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(Circulation. 2004;110:2869-2874.)
© 2004 American Heart Association, Inc.

Heart Failure

Modulation of Doxorubicin-Induced Cardiac Dysfunction in Toll-Like Receptor-2–Knockout Mice

Naoki Nozaki, MD, PhD^*; Tetsuro Shishido, MD, PhD^*; Yasuchika Takeishi, MD, PhD; Isao Kubota, MD, PhD

From the First Department of Internal Medicine, Yamagata University School of Medicine, Yamagata, Japan.

Correspondence to Yasuchika Takeishi, MD, PhD, First Department of Internal Medicine, Yamagata University School of Medicine, 2-2-2 Iida-Nishi, Yamagata, Japan 990-9585. E-mail takeishi{at}med.id.yamagata-u.ac.jp

Received September 19, 2003; de novo received February 14, 2004; revision received June 4, 2004; accepted June 7, 2004.

	Abstract

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Background— Toll-like receptors (TLRs) are members of the interleukin-1 receptor family and are involved in the responsiveness to pathogen-associated molecular patterns. Recent studies have demonstrated that TLRs are activated by endogenous signals, such as heat shock proteins and oxidative stress, which may contribute to congestive heart failure. Oxidative stress is one of the major factors in doxorubicin (Dox)-induced cardiac dysfunction. Thus, we hypothesized that TLRs contribute to the pathogenesis of Dox-induced cardiac dysfunction.

Methods and Results— Cardiac dysfunction was induced by a single injection of Dox (20 mg/kg IP) into wild-type (WT) mice and TLR-2–knockout (KO) mice. Five days after Dox injection, left ventricular dimension at end-diastole was smaller and fractional shortening was higher in KO mice compared with WT mice (P<0.01). Nuclear factor-B activation and production of proinflammatory cytokines after Dox were suppressed in KO mice compared with WT mice (P<0.01). The numbers of TUNEL-positive nuclei and Dox-induced caspase-3 activation were less in KO mice than in WT mice (P<0.01). Survival rate was significantly higher in KO mice than in WT mice 10 days after Dox injection (46% vs 11%, P<0.05).

Conclusions— These findings suggest that TLR-2 may play a role in the regulation of inflammatory and apoptotic mediators in the heart after Dox administration.

Key Words: apoptosis • cardiomyopathy • receptors • interleukins • fee radicals

	Introduction

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Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns such as lipopolysaccharide, peptidoglycan, bacterial lipoprotein, and oligonucleotides during the inflammatory response.^1,2 TLRs have interleukin-1 (IL-1) receptor–like intracellular signaling pathways that lead to nuclear localization of nuclear factor (NF)-B/Rel-type transcription factors.^1–3 Furthermore, TLRs are expressed in various organs such as the lung, brain, kidney, and heart.^2,4 Recent studies have suggested that myocardially activated, TLR-mediated signaling pathways in response to exogenous ligands induce cardiac dysfunction.^5–7 Other studies have also demonstrated that TLR-mediated signaling pathways are activated by endogenous signals such as heat shock protein and oxidative stress in isolated ventricular cardiomyocytes.^8,9 We have recently demonstrated that TLR-2 plays an important role in ventricular remodeling after myocardial infarction.¹⁰

Doxorubicin (Dox) is an effective antitumor anthracycline antibiotic. However, Dox also induces cardiomyopathy that leads to congestive heart failure,^11,12 thereby limiting its clinical use. Dox-induced cardiomyopathy is mainly caused by increased oxidant production in the heart.^13,14 A previous study has also reported that Dox treatment causes cytochrome c release and results in caspase-3 activation and apoptosis.¹⁵ Furthermore, several studies have demonstrated that Dox-induced cardiotoxicity can largely be reduced by the overexpression of the antioxidant enzymes manganese superoxide dismutase and catalase.^16,17 These studies indicate that free radicals play an important role in Dox-induced cardiotoxicity.

Because oxidative stress after Dox administration was identified to play a pivotal role in cardiac dysfunction, we hypothesized that TLR-2 contributed to the pathogenesis of Dox-induced cardiac dysfunction. To test this hypothesis, we examined cardiac function, histologic aspects, cytokine production, lipid peroxidation, and survival in TLR-2–knockout (KO) mice after Dox injection.

