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European Journal of Heart Failure 2008 10(3):233-243; doi:10.1016/j.ejheart.2008.01.004
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© 2008 European Society of Cardiology

Toll-like receptor-4 deficiency attenuates doxorubicin-induced cardiomyopathy in mice

Alexander Riada, Sandra Bienb, Matthias Gratzb, Felicitas Eschera, Markus M. Heimesaatc, Stefan Bereswillc, Thomas Kriegd, Stephan B. Felixd, Heinz P. Schultheissa, Heyo K. Kroemerb and Carsten Tschöpea,*

a Charité – Universitätsmedizin Berlin, Germany, Campus Benjamin Franklin, Department of Cardiology and Pneumology Hindenburgdamm 30, 12200 Berlin, Germany
b University of Greifswald, Department of Pharmacology Germany
c Charité – Universitätsmedizin Berlin, Germany, Campus Benjamin Franklin, Department of Microbiology Hindenburgdamm 30, 12200 Berlin, Germany
d University of Greifswald, Department of Cardiology Germany

* Corresponding author. Tel.: +49 30 8445 2349; fax: +49 30 8445 4648. E-mail address: carsten.tschoepe{at}charite.de (C. Tschöpe).


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Background: Cardiac inflammation and generation of oxidative stress are known to contribute to doxorubicin (Dox)-induced cardiomyopathy. Toll-like receptors (TLRs) are a part of the innate immune system and are involved in cardiac stress reactions. Since TLR4 might play a relevant role in cardiac inflammatory signalling, we investigated whether or not TLR4 is involved in Dox-induced cardiotoxicity.

Methods and results: Five days after a single injection of Dox (20 mg/kg; i.p.), left ventricular pressure–volume loops were measured in wild-type and TLR4-deficient mice (TLR4–/–) Dox-treated and control mice. Analyses of possible pathophysiological mechanisms were performed in left ventricular tissue and isolated myocytes, respectively. Dox injection resulted in an impairment of left ventricular function and neurohumoral activation, indexed by increased ET-1 expression. This was further associated with an increase in cardiac oxidative stress, inflammation and apoptosis, as indicated by an up-regulation of cardiac lipid peroxidation, TNF-{alpha} expression and enhanced content of TUNEL-positive cells. In contrast, TLR4–/–Dox mice showed improved left ventricular function with reduced oxidative and inflammatory stress response including reduced cardiac apoptosis. These results were found to be associated with an increase of GATA-4 expression.

Conclusions: TLR4 deficiency improves left ventricular function and attenuates pathophysiological key mechanisms in Dox-induced cardiomyopathy.

Key Words: Experimental study • Heart failure • Left ventricular dysfunction • Inflammation • Doxorubicin

Received July 8, 2007; Revised November 19, 2007; Accepted January 7, 2008


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
The anthracycline doxorubicin (Dox) is widely used as an effective antitumour drug. Cardiotoxicity leading to congestive heart failure is the primary factor limiting the clinical use of Dox [1-3]. Conventional heart failure therapy has been shown to attenuate Dox-induced cardiotoxicity [4]. Both anti-adrenergic therapy using β-blockers and inhibition of the renin-angiotensin-system, at least at the early stage of Dox-induced cardiotoxicity, can reduce the progression of this disease [5-7]. Cardiac transplantation remains a vital option for patients with end-stage Dox-induced heart failure [8]. However, although a variety of approaches to protect the heart against Dox-induced cardiotoxicity have been attempted, treatment to prevent short and long term Dox-induced cardiac damage remains limited [9]. Currently, there is no specific therapeutic strategy against this severe disease.

