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European Journal of Heart Failure 2007 9(10):986-994; doi:10.1016/j.ejheart.2007.07.016
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© 2007 European Society of Cardiology

Dystrophin-deficiency increases the susceptibility to doxorubicin-induced cardiotoxicity

Shiwei Denga, Bettina Kulleb, Mehdi Hosseinic, Gregor Schlüterc, Gerd Hasenfussd, Leszek Wojnowskia,* and Albrecht Schmidtd

a Department of Pharmacology, Johannes Gutenberg University 55101 Mainz, Germany
b Department of Biostatistics and Department of Mathematics, University of Oslo Norway
c Department of Human Genetics, University of Göttingen 37075 Göttingen, Germany
d Department of Cardiology and Pneumology, University of Göttingen 37075 Gottingen, Germany

* Corresponding author. Department of Pharmacology, Johannes Gutenberg University Obere Zahlbacher Strasse 67, 55101 Mainz, Germany. Tel.: +49 6131 3933460; fax: +49 941 5992 36727. E-mail address: wojnowski{at}uni-mainz.de (L. Wojnowski).


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Appendix A. Supplementary data
 References
 
Background and aim: The clinical use of doxorubicin (DOX) and other anthracyclines is limited by a dosage-dependent cardiotoxicity, which can lead to cardiomyopathy. The role of the individual genetic makeup in this disorder is poorly understood. Alterations in genes encoding cardiac cytoskeleton or sarcolemma proteins may increase the susceptibility to doxorubicin-related cardiotoxicity.

Methods: Female dystrophin-deficient mice (MDX) and age-matched wild-type mice underwent chronic treatment with doxorubicin. Cardiac function and tissue damage were assessed by echocardiography and histopathology, respectively. Gene expression changes were investigated using microarrays.

Results: DOX treatment resulted in mortality, cardiac insufficiency, and cardiac interstitial fibrosis. These alterations were more pronounced in DOX-treated MDX mice than in DOX-treated wild-type mice. Changes in gene expression were more numerous in MDX mice, including genes involved in cell adhesion, oxidative stress, cytoskeleton organization, inflammatory and immune response and cell death.

Conclusions: Dystrophin deficiency facilitates the development and progression of doxorubicin-induced cardiac injury. The underlying mechanisms may involve changes in cell adhesion, in cytoskeleton, as well as in inflammatory and immune responses. Genetic variants of cytoskeletal proteins in humans may affect the individual susceptibility to doxorubicin. Cardiotoxic drugs may accelerate the manifestation of pre-clinical cardiomyopathies caused by deficiencies in cytoskeletal or sarcolemma proteins.

Key Words: Doxorubicin • Dystrophin • Cardiotoxicity

Received December 22, 2006; Revised June 15, 2007; Accepted July 17, 2007


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Appendix A. Supplementary data
 References
 
Doxorubicin (DOX) and other anthracyclines are potent and effective chemotherapeutic agents, but they are associated with a dose-dependent cardiotoxicity, which is the most important factor limiting their clinical use [1,2]. The cardiotoxicity can take two major forms. Acute or sub-acute injury, which manifests as depression of contractile function or as arrhythmias, may occur immediately after treatment. Chronic cardiotoxicity is clinically more relevant, and is characterized by dilated cardiomyopathy (DCM) and heart failure [3].

It is usually assumed that the molecular and cellular mechanisms of chronic cardiotoxicity are quite different from the mechanisms which account for the antineoplastic effect. The latter is attributed to topoisomerase II inhibition [4]. Mechanisms responsible for cardiotoxicity are incompletely understood, but they are likely multifactorial and may include reactive oxygen species generation [5], calcium overload [6], release of vasoactive amines [7], and disturbances in myocardial adrenergic function [8]. Additionally, doxorubicin, the most prescribed and best investigated anthracycline, is capable of regulating transcriptional levels of certain genes in the heart, for instance, {alpha}-actin, troponin, myosin light-chain 2, and the M isoform of creatine kinase [9].