	Methods

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Animals and Treatments
The TLR-2–KO mice were kindly supplied by Drs Shizuo Akira and Osamu Takeuchi (Osaka University, Osaka, Japan).¹⁸ Male KO mice and normal wild-type (WT) mice of the same C57BL/6 background were used. Mice were housed in a facility with a 12-hour/12-hour light/dark cycle and were given free access to water and standard rodent chow. The room was kept specific pathogen-free. The animals were handled according to the animal welfare regulations of Yamagata University, and the study protocol was approved by the Animal Subjects Committee of Yamagata University. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. Both WT and KO mice were randomly assigned to either the control group or the Dox-treated group. Dox HCl (Sigma Chemical Co) was dissolved in saline and administered by intraperitoneal injection at a dose of 20 mg/kg.^15–17,19 Control mice received injections of saline of comparable volume.

Two-Dimensional Echocardiography
Mice were anesthetized intraperitoneally with 2.5% tribromoethanol, and additional doses were given as needed. We performed transthoracic echocardiography 5 days after Dox or vehicle injection with use of an FFsonic 8900 (Fukudadenshi) with a 13-MHz, phased-array transducer. Left ventricular internal dimensions at end-diastole (LVEDD) and at end-systole (LVESD) were measured digitally on the M-mode tracings and were averaged from 3 cardiac cycles.^7,10 LV fractional shortening (%FS) was calculated as [(LVEDD–LVESD)/LVEDD]x100.

Morphological Examination and TUNEL Analysis
At 5 days after Dox injection, the heart was excised and fixed with a 10% solution of formalin in PBS. The heart was embedded in paraffin and serially cut from the apex to the base. Sections were stained with either hematoxylin and eosin or Azan. Sections were also used for a TdT-mediated dUTP nick end-labeling (TUNEL) procedure for detecting apoptotic cardiomyocytes, performed with a CardioTACS kit (R&D Systems) according to the manufacturer’s instructions. The percentage of TUNEL-positive myocytes was determined by counting 10 random fields per section under a microscope (BX50, Olympus).

Proinflammatory Cytokine Production
Five days after injection, hearts from WT and KO mice were excised, rinsed in PBS, frozen in LN₂, and stored at –70°C. Protein extraction was performed as described elsewhere.^20,21 Myocardial proinflammatory cytokine levels were measured by a commercially available ELISA (R&D Systems) for murine tumor necrosis factor (TNF)-, IL-6, and IL-1ß according to the manufacturer’s instructions.

Lipid Peroxidation
Lipid peroxidation in myocardial tissues was measured with a commercially available colorimetric assay kit for malondialdehyde (MDA; LPO-586, Bioxytech, Oxis).¹⁷ Protein extracts from myocardial tissues were used for this assay. The measurement of MDA was used as an indicator of lipid peroxidation.

Caspase-3 Activity
Caspase-3 activity in myocardial tissues was measured with a CPP32/caspase-3 colorimetric protease assay that recognizes the sequence DEVD. The assay was from the labeled substrate DEVD-pNA (CPP32/caspase-3 colorimetric protease kit, MBL).

DNA-p65 NF-B Binding Assay and EMSA
Nuclear protein extracts were prepared as described previously^6,9 with minor modifications. DNA-p65 NF-B binding activity was measured with a BD Mercury TransFactor kit (BD Biosciences, Clontech), which detects DNA binding by specific transcription factors.²² Electrophoretic mobility shift assay (EMSA) was performed by a nonradioactive method (Pierce) according to the manufacturer’s instructions. The oligonucleotides where the 5' end of the probe was labeled with biotin contained the NF-B binding site (5'-AGT TGA GGG GAC TTT CCC AGC C-3').

Extraction of Total RNAs and RT-PCR
RNA extraction and the reverse transcriptase–polymerase chain reaction (RT-PCR) study were performed as described previously.¹⁰ PCR primers for Bax were 5'-CCAGCTCTGAACAGATCATG-3' (forward) and 5'-AGCTCCATATTGCTATCCAG-3' (reverse); for Bcl-2 were 5'-CCAGCTCTGAACAGATCATG-3' (forward) and 5'-CCAAACATCCAGAGACAA-3' (reverse); for Bcl-xl were 5'-AGACCCCCAGTGCCATCAAT-3' (forward) and 5'-CCCGCCAAAGGAGAAAAA-3' (reverse); for p53 were 5'-TCTGGGACAGCCAAGTCTGT-3' (forward) and 5'-CAGCATCTTATCCGAGTGGA-3' (reverse); and for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were 5'-ACTCCACTCACGGCAAATTCAACGG-3' (forward) and 5'-AGGGGCGGAGATGATGACCC-3' (reverse).^10,23 The PCR products were fractionated on a 1% to 2% agarose gel and visualized by ethidium bromide staining. The intensities of the bands were normalized for GAPDH and were expressed as fold increase over control WT.