It is notable that, cytokine release mediated by activation of the innate immune system is believed to be involved in the pathogenesis of Dox-induced cardiotoxicity [10-12]. Toll-like receptors (TLRs) are a part of the innate immune system and are probably also involved in the development of Dox-induced cardiomyopathy, as has been shown in TLR2-deficient mice [11]. However, the TLR-system is complex and includes several receptors, including TLR4, whose role in Dox-induced cardiomyopathy had not yet been investigated. TLR4 is expressed on the cell surface of cardiac cells, including cardiomyocytes, smooth muscle cells and endothelial cells. TLR4 activation, resulting from a variety of ligands such as pathogen-associated molecular patterns, heat shock proteins and ox-LDL may lead to a pro-inflammatory response in the heart [13-15]. Recently, a study in humans demonstrated an enhancement of TLR-4-positive circulating monocytes in patients with acute coronary syndrome [16]. Furthermore, increased TLR4 expression was observed in isolated cardiomyocytes from humans and animals with cardiomyopathies.[17] Growing evidence of a causal link between TLRs and the development of heart failure has been derived mostly from studies in knock-out mice supporting a relevant role of this receptor family. It had been shown that TLR4 can modulate LV hypertrophy, myocyte contractility, myocardial ischaemia-reperfusion injury, and plays a role in inflammatory responses including septic shock syndrome [18-22]. We therefore suggested that TLR4 may also contribute to the mechanisms involved in Dox-induced cardiomyopathy. To prove this hypothesis, we studied the development of Dox-induced cardiomyopathy in TLR4-deficient mice (TLR4–/–) including the cardiac inflammatory stress response and its functional consequences.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Experimental animals
C57BL/10ScSn wild-type (WT)[23] and TLR4-deficient mice ([TLR4–/–] C57BL/10ScN, carrying a deletion of the TLR4 gene [23]) were obtained from the breeding stocks of the Max-Planck-Institut für Immunologie (Freiburg, Germany). All mouse strains were bred in the Forschungsinstitut für Experimentelle Medizin (Berlin, Germany). WT and TLR4–/– mice, aged 8 to 10 weeks, were randomly selected into four groups for treatment with Dox (Doxo Cell®, Cell Pharm, Germany, 20 mg/kg; intraperitoneally) (WTDox and TLR4–/–Dox) at a dose shown to be cardiotoxic [11], or with the same volume of saline (WT and TLR4–/–). Five days after Dox injection, mice were haemodynamically characterized. Finally, hearts were excised and prepared for molecular, biological and immuno histochemical analyses as described below. To investigate the early regulation of nuclear factor kappa B (NF-kB) in Dox-induced cardiomyopathy, we performed additional experiments (see below) using cardiac tissue from WT and TLR4–/– mice one hour after Dox-injection. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

2.2. Surgical procedures and haemodynamic measurements
Animals were anaesthetized (thiopental 125 µg/g; i.p.), intubated and artificially ventilated. As described previously [24,25], a 1.4 F microconductance pressure catheter (ARIA SPR-719; Millar-Instruments, Inc., Texas, USA) was positioned in the LV for continuous registration of LV pressure-volume (PV) loops in a closed-chest model.

Systolic function was quantified by LV end-systolic pressure (LVP, mm Hg), and dP/dtmax (mm Hg/s) as an index of LV contractility. Diastolic function was measured by dP/dtmin (mm Hg/s), and the end-diastolic-pressure-volume-relationship (stiffness, mm Hg/{varepsilon}l), determined from an exponential fit to the end-diastolic pressure-volume points. Global cardiac function was quantified by ejection fraction (EF, %), stroke volume (SV, {varepsilon}l), heart rate (HR, beat/min), and cardiac output (CO, ml/min) [24].

2.3. Tissue preparation
For immuno histochemical analyses, cardiac tissues were embedded in OCT compound (Tissue Tec®, Sakura Finetek). For molecular biological analyses, the isolated heart tissues were snap frozen immediately in liquid nitrogen and stored at –80 °C.

2.4. Isolation of mouse cardiomyocytes
Hearts of WT, WTDox, TLR4–/– and TLR4–/–Dox mice (n=6 per group, anaesthetised with Trapanal, 667 mg/kg, i.p.) were excised, mounted on a Langendorff-apparatus and perfused with modified Krebs-Henseleit-buffer containing 110 mM NaCl, 2.6 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 11 mM Glucose, 25 mM Hepes, pH=7.4. For digestion, collagenase type II (Worthington, Lakewood, USA) and 33 mM CaCl2 were added. Perfusion took place over 30 min at a constant pressure of 65 mm Hg. The media were maintained at 37 °C and saturated with oxygen. Ventricles were minced in the same buffer, dispersed for a further 10 min and filtered through a mesh (200 {varepsilon}m) to remove undigested tissue. Viable myocytes were separated by centrifugation in a 4 % bovine serum albumin gradient, immersed into liquid nitrogen, and used for further measurements.

2.5. Quantitative real-time reverse transcriptase PCR (TaqMan®)
For RNA preparation, cardiac tissue samples were homogenized and total RNA was prepared using TRIZOL® reagent (Invitrogen) according to the manufacturer’s protocol. Quantitative real-time RT-PCR (qPCR) was performed on an ABI PRISM 7700 sequence detection system (Applied Biosystems). For detection of murine ppET-1, Primer and probe oligonucleotides (forward primer: 5'-TGTTCGTGACTTTCCAAGG-3'; reverse primer: 5'-AGCTCCGGT GCTGAGTTCGG-3'; probe: 5'-6FAM-CTCCAGAAACAGCTGTC-3') were designed as based on the murine ppET-1 cDNA sequence (accession number U35233 [GenBank] ). For normalization, 18S rRNA was purchased by a commercial TaqMan PreDeveloped Assay Reagent (Applied Biosystems). Measurement of GATA-4 mRNA levels was performed using a commercially available kit (Applied Biosystems).

2.6. Lipid peroxidation in cardiomyocytes and the left ventricle
Cardiomyocyte and left ventricular lipid peroxidation was measured using the commercially available colorimetric assay kit Bioxytech® LPO-586 (Oxis International) [26]. Briefly, 150 {varepsilon}l of protein extracts were used for measurement of malondialdehyde (MDA) and 4-hydroxyalkenals (HAE) considered as indicators of lipid peroxidation as described in the manufacturer's directions for use.