The cytoskeletal apparatus has been linked to doxorubicin-related cardiotoxicity [10] and abnormalities are often concomitant with other pathological lesions found in the failing human myocardium [11]. Cytoskeletal and extracellular matrix proteins are involved in the cardiac response to a single therapeutic dose of doxorubicin [12]. In a recent study, deficiency of the cytoskeletal protein dystrophin markedly increased the susceptibility to enterovirus-induced cardiomyopathy [13]. Dystrophin is a part of the dystrophin-glycoprotein complex (DGC) which links the myofiber cytoskeleton to the extracellular matrix. The impairment of any component of DGC may break the mechanical link vital for sarcolemmal integrity. Mutations in the dystrophin gene may result in cardiomyopathy, in addition to the well known Duchenne phenotype.

Whereas doxorubicin treatment and dystrophin deficiency may each result in a dilated cardiomyopathy, nothing is known about the combined effect of these two factors. We hypothesized that dystrophin-deficiency may affect the presentation of DOX-related myocardial injury. In the present study, we tested if mice deficient in dystrophin were more susceptible to doxorubicin-induced cardiotoxicity in comparison to wild-type animals. Young C57BL/10 mice and dystrophin-deficient C57BL/10ScSn-Dmdmdx/J (MDX) mice, which are the most commonly used laboratory model of human Duchenne muscular dystrophy, were chronically treated with doxorubicin. Left ventricular function and cardiac pathological alterations were evaluated by echocardiography and by histological methods, respectively. High density oligonucleotide RNA microarrays were applied to profile global gene expression and achieve a better understanding of the mechanisms for cardiotoxicity. Since the early transcriptional response to DOX has been extensively investigated elsewhere [14] and the clinically-relevant form of DOX-induced cardiotoxicity is the cumulative, dose-dependent chronic form, we focus in the present work on transcriptional changes associated with late phase cardiomyopathy.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Appendix A. Supplementary data
 References
 
2.1. Experimental protocol
Eight-week-old female heterozygous dystrophin-deficient mice (MDX) and age-matched wild-type (WT) C57BL/10 mice were used. Animals were randomised into four groups (20 animals per group): WT-CON, WT-DOX, MDX-CON and MDX-DOX. Mice in WT-DOX and MDX-DOX group were chronically treated with doxorubicin (Pfizer, Germany) by 8 intraperitoneal injections of 4 mg/kg (cumulative dose of 32 mg/kg). Injections were performed twice a week, but after the first four injections the treatment was interrupted for two weeks to allow recovery from bone marrow depression. Mortality and body weight were recorded daily and the study was terminated when the mortality in at least one group reached 75%. The investigations conformed with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health and were approved by the responsible ethics committee.

2.2. Mouse echocardiography
Left ventricular (LV) function was assessed serially using in vivo echocardiography. 2D guided M-mode echo (30 MHz) was obtained from short- and long-axis views at the level of the largest LV-diameter using a VS-VEVO 660/230 High Resolution Imaging System (VisualSonics, Toronto, Canada). Animals were lightly anesthetized intraperitoneally with tribromoethanol and allowed to breathe spontaneously. The chest was shaved, acoustic coupling gel was applied and a warming pad was used to maintain normothermia. Mice were imaged in a shallow left lateral decubitus position. Left ventricular end-diastolic dimension (LVEDD) and left ventricular end-systolic dimension (LVESD) were measured from original tracings with the use of the leading edge convention of the American Society of Echocardiography. Left ventricular percent fractional shortening (FS), left ventricular mass (LVM), and relative wall thickness (h/r) were calculated.

2.3. Histological studies
Heart specimens were excised, fixed in buffered 4% formaldehyde, embedded in paraffin and cut into 5-µm sections. Azan staining [15] was performed to detect cardiac collagen distribution according to standard procedures. Histopathological changes were examined using light microscopy by two independent investigators who were blinded to the identity of experimental groups. In addition, in each slice 10-15 fields were randomly selected and collagen volume fraction (CVF) in each field was determined using a computer image analyzing system NIH ImageJ (http://rsb.info.nih.gov/ij/index.html). CVF was calculated as the sum of all areas stained positive divided by the sum of all myocardial areas in each field. For immunohistochemistry study, after antigen retrieval and blocking of endogenous peroxidase, biotinylated mouse monoclonal antibody for secreted phosphoprotein 1 (Spp1, 1:100, Santa Cruz Biotechnology) and atrial natriuretic peptide (ANP, 1:500, Biomol) were applied to the sections using DakoCytomation ARK kit. Chromogen was developed with 3,3-diaminobenzidine (DAB, Dako). Nuclei were counterstained with Mayer's hematoxylin.