Statistics Analysis
All values are expressed as mean±SE. To compare echocardiographic data, lipid peroxidation, nuclear translocation of p65, cytokine production, percentage of apoptotic nuclei, and caspase-3 activity, 1-way ANOVA followed by a post hoc procedure was performed. Survival curves after Dox injection were created by the Kaplan-Meier method and compared by a log-lank test. Statistical significance was accepted at a value of P<0.05.

	Results

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Effects of Dox Administration on Cardiac Function
Figure 1 shows representative echocardiograms after vehicle or Dox administration in WT and KO mice. The LVEDD (P<0.01) and LVESD (P<0.01) were markedly increased in WT mice after Dox injection, as shown in Figures 1 and 2. However, in TLR-2–KO mice, LV dilatation after Dox was prevented, and LVEDD (P<0.01) and LVESD (P<0.01) after Dox were significantly smaller than in WT mice (Figure 2A and 2B). Dox caused a reduction of LVFS in both WT and KO mice (P<0.01). However, the decrease in LVFS was significantly less in KO mice (P<0.01) compared with WT mice, as shown in Figure 2C. Heart rate after Dox was similar between WT and KO mice (473±7 vs 470±6 bpm).

View larger version (94K): [in this window] [in a new window]   Figure 1. Representative M-mode echocardiograms of WT and KO mice given vehicle or Dox. Abbreviations are as defined in text.

View larger version (17K): [in this window] [in a new window]   Figure 2. Quantitative group data for echocardiographic measurements: A, LVEDD; B, LVESD; and C, %FS. Data were obtained from 10 mice in each group. WT-Con and KO-Con indicate WT and KO mice given vehicle, respectively. WT-Dox and KO-Dox indicate WT and KO mice treated with Dox, respectively. *P<0.05 and **P<0.01 compared with WT-Con. Abbreviations are as defined in text.

After echocardiography, mice were killed with a lethal injection of sodium pentobarbital (80 mg/kg) intraperitoneally, and LV and lung weights after Dox were measured in WT and KO mice. Although body weight was the same (27.4±0.7 vs 27.4±0.5 g), LV weight (80±2 vs 92±6 mg, P<0.01) and lung weight (147±4 vs 181±8 mg, P<0.01) after Dox administration were less in KO mice than in WT mice.

Lipid Peroxidation and p65 of NF-B Activity in Myocardium
The levels of the lipid peroxidation product MDA in myocardium were markedly elevated by Dox treatment in both WT and KO mice (P<0.01), but there was no significant difference between WT and KO mice (Figure 3A).

View larger version (31K): [in this window] [in a new window]   Figure 3. Dox-induced lipid peroxidation and NF-B activation. A, MDA levels were measured to estimate extent of lipid peroxidation in tissue. B, EMSA was performed with nuclear extracts from mouse hearts to examine NF-B activity in response to Dox: lane 1, WT control; lane 2, KO control; lane 3, WT Dox; lane 4, KO Dox; and lane 5, WT Dox+cold probe. Nuclear protein extracted from WT mice 1 hour after Dox injection (lane 3) showed strong binding activity for NF-B oligonucleotide probe compared with that from KO mice (lane 4). Binding was specifically inhibited by excess unlabeled NF-B oligonucleotide (lane 5). Experiment was done in triplicate. C, DNA binding activity of NF-B p65 after Dox treatment was suppressed in KO mice compared with WT mice. Data were obtained by DNA-p65 binding assay from 5 animals for each group. Abbreviations are as defined in text and legend to Figure 2. *P<0.05 compared with WT-Con.