2.7. DNA-p65 NF-{kappa}B binding assay
As described previously [11], DNA-p65 NF-{kappa}B binding activity in myocardial tissue was measured using a BD Mercury TransFactor kit (BD Biosciences, Clontech), which detects DNA binding by specific transcription factors one hour and five days after Dox injection. Values were normalized in respect to the protein content.

2.8. Determination of ET-1 and TNF-{alpha} by ELISA
Myocardial ET-1 and TNF-{alpha} were measured using commercially available ELISA kits (R and D Systems) according to the manufacturer's instructions. For determination of ET-1 and TNF-{alpha}, protein extracts from tissues were used.

2.9. Western blot analysis
Western blot analyses were performed using primary antibodies raised against Bax (Santa Cruz, diluted 1:500), Bcl-2 (Santa Cruz, diluted 1:500) and GATA-4 (Santa Cruz 1:1000). GAPDH (Biodesign International, diluted 1:1500) served as loading control. Detection of the signals was performed using the LumiPhosTM reagent (Pierce) and chemiluminescence was detected using x-ray films.

2.10. Cardiac immunostaining
Serial, 7-{varepsilon}m thick, transverse cryosections of Tissue Tec® embedded heart tissue were used. As previously described [27], for measurement of cardiac infiltration of immune cells, sections were incubated with goat anti-CD3 (1:25; Santa Cruz), rat anti-CD8a (1:50; Pharmingen) and rat anti-CD11b (1:50; Pharmingen). Then, sections were exposed to biotinylated anti-rat and anti-rabbit IgG, respectively, diluted to 1:100 (DAKO) or rabbit anti-goat IgG diluted to 1:200 (ABC-Kit). Subsequently, the sections were incubated with VECTASTAIN® ABC reagent (Vector). Cardiac expression of nitrotyrosine was performed using a rabbit anti-nitrotyrosine (1:75; Sigma) antibody in conjunction with the EnVison+® system (DAKO). For quantitative analysis of infiltrating cells and nitrotyrosine expression, the number per mm2 of CD3+, CD8a+ and CD11b+ cells as well as the area fraction of nitrotyrosine, were measured by digital image analysis as described previously [28].

2.11. TUNEL assay
Apoptotic cells were detected in paraffin tissue sections by end-labelling the fragmented DNA using the DeadEndTM Colorimetic TUNEL System (Promega, USA) according to the manufacturer’s instructions. TUNEL positive cells were calculated as cells per area of heart tissue.

2.12. Statistical analysis
Statistical analysis was performed using SPSS Version 12.0. Data are expressed as the mean±SEM. Statistical differences were assessed by using the Kruskal-Wallis test in conjunction with the Mann Whitney U post-hoc test. To analyse the relationship between LV function and TNF-{alpha} expression, data were compared by multiple bivariate analysis, and the Spearman Rho correlation coefficient was calculated as described previously [29]. Differences were considered statistically significantly at a value of P<0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. Left ventricular function
LV dysfunction is a hallmark of acute Dox-induced cardiotoxicity. We measured LV function by assessing pressure-volume loops using a microconductance catheter. Haemodynamic data are shown in Table 1. No parameter of systolic, diastolic, and global LV function was found to differ between WT and TLR4–/– mice. Five days after Dox injection, WTDox mice displayed significantly impaired systolic (LVP –31%, dP/dtmax –45%, P<0.01), diastolic (dP/dtmin –41%, stiffness +300%; P<0.05) and global (EF –27%, SV –39%, HR –18%, CO –54%; P<0.05) LV function. In contrast, among TLR4–/–Dox mice, parameters of systolic (LVP +27%, dP/dtmax +44%, P<0.05), diastolic (dP/dtmin +30%, stiffness –63%; P<0.05), and global (EF +29%, SV +57%, Co +84%; P<0.05) LV function were significantly improved compared to the WTDox mice.


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  		Table 1 Haemodynamic parameters

 
3.2. TNF-{alpha} protein expression and cardiac cell infiltration
To characterize the cardiac inflammatory response, we determined cardiac TNF-{alpha} protein expression and cell infiltration of CD3-, CD4- and CD11b-positive cells; this did not differ between untreated WT and TLR4–/– mice. Dox injection led to an increased (1.9-fold; P<0.05) expression of TNF-{alpha} expression in WT mice compared to untreated controls. TLR4–/–Dox mice did not display a significant difference in comparison to untreated TLR4–/– mice (Fig. 1). In addition, there was a significant correlation between decreased parameters of systolic LV function and cardiac TNF-{alpha} expression (dp/dtmax: P=0.0046, LVP: P=0.0052).