2.4. Microarray experiments
Genome-wide gene expression profiles were obtained using oligonucleotide RNA microarrays according to standard procedures. Briefly, at the end of the study three animals in each group were sacrificed by cervical dislocation and the whole heart was excised. Total RNA from the left ventricle was extracted using Trizol reagent (Gibco). RNA specimens within the same experimental group were pooled and used for double-strand complementary DNA synthesis using reverse transcription. Biotin-labelled cRNA was in vitro synthesized, purified and fragmented. 15 µg cRNA was used for hybridization to Affymetrix mouse genome U74av2 GeneChips (Affymetrix, Santa Clara, CA) according to the manufacturer's instructions. As a summary measure of the gene expression level, we used the Robust Multi-array Average (RMA) measurement [16] by fitting a linear model using log-transformed PM values after carrying out a global background adjustment and across-array (quantile) transformation [17]. A robust procedure (median polish) was used to protect against outlier probes. Preprocessing was done using the affy-package from Bioconductor (www.bioconductor.org). Genes with expression levels which changed at least 2-fold in inter-group comparisons were defined as differentially expressed genes. They were functionally categorized using the web-based tool Onto-express [18].

In a preliminary study, the reproducibility of microarray data was estimated by repeated hybridizing of RNA samples from the same RNA pool or from two groups of animals with the same genetic background (wild-type C57BL/6J mice, 5 animals per group). These results consistently showed approximately 30 genes with differential expression level (>2-fold) which we considered as a background value. Additionally, we validated microarray results on pooled RNA using quantitative real-time PCR in a parallel study in which wild-type mice were exposed to a single large dose of doxorubicin (15 mg/kg) and compared 4 days later to untreated mice (unpublished experiment). The measurements were conducted on the same batch of microarrays as those used throughout the study. All ten genes were found to be over-expressed in real-time PCR measurements, in agreement with microarray data. The increases correlated very well between microarray and real-time PCR results (r2=0.83).

2.5. Statistical analysis
Results are expressed as mean±S.E.M. Echocardiographic and histological data between groups were compared by one-way ANOVA followed by Newman-Keuls post hoc test. The Kaplan-Meier survival curves were compared by log-rank test. A P value of less than 0.05 was considered to indicate statistical significance.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Appendix A. Supplementary data
 References
 
3.1. Survival analysis
None of the untreated animals died during the study, irrespective of the MDX genotype status. In contrast, doxorubicin reduced the survival both in WT mice and in MDX mice (both P values <0.01 compared to genotype-matched untreated mice). As shown in Fig. 1, treated MDX mice exhibited a significantly worse survival in comparison to treated WT mice (log-rank test, P=0.045), as 15 out of 20 animals (75%) died in this group, in comparison with 8 out of 20 (40%) in the WT-DOX group.


Figure 01
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  		Fig. 1 Kaplan-Meier curves of overall survival in untreated and DOX-treated WT and MDX mice. P=0.045 (log-rank test), WT-DOX vs. MDX-DOX. P<0.01 (log rank test), DOX-treated group vs. genotype-matched control group.