We then examined NF-B activity in the mouse hearts after Dox treatment. We performed EMSA with specific oligonucleotide probes for the NF-B binding-site regions, as shown in Figure 3B. Nuclear protein extracted from Dox-stimulated WT mouse hearts (lane 3) showed strong binding activity for the NF-B oligonucleotide probe compared with that from Dox-treated KO mice (lane 4). This binding was specifically inhibited by an excess of unlabeled NF-B oligonucleotide (lane 5). As shown in Figure 3C, DNA binding activity of NF-B p65 was increased and peaked 1 hour after Dox in WT mice. However in TLR-2–KO mice, Dox did not induce significant NF-B activation, and p65-DNA binding activity was suppressed in KO mice compared with that in WT mice 1 hour after Dox injection (P<0.01).

Cytokine Production in Myocardium
We examined proinflammatory cytokine production in myocardial tissue after Dox injection (Figure 4). Protein expressions of TNF- and IL-6 were markedly elevated after Dox treatment (P<0.01) in WT mice. However, these increases in TNF- and IL-6 production after Dox treatment were suppressed in KO mice compared with WT mice (P<0.01). The level of IL-1ß was significantly elevated after Dox in WT mice (P<0.05) but not in KO mice.

View larger version (19K): [in this window] [in a new window]   Figure 4. Production of TNF-, IL-6, and IL-1ß in mouse hearts after Dox administration. Abbreviations are as defined in text and legend to Figure 2.

Effect of Dox Administration on Myocardial Infrastructure
To determine the mechanisms of the preservation of LV function in TLR-2–KO mice, we compared the myocardial histologic features of WT and KO mice after Dox treatment. The histologic study of hearts from WT and KO mice demonstrated no remarkable change after Dox by hematoxylin and eosin or Azan staining. There was also no significant difference in inflammatory infiltrates between WT and KO mice after Dox (data not shown). Then, we performed TUNEL staining with the myocardial sections. The numbers of TUNEL-positive nuclei were markedly increased in WT mice after Dox treatment (Figure 5A). Importantly, numbers of TUNEL-positive nuclei after Dox were much less in KO mice (P<0.01) than in WT mice (Figure 5B and 5C).

View larger version (76K): [in this window] [in a new window]   Figure 5. Dox-induced myocardial apoptosis in WT and TLR-2–KO mice. A and B show representative TUNEL-stained sections from WT and KO mice, respectively, with Dox (20 mg/kg IP). C shows numbers of TUNEL-positive myocardial nuclei in WT and KO mice. D shows caspase-3 activity in myocardial tissues. Abbreviations are as defined in text and legend to Figure 2. *P<0.05 compared with WT-Con.

Dox-Induced Caspase-3 Activation and Alteration of Apoptotic Factor Genes
We compared caspase-3 activity in WT and KO mice after Dox injection. As shown in Figure 5D, caspase-3 activity was markedly increased in WT mice after Dox treatment (P<0.01). However, the activation of caspase-3 by Dox was abolished in KO mice (P<0.01 vs WT mice).

We performed RT-PCR to examine the changes in gene expression of proapoptotic factors such as Bax and p53 and antiapoptotic factors such as Bcl-2 and Bcl-xl after Dox. There was no difference in gene expression of p53 between WT and KO mice after Dox or vehicle injection (data not shown). Gene expression of Bax (WT, 2.0±0.4-fold increase over control,P<0.01 vs control; KO, 1.8±0.6-fold, P<0.01), Bcl-2 (WT, 1.9±0.4-fold, P<0.01; KO, 1.7±0.3-fold, P<0.01), and Bcl-xl (WT, 1.5±0.5-fold, P<0.05; KO, 1.8±0.3-fold, P<0.01) was significantly increased after Dox administration in both WT and TLR-2–KO mice, but the upregulation of these genes was not different between WT and KO mice. The ratio of Bax to Bcl-xl after Dox treatment in KO mice tended to be smaller than in WT mice (P=0.09).

Survival Rates
Survival rates were compared between WT and KO mice up to 10 days after Dox injection, as shown in Figure 6. As a consequence of preserved LV function in KO mice after Dox, survival was significantly higher in KO mice than in WT mice (46% vs 11%, P<0.05).

View larger version (11K): [in this window] [in a new window]   Figure 6. Survival curves after Dox injection in WT and KO mice. Survival curves were created by Kaplan-Meier method and compared by log-lank test. Percentages of surviving WT and KO mice were plotted. Abbreviations are as defined in text.