Figure 01
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  		Fig. 1 Cardiac protein expression of TNF-{alpha} in doxorubicin-induced cardiomyopathy. Protein expression was measured using ELISA. Data are expressed as mean±SEM. * = P<0.05; # = non significant vs. TLR4–/– (n=6 per group). WT, wild-type; Dox, doxorubicin; TLR4, Toll-like receptor 4.

 
WTDox mice displayed significantly enhanced infiltration of lymphocytes and macrophages indexed by CD3-, CD8a- and CD11b-positive cells compared to WT mice (Fig. 2). In contrast, TLR4–/–Dox mice showed significantly decreased content of cardiac infiltrating cells compared to WTDox mice.


Figure 02
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  		Fig. 2 Cardiac cell infiltration in doxorubicin-induced cardiomyopathy. Infiltration of cardiac CD3+, CD8a+ and CD11b+ cells were quantified (A) using immunostaining and digital image analysis. Data are expressed as mean±SEM. * = P<0.05; # = P<0.05 vs. TLR4–/– and WTDox (n=6 per group). Representative pictures of cardiac cell infiltration (red) are shown in (B). WT, wild-type; Dox, doxorubicin; TLR4, Toll-like receptor 4. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

 
3.3. Regulation of oxidative stress in isolated cardiomyocytes and heart tissue
Lipid peroxidation as a marker for oxidative stress was evaluated in isolated cardiomyocytes as well as in LV tissue. As shown in Fig. 3A, lipid peroxidation was enhanced in both isolated cardiomyocytes (+45%, P<0.05) and LV tissue (+37%, P<0.05) from WTDox mice when compared to WT mice. In contrast, in TLR4–/–Dox mice, lipid peroxidation was not significantly enhanced when compared to TLR4–/– mice. As shown in Fig. 3B, LV tissue from WTDox mice displayed significantly enhanced expression of nitrotyrosine compared to WT controls (+150%, P<0.05). In contrast, TLR4–/–Dox mice displayed significantly reduced cardiac expression of nitrotyrosine compared to WTDox mice (–82%, P<0.05).


Figure 03
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  		Fig. 3 Cardiac lipid peroxidation and nitrotyrosine in doxorubicin-induced cardiomyopathy. Lipid peroxidation from both isolated primary cardiomyocytes and heart tissue (A) were quantified using a commercial kit (n=6 per group). Cardiac nitrotyrosine expression (B) was quantified by immunostaining and digital image analysis. Data are expressed as mean±SEM. Representative pictures of nitrotyrosine staining (red) are depicted above the quantification graph. * = P<0.05 (n=6 per group). WT, wild-type; Dox, doxorubicin; TLR4, Toll-like receptor 4. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

 
3.4. Apoptosis, Bax and Bcl-2 protein expression
As shown in Fig. 4C, WTDox mice displayed significantly more TUNEL-positive apoptotic cells when compared to WT controls (5-fold, P<0.05). In contrast, the content of TUNEL-positive apoptotic cells did not differ significantly between TLR4–/– and TLR4–/–Dox mice. Protein expression of the pro-apoptotic protein Bax was markedly increased in WTDox mice when compared to WT mice, whereas TLR4–/–Dox mice displayed no up-regulation in comparison with TLR4–/– mice (Fig. 4A). The expression of the anti-apoptotic protein Bcl-2 did not differ between WT and WTDox mice (Fig. 4B). In contrast, Bcl-2 expression in TLR4–/–Dox mice was 6.5-fold (P<0.01) enhanced in comparison with untreated TLR4–/– mice.


Figure 04
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  		Fig. 4 Apoptosis in doxorubicin-induced cardiomyopathy. Representative western blots with specific bands of Bax (A) and Bcl-2 (B) expression and quantification were shown. Cardiac apoptotic cells were quantified using TUNEL staining (red coloured) and digital image analysis (C). Data were expressed as mean±SEM. * = P<0.05 (n=6 per group). WT, wild-type; Dox, doxorubicin; TLR4, Toll-like receptor 4. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

 
3.5. Endothelin-1 expression
LV dysfunction leads to neurohumoral activation. Hence we measured cardiac ET-1 mRNA- and protein expression five days after Dox injection (Fig. 5A and B) using molecular biological techniques. Five days after Dox administration, WT mice displayed a significant increase in ET-1 mRNA (5.2-fold; P<0.01) and protein (2.1-fold; P=0.02) content when compared to WT controls. In comparison with untreated TLR4–/– mice, Dox administration into TLR4–/– mice did not result in any significant enhancement of either ET-1 mRNA or protein expression.


Figure 05
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  		Fig. 5 Cardiac endothelin-1 regulation in doxorubicin-induced cardiomyopathy. Expression of endothelin-1 mRNA (A) and protein expression (B) were measured by RT-PCR and ELISA, respectively. Data are expressed as mean±SEM. n. s. = non significant; * = P<0.05 (n=6 per group). WT, wild-type; Dox, doxorubicin; TLR4, Toll-like receptor 4.