 
3.2. Alterations in cardiac function resulting from DOX treatment
Echocardiography was performed on all animals surviving at the end of the observation period to determine the impact of DOX on cardiac function and geometry. Animals in both DOX-treated groups exhibited an enlarged cardiac cavity, which was more pronounced in MDX mice (Fig. 2). Reduced left ventricular FS (Fig. 3A) and enhanced LVESD (Fig. 3C) were found in both DOX-treated groups as compared to the genotype-matched, untreated controls. Moreover, these alterations were significantly more pronounced in MDX-DOX group compared to those in WT-DOX group (P<0.05). Additionally, compared with the untreated MDX mice, DOX-treated MDX mice exhibited significantly increased LVEDD (Fig. 3B) and left ventricular mass/body mass (Fig. 3D) as well as reduced relative wall thickness (Fig. 3E). All these changes are indicative of a dilated cardiomyopathy. Relative to the control WT group, no impairment of cardiac function was observed in the MDX control group, which is in accordance with a previous study [19]. The above observations suggest a worse cardiac performance and a more pronounced cardiomyopathy phenotype in the MDX-DOX group compared to the WT-DOX group.


Figure 02
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  		Fig. 2 Representative M-mode images showing cardiac dimensions in WT-CON, WT-DOX, MDX-CON and MDX-DOX mice. SW, septal wall; PW, posterior wall; LVESD, left ventricular end systolic dimension; LVEDD, left ventricular end diastolic dimension.

 


Figure 03
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  		Fig. 3 Echocardiographic measurements of left ventricular function. FS, fractional shortening (A), LVEDD, left ventricular end diastolic dimension (B), LVESD, left ventricular end systolic dimension (C), LV mass/body mass (D), and h/r, relative wall thickness (E). Values are presented as mean±S.E.M. *P<0.01 vs. genotype-matched control group, #P<0.05 vs. WT-DOX.

 
3.3. Increased interstitial fibrosis in heart tissues after DOX exposure
Cardiac fibrosis was estimated by Azan staining, which detects collagen. Representative micrographs show extensive interstitial fibrosis (blue staining) in DOX-treated MDX mice (Fig. 4B). Fibrosis was also significant in the WT-DOX group (Fig. 4D) compared to the control WT animals (Fig. 4C); however, the interstitial fibrosis was significantly less extensive in comparison with that in MDX-DOX group. Fibrosis in hearts from both control WT and control MDX mice (Fig. 4A) was barely discernible, this observation is consistent with previous investigations which showed little pathology at 2-3 months of age among MDX mice [20]. Fig. 4E shows the collagen volume fraction in each group.


Figure 04
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  		Fig. 4 Representative micrographs of interstitial fibrosis stained with Azan in hearts of (A) MDX-CON, (B) MDX-DOX, (C) WT-CON, and (D) WT-DOX mice. Interstitial fibrosis (i.e. collagen) is stained blue. Scale bar=25 µm. (E) Collagen content as quantified by image analysis of Azan-stained heart sections. *P<0.01 vs. genotype-matched control group, #P<0.05 vs. MDX-DOX.

 
3.4. Identification of differentially expressed genes in response to DOX treatment
We used high-density oligonucleotide microarray, which contains oligonucleotide probes for approximately 12,000 genes. A number of differentially expressed genes substantially exceeding the background value (30 genes) was found in only one of the three inter-group comparisons performed: We identified 46 up-regulated and 27 down-regulated (73 total) genes between treated and untreated MDX mice (see Table in Appendix A for supplemental data). The corresponding number from a comparison of treated and untreated WT mice was 33, with 5 genes up-regulated and 28 down-regulated (Table 1). 17 genes, including several genes encoding cytoskeletal proteins, were differentially expressed between untreated MDX and WT mice (Table 2). In the MDX and in the WT group, DOX treatment did not change the expression level of dystrophin mRNA (data not shown), which was lower in control heterozygous MDX mice compared to control WT mice (0.761 fold) [21].


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  		Table 1 Differentially expressed genes in WT-DOX group compared with WT-CON group

 


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  		Table 2 Differentially expressed genes in MDX-CON group compared with WT-CON group

 
3.5. Functional clustering of differentially expressed genes
The differentially expressed genes were functionally classified using the web-accessible and ontology-based Onto-Express software [18]. Onto-Express is a novel tool able to automatically categorized the differentially regulated genes into biological processes. The statistically significant biological processes associated with dysregulated genes were defined by a P value less than 0.05. Only those significant processes containing at least 2 differentially expressed genes were taken into account.