	Discussion

Top Abstract Introduction Methods Results Discussion Conclusions References

 
In the present study, TLR-2–KO mice showed less NF-B activation, lower production of proinflammatory cytokines, fewer TUNEL-positive nuclei in the myocardium, and less caspase-3 activation after Dox than did WT mice. Consequently, cardiac function was preserved and survival rate was higher in TLR-2–KO mice compared with WT mice. This study provides direct evidence for the involvement of TLR-2–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.¹⁶ Olson et al¹⁹ 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 reactive oxygen species.^13–17,21 On the other hand, a previous study suggested that TLR-4 is necessary for upregulating cytokine expression and myocardial dysfunction in the adult mammalian heart. Recently, Frantz et al⁹ have demonstrated that TLR-2 is involved in the oxidative stress–induced activation of NF-B in neonatal rat cardiomyocytes. 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 productions, and apoptosis 5 days after Dox treatment in this study.

In the present study, survival rates 10 days after 20 mg/kg Dox injection were 11% in WT mice and 46% in TLR-2–KO 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.^19,24 Although we did not 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.^19,24 Our observation suggests that significant pathophysiologic changes in WT mice 5 days after Dox injection may contribute to the 10-day survival rate.

NF-B activation by Dox was observed very early (1 hour) in WT mice, and this activation was abolished in TLR-2–KO mice. This timing of NF-B activation was consistent with that found in a previous study,⁶ although no NF-B activation was detected in WT and TLR-2–KO mice 5 days after Dox injection (data not shown). Cytokine production was increased in WT mice in the early phase (2 to 4 hours; data not shown) and 5 days after Dox injection, and these increases in cytokine production in response to Dox were suppressed in TLR-2–KO mice during the observation period. Additional studies are needed to explore the precise mechanisms for these chronological discrepancies between NF-B activation and cytokine production.

Dox is reported to produce free radicals, cause lipid peroxidation, and disrupt cell membrane functions.^13–17 In this study, cardiac dysfunction, the expression of proinflammatory cytokines, and the apoptosis that were observed after Dox injection were affected by interaction between reactive oxygen species and the TLR-2 pathway. Therefore, these results provide a new insight into Dox-induced cardiomyopathy in the clinical setting.

Several studies have demonstrated that the oxidative stress evoked by Dox activates apoptotic signaling that leads to cardiomyocyte apoptosis in isolated cardiomyocytes.²¹ A recent study has reported that TLR-2 activates the apoptotic signaling pathway via cytokine production.²⁵ In this study, there were fewer TUNEL-positive nuclei and less caspase-3 activity in TLR-KO mice than in WT mice. We also showed that activation of NF-B and production of proinflammatory cytokines were suppressed in TLR-2–KO mice. Wang et al²⁶ have recently demonstrated that NF-B activation is necessary for cardiomyocyte apoptosis evoked by Dox. Taken together, these results suggest that not only the direct effect of oxidative stress but also the inflammatory response induced by the TLR-2–mediated pathway plays a significant role in cardiomyocyte apoptosis evoked by Dox. In this study, we also examined proapoptotic and ant-apoptotic gene expression, and there were no significant differences between WT and TLR-2–KO mice.

Total dose, the single dose of Dox, administration intervals, and the observation periods after treatment in experimental studies of Dox vary widely among studies. Other routes of administration might cause a difference between proapoptotic and antiapoptotic gene expression levels. A recent study reported that Dox administration upregulated mRNA levels and protein production of Bax and p53 in ICR mice.²⁷ It is possible that a different mouse strain might display different gene expression or a different time course of gene expression.

	Conclusions

Top Abstract Introduction Methods Results Discussion Conclusions References

 
We have shown the preservation of cardiac function, increase in survival rate, and attenuation of myocardial apoptosis after Dox in TLR-2–KO mice. In conclusion, this study has demonstrated for the first time that TLR-2–mediated signaling is involved in cytokine production, apoptosis, and cardiac dysfunction after Dox treatment in vivo.

	Acknowledgments

 
This study was supported in part by grants-in-aid for scientific research (No. 14770306 to N.N. and No. 14570635 to Y.T.) from the Ministry of Education, Science, Sports and Culture, Japan, and grants from the Japan Foundation of Cardiovascular Research and the Japan Heart Foundation (to Y.T.). We thank Eiji Tsuchida, Yukiko Takahashi, and Sachi Adachi for their excellent technical assistance.

	Footnotes

 
*The first 2 authors contributed equally to this work.

	References

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