 
3.6. NF{kappa}B and GATA-4 expression
We measured NF-kB binding activity five days after Dox injection in cardiac tissue using a binding assay. Binding activities of untreated WT and TLR4–/– mice did not differ (Fig. 6A). In addition, we could not observe a significant alteration of NF-kB binding activity in both WTDox and TLR4–/–Dox mice compared to untreated mice at five days and at one hour after Dox injection (Fig. 6A). Since GATA-4 is known to be involved in TLR4 signalling, we measured mRNA levels using a commercial kit. Both mRNA and the protein level of GATA-4 were significantly reduced (mRNA: –50%, protein: –69%; P<0.05) in WTDox mice compared to WT mice (Fig. 6B and C). In contrast, mRNA content was not regulated in TLR4–/–Dox mice when compared with TLR4–/– mice, protein expression of GATA-4 was significantly enhanced in TLR4–/–Dox mice when compared to WTDox mice (+135%; P<0.05).


Figure 06
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  		Fig. 6 Gene activation in doxorubicin-induced cardiomyopathy. NF-{kappa}B binding activity from cardiac nuclear extracts one hour and five days after Dox injection (A), GATA-4 mRNA (B) and protein (C) expression were measured using commercial kits and western blot analysis, respectively. Data are expressed as mean±SEM. * = P<0.05 (n=6 per group). WT, wild-type; Dox, doxorubicin; TLR4, Toll-like receptor 4; h, hour; d, days.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
Here we show for the first time that TLR4 contributes to cardiac inflammation, oxidative stress, and apoptosis as well as LV function in experimental, Dox-induced cardiomyopathy.

In agreement with others, WTDox mice were found to display severe systolic and diastolic LV dysfunction, resulting in impaired cardiac output as measured by the assessment of pressure-volume loops in vivo [11,24,30]. In TLR4–/–Dox mice, global LV function as indexed by stroke volume and cardiac output was improved as a result of enhanced systolic and diastolic performance. This was associated with a reduction in neurohumoral activation, with a reduction in cardiac ET-1 which is known to contribute to the development of heart failure [31-34]. Thus, we conclude that TLR4 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, and apoptosis, which are all known to be relevant in this disease.

Oxidative stress can directly damage cells, trigger cytokine expression, increase leukocyte chemotaxis, and initiate complement activation [35]. In agreement with others[11,36], we found that lipid peroxidation and protein expression of nitrotyrosine as an index of oxidative stress were markedly increased in cardiac tissue of WTDox mice when compared to that of WT controls. Since lipid peroxidation is active in a variety of cell types possibly present in the LV, we measured lipid peroxidation activity in myocytes isolated from WT and TLR4–/– mice which were treated with doxorubicin in order to identify cardiac specific stress. In line with our findings from LV tissue, lipid peroxidation activity was also found to be enhanced in myocytes isolated from WTDox mice in comparison with myocytes from WT mice. In contrast, in both LV tissue and isolated myocytes, no significant regulation in TLR4–/–Dox mice was seen when compared to TLR4–/– mice. In line with these findings, cardiac expression of nitrotyrosine was significantly reduced in TLR4–/–Dox mice compared to WTDox mice. Our data suggest - the other way round - that TLR4 deficiency is able to attenuate the generation of oxidative stress in the heart implying that TLR4 is not only able to be activated by oxidative stress, but also contributes to its development as has also recently been demonstrated in an animal model of cardiac ischaemia/reperfusion [37]. The precise mechanisms involved in the interaction between TLRs and oxidative stress have not yet been completely defined. In murine leukocytes, oxidative stress is involved in TLR4-mediated intracellular pro-inflammatory gene activation in response to lipopolysaccharide [38]. This is in agreement with our findings showing that Dox did not lead to any significant TNF-{alpha} up-regulation in TLR4–/–Dox mice in comparison with TLR4–/– mice. Concordant with these findings, cardiac infiltration of activated lymphocytes, monocytes and macrophages were significantly attenuated in TLR4–/–Dox mice when compared to WTDox mice as indicated by decreased expressions of CD3-, CD8a-, and CD11b-positive cells [27,39].

Oxidative stress and inflammation might be associated with an induction of apoptosis, as is also known for Dox-induced cardiomyopathy [12]. In agreement with in vitro and in vivo studies using myocytes, we found a marked up-regulation of the pro-apoptotic protein Bax five days after Dox administration in our model [40,41]. This effect was blunted in TLR4–/–Dox mice, suggesting that TLR4 contributes to pro-apoptotic activation in Dox-induced cardiomyopathy. The activation of this typical pro-apoptotic protein was associated with a significant increase of cardiac TUNEL-positive apoptotic cells five days after Dox injection in WT mice as previously demonstrated in this model [11], which was significantly attenuated in TLR4–/– mice. However, the amount of these cells was moderate, which might be explained by the early time point of investigation in our model. To investigate possible mechanisms, which may lead to attenuated apoptosis in Dox-treated TLR4–/– mice, we measured the anti-apoptotic protein Bcl-2 [42], which is a pivotal regulator of mitochondrial apoptosis [43]. Although others have found a reduction in the anti-apoptotic protein Bcl-2 in chronic Dox-induced cardiomyopathy [41], we found no regulation of this protein in our acute model. Interestingly, despite the non-regulation of Bcl-2 due to Dox in WT mice, the Bcl-2 content of TLR4–/–Dox mice were increased more than 6-fold when compared to TLR4–/– controls (P<0.01) suggesting anti-apoptotic protection due to TLR4 deficiency. Very recent investigations support an emerging role for Bcl-2 in protecting cardiac cells against death, including apoptosis and non-apoptotic cell death, depending on autophagy genes [44].