As shown in Table 3, untreated MDX and WT mice differed in biological processes related to cytoskeleton organization and biogenesis, cell adhesion, and inflammatory response. In DOX-treated WT mice, the only significantly induced biological process was the immune response. Additional 7 biological processes were significantly induced by DOX in MDX mice. These are related to the processes of cell death (cytolysis, negative regulation of apoptosis) and to remodelling of heart tissue architecture (extracellular matrix, angiogenesis, heart development). The over-expression of metallothionein 1 and 2 likely reflects defence response against DOX-induced oxidative stress [22].


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  		Table 3 Significant biological processes associated with differentially expressed genes by inter-group comparison

 
3.6. Validation of microarray data by immunohistochemistry
Expression levels of two genes, relevant to cardiac fibrosis: Spp1 and ANP, were confirmed by immunohistochemistry. The direction of protein expression changes of these genes was consistent with their gene expression changes in microarray measurements, i.e. they were increased in MDX mice following DOX treatment. In contrast, no such changes were detected in DOX-treated WT mice. The representative micrographs are shown in Fig. 5.


Figure 05
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  		Fig. 5 Representative micrographs showing protein expression of Spp1 (panel A to D) and ANP (panel E to H) in the heart by immunohistochemistry. A and E: MDX-CON, B and F: MDX-DOX, C and G: WT-CON, D and H: WT-DOX. Positive staining is indicated with arrows (brown colour). Scale bar=25 µm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Appendix A. Supplementary data
 References
 
In humans, 30% of dystrophin-deficient males die from cardiac failure [23], whereas more than 50% of females heterozygous for Duchenne mutations experience clinically relevant cardiomyopathy [24]. Simultaneously, the cytoskeletal apparatus appears to be a sensitive target for DOX, with loss of contractile elements seen after a single injection of the drug [12]. Therefore, we hypothesized that dystrophin deficiency may enhance the development and progression of doxorubicin-induced cardiotoxicity, which was investigated in a murine model of this disorder.

In agreement with our hypothesis, MDX mice were more susceptible to doxorubicin-induced cardiotoxicity than WT mice. This was evidenced by more extensive deterioration in cardiac function, more pronounced changes in heart geometry, and by a higher degree of cardiac interstitial fibrosis in MDX mice than in WT mice. In addition, MDX-DOX animals had a higher mortality, although this may have been caused by DOX effects on other tissues. In addition to heart dysfunction, DOX causes toxicities in several other tissues, involving e.g. bone marrow, kidney, and small intestine. The cause of death in DOX-treated mice is, therefore, very difficult to assess. The higher mortality in DOX-treated MDX mice in comparison to WT mice indicates a role of dystrophin, but other factors or organs must also play a role, since lethality was also observed in DOX-treated WT mice. In contrast, untreated MDX mice had cardiac function and histology comparable to untreated WT mice, which is in agreement with previous investigations of young MDX mutants [19].

To elucidate the mechanism responsible for the increased sensitivity of MDX mutants to DOX, we measured and compared DOX-induced global transcriptional changes in WT and MDX mice. Expression between untreated MDX and WT mice differed in genes encoding for cell adhesion, cytoskeleton, and inflammatory response. Changes in the expression of cell adhesion and cytoskeleton genes in MDX mice were consistent with previous observations [25] and were a compensation for dystrophin deficiency. In humans, cell adhesion proteins are elevated in the failing heart [26], i.e. in phenotypes observed in old MDX mice. The cell adhesion gene Spp1 (see Table in Appendix A) regulates collagen synthesis and accumulation after myocardial infarction [27]. Increased cardiac Spp1 expression has been reported at the onset and during progression of heart failure [28]. In addition, Spp1 plays a role in chemotaxis, indicating a role in the inflammatory response [29]. Inflammation may lead to dilated cardiomyopathy [30] and has been described in old MDX mice together with necrosis and fibrosis [19].

