γ, and δ [10]. p38α MAPK is the major isoform and highly detectible in human heart [9]. Though previous studies [12,13] have indicated a role for p38 MAPK in Dox-induced cardiomyopathy, the specific role of p38α MAPK is not known. Previous study has demonstrated that Dox activates p38 MAPK in cultured cardiomyocytes [13]. Consistent with these findings, we have also found that activation of p38 MAPK and its downstream effector MAPKAPK-2 was observed in hearts of WT mice after Dox injection, and this activation was suppressed in TG mice. Doxinduced cardiomyopathy is associated with increased levels of the proinflammatory cytokines TNF-α, IL-1β, and IL-6 [34]. This cardiac

inflammation is linked with decreased LV function, not only in Dox cardiomyopathy, but also in diabetic cardiomyopathy, pressure overload, and dilated cardiomyopathy [39,40]. p38 MAPK is a key regulatory pathway for many genes, including genes regulating TNF-α, IL-1β, and TGF-β [41]. In our study, cytokine production was increased in WT mice 5 days after Dox injection, and these increases in cytokine production in response to Dox were suppressed in TG mice. These results demonstrate that p38α MAPK plays an important role in cardiac inflammation and dysfunction after Dox injection and the precise mechanism remains to be determined. Oxidative stress and inflammation might be associated with an induction of DNA damage and apoptosis, as is also known for Doxinduced cardiomyopathy [4,26]. Recently, we reported that p38α MAPK is associated with the onset of apoptosis, ROS production, and cardiac remodeling in diabetic mouse hearts [18]. ROS generated from the mitochondria during ischemia and reperfusion activate p38α MAPK, and inhibition of p38α significantly prevented cell death arising from ischemia–reperfusion [32]. DNA damage plays an important early role in anthracycline-induced lethal cardiac myocyte injury through a p53 pathway [42]. p38 MAPK has been reported to phosphorylate p53 in

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Fig. 7. 5 days after Dox injection the myocardial ROS and oxidative stress are elevated. (A, B) Representative ESR spectra and analysis of the hydroxyl radical signal relative to the internal standard of manganese ion. Hydroxyl radical signals were not detected (ND) in the hearts of control animals. Mn (3) and Mn (4) indicate the internal standard signals of manganese ion (Mn2+). (C) Superoxide production by LV homogenates of control and Dox animals. Each bar represents the mean ± SE (n = 4 or 5). **P b 0.01 vs WT-control mice; # P b 0.05, ##P b 0.01 vs TG-control mice; $P b 0.05 vs WT-Dox mice; $$P b 0.01 vs WT-Dox mice.

various models [43,44]. Previous studies have implicated the p53 pathway in Dox-induced cardiotoxicity. For example, reduced levels of cardiomyocyte apoptosis and concomitant improvements in cardiac function were observed in Dox-treated p53 null mice compared with their wild-type littermates [45]. We observed less DNA damage and a marked reduction in cardiomyocyte apoptosis and caspase-3-positive cells in TG mice after Dox injection. The reduction in apoptosis and DNA damage observed in TG cardiac tissue may be a consequence of reduced phosphorylation of p53. The precise mechanism by which p38α MAPK modulates p53 phosphorylation remains to be determined. Moreover, Toko et al. reported that Dox-induced myocyte death is angiotensin (Ang) II dependent [46], and we have also observed marked elevation in Ang II levels (data not shown), with significant cardiac apoptosis after Dox injection and p38 MAPK phosphorylation (activation). It is therefore possible that DNA damage and cardiac apoptosis associated with Dox cardiomyopathy may also be mediated by the Ang II–p38 MAPK–p53 axis. Dox-induced cardiac cell apoptosis has been attributed to the production of ROS [47–49]. Kalyanaraman and colleagues have reported that scavenging of ROS protects against Dox-induced cardiac apoptosis [48,50]. Similarly, cardiac-specific overexpression of antioxidant genes protected mice from Dox-induced cardiac dysfunction [51]. NADPH oxidase is a multicomponent enzyme and the major source of oxidative stress in various diseases. Upon stimulation, the cytosolic complex