To get more insights into the intracellular mechanisms induced by any TLR4 deficiency possibly involved, we determined cardiac NF{kappa}B and GATA-4 regulation in our model. Specifically, a down-regulation of GATA-4 in Dox-induced cardiomyopathy was identified as belonging to a disease-aggravating mechanism. We showed that TLR4 deficiency prevents GATA-4 down-regulation, suggesting that direct or indirect effects of TLR4 signalling are involved in the regulation of this transcription factor. In contrast, NF{kappa}B, which is known not be a classical down-stream target for TLR4 signalling was not regulated in WT or TLR4–/– mice, indicating that NF{kappa}B regulation is, at least during the investigated time frame, also not under the control of TLR4 in our model [45].

In summary, acute Dox-induced cardiomyopathy included cardiac generation of oxidative stress, inflammatory response, apoptosis, and ET-1 up-regulation leading to LV dysfunction. TLR4 deficiency attenuated these mechanisms known to be crucial for the development of Dox-induced heart failure. Our data demonstrate for the first time a relevant role of TLR4 in the development of this disease under experimental conditions. Further studies using therapeutic interventions such as pharmacological TLR4 inhibition as currently being investigated in patients with severe sepsis are now required to prove whether or not these findings can indeed be transformed into clinical practice [37].


   Acknowledgement
 
This study was funded by a grant from the Deutsche Forschungsgesellschaft (DFG; TR-SFB 19, project Z3) to Carsten Tschope. We thank Kerstin Puhl, André Fischer and Gernot Reifenberger for excellent technical assistance. The TLR4–/– mice were kindly provided by Marina Freudenberg (Max-Planck-Institute for Immunobiology, Freiburg, Germany).