DOX treatment of WT mice results in differential expression of two genes implicated in various aspects of the immune response, including antigen processing and presentation, and T cell differentiation. Immune response may play a role in the development of heart failure, since immunomodulating agents such as a serine elastase inhibitor and vesnarinone improve function and histology in animal models of this disorder [26,31]. The number of genes differentially expressed between treated and untreated WT mice is slightly higher than the 23 genes reported by Yi and colleagues [14]. This discrepancy may be a result of the different mouse strains used and different time course for microarray experiments, despite the same cumulative dose of DOX in both experiments.

In MDX mice, DOX treatment resulted in the differential expression of genes representing 7 additional biological processes. Expression changes of cytolysis-related genes indicate that myocardial death induced by doxorubicin in MDX mice may be mediated through necrosis in addition to apoptosis. Although apoptosis occupies a more prominent place in the pathogenesis of DOX-related cardiotoxicity [10,32], there is also substantial evidence supporting a role of necrosis in this process [33]. Necrotic cells may release subcellular membrane components rich in mitochondria which are capable of triggering complement-mediated inflammation [34], conversely, the aforementioned inflammation or immune response may lead to cell death via necrosis or apoptosis. In addition, genes which inhibit apoptosis are up-regulated in DOX-treated MDX mice. Down-regulation of apoptosis may reflect a protective response of the heart tissue to excessive cell death.

Changes in genes related to angiogenesis and heart development observed in MDX mice following DOX treatment are consistent with the activation of tissue repair processes. The two metallothionein genes likely reflect a protection against DOX-induced oxidative stress [22]. In contrast, differential expression of the oxygen transport hemoglobin genes most likely represents a chance finding, since these genes are not expressed in the heart tissue.

At first glance, changes in cell adhesion and cytoskeletal genes between treated and untreated MDX mice appear to be unrelated to DOX exposure, since they are also present in untreated MDX and WT animals. However, the number of differentially expressed cell adhesion genes is much higher following DOX treatment (7 vs. 3 genes, Table 3). More importantly, of the two cytoskeletal genes, the expression of Myl7 is repressed in treated MDX mice, as opposed to the over-expression observed in untreated MDX mice. An opposite relationship is observed for the cell adhesion genes Spp1 and Postn (periostin, osteoblast specific factor), where repression in untreated MDX mice is followed by a strong over-expression in response to DOX. Therefore, we postulate that changes in the expression of these two genes are DOX-specific, at least in part. Changes in cytoskeleton and cell adhesion genes have previously been reported in rats [12] and mice [14] treated with DOX.

The above observations are consistent with an accelerated development of cardiomyopathy in DOX-treated MDX mutants rather than with a distinct, MDX-specific process. Indeed, changes seen in MDX-DOX mice such as necrosis, inflammation, and fibrosis also develop in untreated old MDX mutants [19]. The higher sensitivity to DOX may be caused by DOX-induced impairment of cytoskeleton/sarcolemma protein, which may augment the effect of dystrophin deficiency on cell membrane integrity. The resulting cell membrane injury, via calcium influx, likely triggers apoptosis, necrosis, and tissue repair mechanisms such as fibrosis.

The significance of our findings may go beyond the rare cases of DOX-treated male and female carriers of Duchenne mutations. Cardiotoxicity is observed only in a fraction of all DOX-treated patients, which may be caused by the genetic makeup of the affected individuals [35]. Data presented here suggest that variants of other cytoskeletal and sarcomeric proteins may affect the individual sensitivity to doxorubicin. Conversely, DOX and other cardiotoxic drugs may enhance the manifestation or increase the progression of mild, subclinical DCM forms. DCM is the most frequent form of primary myocardial disease and the third most common cause of heart failure. Strongly penetrant mutations, mostly in cytoskeleton and sarcolemma genes, are responsible for at least a third of manifest DCM cases [36]. An unknown number of less penetrant but more common alleles may interact with cardiotoxic drugs such as DOX, leading to clinically manifest DCM.


   Appendix A. Supplementary data
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Appendix A. Supplementary data
References
 
Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ejheart.2007.07.016.


   Acknowledgements
 
This study was partially funded by the German Federal Ministry for Education and Science (BMBF) grant 01GS0421.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Appendix A. Supplementary data
 References
 

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