migrates and assembles with the membrane subunits to form an active oxidase capable of producing superoxide anion [52]. Recent studies in human endothelial cells demonstrated that p38 MAPK activates NADPH oxidase by enhancing phosphorylation and assembly of NADPH oxidase subunits [53], and the activation of NADPH oxidase is suppressed by p38 MAPK inhibitors [54,55]. Recently, we have reported that NADPH oxidase is enhanced in diabetic heart and it is inhibited by DN p38α MAPK [18]. In this study, we observed that the myocardial expression of p22-phox, p67-phox, and Nox4 was significantly suppressed in TG mice relative to WT mice at 5 days after Dox injection. Moreover, WT mice had a higher level of ROS content after Dox injection relative to TG mice. In these regards, here, it is noteworthy that the p38 MAPK pathway may regulate NADPH oxidase activation as well as ROS production in Doxinduced cardiomyopathy and the precise mechanism remains to be determined. In this study, cardiac dysfunction, the expression of proinflammatory cytokines, and the apoptosis that were observed after Dox injection were affected by interaction between oxidative stress and the p38α MAPK pathway. Therefore, these results provide a new insight into Dox-induced cardiomyopathy in the clinical setting. Taken together our results suggest that inhibition of p38α MAPK may represent a useful therapeutic target to ameliorate cardiac dysfunction, oxidative stress, DNA damage, apoptotic cell death, and cardiac inflammation associated with Dox-induced cardiomyopathy. Our data support the pharmacological inhibition of p38 MAPK as a new

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Fig. 8. 5 days after Dox injection the NADPH oxidase subunits are elevated. (A–D) Representative Western immunoblots and densitometry analysis using Scion image software for p22-phox, p67-phox, and Nox4 in control and Dox mice. Blots were normalized against GAPDH. Each bar represents the mean ± SE (n = 4 or 5). *P b 0.05 vs WT-control mice; **P b 0.01 vs WT-control mice; #P b 0.05 vs TG-control mice; ##P b 0.01 vs TG-control mice; $P b 0.05 vs WT-Dox mice.

promising therapeutic strategy regarding the prevention of Dox complications. However, further studies have to prove whether these findings can be translated to human conditions. Acknowledgments This research was supported by grants from the Yujin Memorial Grant; the Ministry of Education, Culture, Sports, Science, and Technology of Japan; the Promotion and Mutual Aid Corporation for Private Schools of Japan; and from Niigata City. References [1] Ganey, P. E.; Carter, L. S.; Mueller, R. A.; Thurman, R. G. Doxorubicin toxicity in perfused rat heart: decreased cell death at low oxygen tension. Circ. Res. 68: 1610–1613; 1991. [2] Safra, T.; Muggia, F.; Jeffers, S.; Tsao-Wei, D. D.; Groshen, S.; Lyass, O., et al. Pegylated liposomal doxorubicin (doxil): reduced clinical cardiotoxicity in patients reaching or exceeding cumulative doses of 500 mg/m2. Ann. Oncol. 11: 1029–1033; 2000. [3] Kluza, J.; Marchetti, P.; Gallego, M. A.; Lancel, S.; Fournier, C.; Loyens, A., et al. Mitochondrial proliferation during apoptosis induced by anticancer agents: effects of doxorubicin and mitoxantrone on cancer and cardiac cells. Oncogene 23: 7018–7030; 2004. [4] Takemura, G.; Fujiwara, H. Doxorubicin-induced cardiomyopathy from the cardiotoxic mechanisms to management. Prog. Cardiovasc. Dis. 49:330–352; 2007. [5] Singal, P. K.; Iliskovic, N. Doxorubicin-induced cardiomyopathy. N Engl J. Med. 339: 900–905; 1998. [6] Li, T.; Singal, P. K. Adriamycin-induced early changes in myocardial antioxidant enzymes and their modulation by probucol. Circulation 102:2105–2110; 2000. [7] Ushio-Fukai, M.; Alexander, R. W.; Akers, M.; Griendling, K. K. p38 mitogenactivated protein kinase is a critical component of the redox-sensitive signaling pathways activated by angiotensin II: role in vascular smooth muscle cell hypertrophy. J. Biol. Chem. 273:15022–15029; 1998. [8] Force, T.; Bonventre, J. V. Growth factors and mitogen-activated protein kinases. Hypertension 31:152–161; 1998.

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