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

  1. Teraoka K., Hirano M., Yamaguchi K., Yamashina A. Progressive cardiac dysfunction in adriamycin-induced cardiomyopathy rats. Eur J Heart Fail (2000) 2(4):373–378.[Abstract/Free Full Text]
  2. Belham M., Kruger A., Mepham S., Faganello G., Pritchard C. Monitoring left ventricular function in adults receiving anthracycline-containing chemotherapy. Eur J Heart Fail (2007) 9(4):409–414.[Abstract/Free Full Text]
  3. Billingham M.E., Mason J.W., Bristow M.R., Daniels J.R. Anthracycline cardiomyopathy monitored by morphologic changes. Cancer Treat Rep (1978) 62(6):865–872.[Web of Science][Medline]
  4. Takemura G., Fujiwara H. Doxorubicin-induced cardiomyopathy from the cardiotoxic mechanisms to management. Prog Cardiovasc Dis (2007) 49(5):330–352.[CrossRef][Web of Science][Medline]
  5. Kalay N., Basar E., Ozdogru I., Er O., Cetinkaya Y., Dogan A., et al. Protective effects of carvedilol against anthracycline-induced cardiomyopathy. J Am Coll Cardiol (2006) 48(11):2258–2262.[Abstract/Free Full Text]
  6. Cardinale D., Colombo A., Sandri M.T., Lamantia G., Colombo N., Civelli M., et al. Prevention of high-dose chemotherapy-induced cardiotoxicity in high-risk patients by angiotensin-converting enzyme inhibition. Circulation (2006) 114(23):2474–2481.[Abstract/Free Full Text]
  7. Vaynblat M., Shah H.R., Bhaskaran D., Ramdev G., Davis W.J. 3rd, Cunningham J.N. Jr., et al. Simultaneous angiotensin converting enzyme inhibition moderates ventricular dysfunction caused by doxorubicin. Eur J Heart Fail (2002) 4(5):583–586.[Abstract/Free Full Text]
  8. Thomas X., Le Q.H., Fiere D. Anthracycline-related toxicity requiring cardiac transplantation in long-term disease-free survivors with acute promyelocytic leukemia. Ann Hematol (2002) 81(9):504–507.[CrossRef][Web of Science][Medline]
  9. Granger C.B. Prediction and prevention of chemotherapy-induced cardiomyopathy: can it be done? Circulation (2006) 114(23):2432–2433.[Free Full Text]
  10. Liu L., Zhang X., Qian B., Min X., Gao X., Li C., et al. Over-expression of heat shock protein 27 attenuates doxorubicin-induced cardiac dysfunction in mice. Eur J Heart Fail (2007).
  11. Nozaki N., Shishido T., Takeishi Y., Kubota I. Modulation of doxorubicin-induced cardiac dysfunction in toll-like receptor-2-knockout mice. Circulation (2004) 110(18):2869–2874.[Abstract/Free Full Text]
  12. Minotti G., Menna P., Salvatorelli E., Cairo G., Gianni L. Anthracyclines: molecular advances and pharmacologic developments in antitumor activity and cardiotoxicity. Pharmacol Rev (2004) 56(2):185–229.[Abstract/Free Full Text]
  13. Baeuerle P.A., Baltimore D. NF-kappa B: ten years after. Cell (1996) 87(1):13–20.[CrossRef][Web of Science][Medline]
  14. Medzhitov R., Preston-Hurlburt P., Janeway C.A. Jr. A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature (1997) 388(6640):394–397.[CrossRef][Web of Science][Medline]
  15. Xu X.H., Shah P.K., Faure E., Equils O., Thomas L., Fishbein M.C., et al. Toll-like receptor-4 is expressed by macrophages in murine and human lipid-rich atherosclerotic plaques and upregulated by oxidized LDL. Circulation (2001) 104(25):3103–3108.[Abstract/Free Full Text]
  16. Methe H., Kim J.O., Kofler S., Weis M., Nabauer M., Koglin J. Expansion of circulating Toll-like receptor 4-positive monocytes in patients with acute coronary syndrome. Circulation (2005) 111(20):2654–2661.[Abstract/Free Full Text]
  17. Frantz S., Kobzik L., Kim Y.D., Fukazawa R., Medzhitov R., Lee R.T., et al. Toll4 (TLR4) expression in cardiac myocytes in normal and failing myocardium. J Clin Invest (1999) 104(3):271–280.[Web of Science][Medline]
  18. Ha T., Li Y., Hua F., Ma J., Gao X., Kelley J., Zhao A., et al. Reduced cardiac hypertrophy in toll-like receptor 4-deficient mice following pressure overload. Cardiovasc Res (2005) 68(2):224–234.[Abstract/Free Full Text]
  19. Boyd J.H., Mathur S., Wang Y., Bateman R.M., Walley K.R. Toll-like receptor stimulation in cardiomyoctes decreases contractility and initiates an NF-kappaB dependent inflammatory response. Cardiovasc Res (2006) 72(3):384–393.[Abstract/Free Full Text]
  20. Oyama J., Blais C. Jr., Liu X., Pu M., Kobzik L., Kelly R.A., et al. Reduced myocardial ischemia-reperfusion injury in toll-like receptor 4-deficient mice. Circulation (2004) 109(6):784–789.[Abstract/Free Full Text]
  21. Nemoto S., Vallejo J.G., Knuefermann P., Misra A., Defreitas G., Carabello B.A., et al. Escherichia coli LPS-induced LV dysfunction: role of toll-like receptor-4 in the adult heart. Am J Physiol Heart Circ Physiol (2002) 282(6):H2316–H2323.[Abstract/Free Full Text]
  22. Timmers L., Sluijter J.P., van Keulen J.K., Hoefer I.E., Nederhoff M.G., Goumans M.J., et al. Toll-like receptor 4 mediates maladaptive left ventricular remodeling and impairs cardiac function following myocardial infarction. Circ Res (2007).
  23. Poltorak A., He X., Smirnova I., Liu M.Y., Van Huffel C., Du X., Birdwell D., et al. Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science (1998) 282(5396):2085–2088.[Abstract/Free Full Text]
  24. Westermann D., Knollmann B.C., Steendijk P., Rutschow S., Riad A., Pauschinger M., et al. Diltiazem treatment prevents diastolic heart failure in mice with familial hypertrophic cardiomyopathy. Eur J Heart Fail (2006) 8(2):115–121.[Abstract/Free Full Text]
  25. Steendijk P., Baan J. Comparison of intravenous and pulmonary artery injections of hypertonic saline for the assessment of conductance catheter parallel conductance. Cardiovasc Res (2000) 46(1):82–89.[Abstract/Free Full Text]
  26. Kang Y.J., Chen Y., Epstein P.N. Suppression of doxorubicin cardiotoxicity by overexpression of catalase in the heart of transgenic mice. J Biol Chem (1996) 271(21):12610–12616.[Abstract/Free Full Text]
  27. Pauschinger M., Rutschow S., Chandrasekharan K., Westermann D., Weitz A., Schwimmbeck L, Peter, et al. Carvedilol improves left ventricular function in murine coxsackievirus-induced acute myocarditis association with reduced myocardial interleukin-1beta and MMP-8 expression and a modulated immune response. Eur J Heart Fail (2005) 7(4):444–452.[Abstract/Free Full Text]
  28. Witzenbichler B., Westermann D., Knueppel S., Schultheiss H.P., Tschope C. Protective role of angiopoietin-1 in endotoxic shock. Circulation (2005) 111(1):97–105.[Abstract/Free Full Text]
  29. Tschope C., Walther T., Escher F., Spillmann F., Du J., Altmann C., et al. Transgenic activation of the kallikrein-kinin system inhibits intramyocardial inflammation, endothelial dysfunction and oxidative stress in experimental diabetic cardiomyopathy. Faseb J (2005) 19(14):2057–2059.[Abstract/Free Full Text]
  30. Takimoto E., Champion H.C., Li M., Belardi D., Ren S., Rodriguez E.R., et al. Chronic inhibition of cyclic GMP phosphodiesterase 5A prevents and reverses cardiac hypertrophy. Nat Med (2005) 11(2):214–222.[CrossRef][Web of Science][Medline]
  31. Daugaard G., Lassen U., Bie P., Pedersen E.B., Jensen K.T., Abildgaard U., et al. Natriuretic peptides in the monitoring of anthracycline induced reduction in left ventricular ejection fraction. Eur J Heart Fail (2005) 7(1):87–93.[Abstract/Free Full Text]
  32. Yang L.L., Gros R., Kabir M.G., Sadi A., Gotlieb A.I., Husain M., et al. Conditional cardiac overexpression of endothelin-1 induces inflammation and dilated cardiomyopathy in mice. Circulation (2004) 109(2):255–261.[Abstract/Free Full Text]
  33. Rothermund L., Paul M. The role of endothelin in hypertension. Curr Opin Nephrol Hypertens (1998) 7(4):451–456.[Web of Science][Medline]
  34. Bien S., Riad A., Ritter C.A., Gratz M., Olshausen F., Westermann D., et al. The endothelin receptor blocker bosentan inhibits doxorubicin-induced cardiomyopathy. Cancer Res (2007) 67(21):10428–10435.[Abstract/Free Full Text]
  35. Dhalla N.S., Elmoselhi A.B., Hata T., Makino N. Status of myocardial antioxidants in ischemia-reperfusion injury. Cardiovasc Res (2000) 47(3):446–456.[Abstract/Free Full Text]
  36. Weinstein D.M., Mihm M.J., Bauer J.A. Cardiac peroxynitrite formation and left ventricular dysfunction following doxorubicin treatment in mice. J Pharmacol Exp Ther (2000) 294(1):396–401.[Abstract/Free Full Text]
  37. Shimamoto A., Chong A.J., Yada M., Shomura S., Takayama H., Fleisig A.J., et al. Inhibition of Toll-like receptor 4 with eritoran attenuates myocardial ischemia-reperfusion injury. Circulation (2006) 114(1 Suppl):I270–I274.[Web of Science][Medline]
  38. Asehnoune K., Strassheim D., Mitra S., Kim J.Y., Abraham E. Involvement of reactive oxygen species in Toll-like receptor 4-dependent activation of NF-kappa B. J Immunol (2004) 172(4):2522–2529.[Abstract/Free Full Text]
  39. Turina M., Mulhall A., Gardner S., Polk H.C. Jr., Miller F.N. Mannitol upregulates monocyte HLA-DR, monocyte and neutrophil CD11b, and inhibits neutrophil apoptosis. Inflammation (2007).
  40. Spallarossa P., Garibaldi S., Altieri P., Fabbi P., Manca V., Nasti S., et al. Carvedilol prevents doxorubicin-induced free radical release and apoptosis in cardiomyocytes in vitro. J Mol Cell Cardiol (2004) 37(4):837–846.[CrossRef][Web of Science][Medline]
  41. Wu S., Ko Y.S., Teng M.S., Ko Y.L., Hsu L.A., Hsueh C., et al. Adriamycin-induced cardiomyocyte and endothelial cell apoptosis: in vitro and in vivo studies. J Mol Cell Cardiol (2002) 34(12):1595–1607.[CrossRef][Web of Science][Medline]
  42. Danial N.N., Korsmeyer S.J. Cell death: critical control points. Cell (2004) 116(2):205–219.[CrossRef][Web of Science][Medline]
  43. Gross A. Mitochondrial carrier homolog 2: a clue to cracking the BCL-2 family riddle? J Bioenerg Biomembr (2005) 37(3):113–119.[CrossRef][Web of Science][Medline]
  44. Kutschka I., Kofidis T., Chen I.Y., von Degenfeld G., Zwierzchoniewska M., Hoyt G., et al. Adenoviral human BCL-2 transgene expression attenuates early donor cell death after cardiomyoblast transplantation into ischemic rat hearts. Circulation (2006) 114(1 Suppl):I174–I180.[Web of Science][Medline]
  45. Akira S., Takeda K. Toll-like receptor signalling. Nat Rev Immunol (2004) 4(7):499–511.[CrossRef][Web of Science][Medline]

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