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Cardiovascular Research 1999 43(2):398-407; doi:10.1016/S0008-6363(99)00142-X
© 1999 by European Society of Cardiology
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Copyright © 1999, European Society of Cardiology

Doxorubicin induces slow ceramide accumulation and late apoptosis in cultured adult rat ventricular myocytes

Eric Delpya, Stéphane N Hatema, Nathalie Andrieua, Cyrille de Vaumasa, Morgana Henaffa, Catherine Rücker-Martinb, Jean-Pierre Jaffrézouc, Guy Laurentc, Thierry Levaded and Jean-Jacques Mercadiera,*

aINSERM U 460, Faculté de Médecine Xavier Bichat, 16 rue Henri Huchard, 75018 Paris, France
bCNRS ERS 566, Centre Chirurgical Marie Lannelongue, Le Plessis Robinson, France
cINSERM CJF 9503, Centre Claudius Régaud, Toulouse, France
dINSERM U 466, CHU de Rangueil, Toulouse, France

* Corresponding author. Tel.: +33-144-856-158; fax: +33-144-856-157 jjmercadier{at}wanadoo.fr

Received 16 December 1998; accepted 23 March 1999


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objectives: Anthracyclines cause apoptotic death in many cell types through activation of the ceramide pathway. We tested the hypothesis that doxorubicin induces cardiac myocyte apoptosis through ceramide generation. Methods: Adult rat ventricular myocytes were grown in the presence of 10% fetal calf serum, and exposed to 0.5 µM doxorubicin (Dox) for 1 h on the day of cell isolation (day 0). We used the membrane-permeant ceramide analog C2-ceramide (C2-cer) to mimic the effects of endogenous ceramide and PDMP to induce endogenous ceramide accumulation. Apoptosis was assessed upon morphological criteria and DNA fragmentation by the TUNEL method and agarose gel electrophoresis. Ceramide concentration was assessed using the DAG kinase assay. Results: Myocyte exposure to Dox was associated with cellular and nuclear alterations typical of apoptosis on day 7 but not on day 3. At day 7, the percentage of TUNEL-positive myocytes was markedly increased in Dox-treated cultures compared to control (Cl) cultures (82±3 vs. 12±1%, n=7; p<0.001); internucleosomal DNA fragmentation was confirmed by the observation of DNA ladders. These alterations were associated with an increase in the intracellular ceramide concentration (1715±243 vs. 785±99 pmol/mg prot, n=5; p<0.01), a phenomenon also detected on day 3 (731±59 vs. 259±37 pmol/mg prot, n=5; p<0.001). Incubation of myocytes at day 0 with 50 µM C2-cer induced rapid cell shrinkage and DNA fragmentation (45±3 vs. 10±1% TUNEL-positive myocytes on day 1 in C2-cer-treated and Cl cultures, respectively; n=6, p<0.001). Myocyte exposure to 10 µM PDMP for 7 days (n=5), caused ceramide accumulation (1.7-fold increase vs. Cl, p<0.01), and a marked increase in the percentage of TUNEL-positive myocytes (62±6 vs. 11±3% in Cl cultures, p<0.001). Ventricles of rats injected intraperitoneally with a cumulative dose of 14 mg/kg Dox over a period of 2 weeks also showed an increased ceramide concentration 2 weeks later (11.01±0.64 vs. 5.24±0.88 pmol/mg prot, n=6; p<0.001). Conclusion: Our study confirms the existence of a functional ceramide pathway related to apoptosis in cardiac myocytes, and points to its possible involvement in doxorubicin-induced cardiomyopathy.

KEYWORDS Apoptosis; Cardiomyopathy; Heart failure; Myocytes


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
The anthracycline drug doxorubicin is widely used to treat a number of cancers, but its use remains limited by both acute and delayed dose-dependent cardiotoxicity. Several hypotheses have been forwarded to account for this cardiotoxicity, including free radical-mediated myocardial injury, myocyte damage induced by calcium overload, impaired myocardial adrenergic regulation, release of vasoactive amines, and cellular toxicity of anthracycline metabolites (reviewed in Refs. [1] and [2]). Surprisingly, the fact that anthracycline cardiotoxicity may result, at least in part, from the induction of cardiac myocyte apoptosis has been little investigated although it has been shown in leukemic cell lines that anthracyclines including doxorubicin exert most of their anti-tumor effect through the induction of cell apoptosis [3,4]. Such an hypothesis is supported by the recent demonstration that doxorubicin associated with serum deprivation induced the apoptosis of neonatal rat cardiomyocytes in primary cultures, a process which was partly prevented by pretreating cells with IGF I [5].

Ceramide has recently emerged as an important lipid second messenger mediating a number of downstream events such as cell proliferation, differentiation, growth arrest and apoptosis, depending on the cell type and physiological context considered [6,7]. Regarding apoptosis, although ceramide generation has been initially restricted to specific apoptotic triggers and pathways, data are accumulating now to suggest that ceramide would be more widely involved in mediating the apoptotic process. For instance, although formation of the death domain adaptor protein complexes following TNF receptor activation appears independent of ceramide, it seems that its ability to confer apoptosis may depend on coordinated signaling via ceramide [8]. In the heart, an activation of the ceramide pathway has been implicated in cardiac myocyte apoptosis induced by TNF [9] and also during the hypoxia–reoxygenation sequence in vitro and the ischemia–reperfusion sequence in vivo [10], demonstrating the existence of a ceramide-mediated apoptotic pathway in these cells.

It has been shown also in leukemic cell lines that daunorubicin, another member of the anthracycline family, generates intracellular ceramide and induce apoptosis [11,12]. Therefore, the aim of the present study was to determine whether ceramide generation could be involved in doxorubicin-induced apoptosis of cardiac myocytes. We used a model of cultured adult rat ventricular myocytes widely used to study a number of molecular changes occurring in cardiac myocytes during various physiological and pathological conditions [13–15]. Fetal calf serum (FCS) was added to the culture medium throughout the culture to avoid the intrinsic pro-apoptotic effect of serum deprivation [5]. We also examined whether doxorubicin injection to rats induced cardiac ceramide accumulation.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
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 1985).

2.1 Isolation and culture of cardiac myocytes
Adult rat ventricular myocytes were isolated as described [16] with slight modifications: male Wistar rats (200–250 g) were anesthetized with sodium pentobarbital (60 mg/kg i.p.) and hearts were rapidly excised and subjected to retrograde perfusion (8 ml/min) in a Langendorff apparatus with Krebs’ solution containing (in mM): KCl 4.75; KH2PO4 1.2; NaCl 35; Na2HPO4 16; NaHCO3 25; HEPES 10; glucose 10 and sucrose 134; pH 7.4 at 37°C for 3 min. Hearts were then perfused with the same solution containing collagenase (0.62 IU/ml, Boehringer Mannheim), and hyaluronidase (147 IU/ml, Sigma–Aldrich) for approximately 15 min. Once the enzymatic digestion was achieved, ventricular myocytes were isolated by several low-speed centrifugation (10xg, 3 min) and sedimentation (5 min) steps. An average of 5.6±0.4x106 (n=44) myocytes per heart were obtained, and 81±0.9% of the isolated myocytes were calcium-tolerant, i.e. rod-shaped and well striated. Myocytes were resuspended in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (Biowhittaker), 4% non-essential amino acids (GIBCO), 1 nM insulin (Serva) and antibiotics (100 IU/ml penicillin; 0.1 mg/ml streptomycin, GIBCO). Myocytes were then plated (day 0) on laminin (10 mg/ml, GIBCO) pre-coated 2-well Labtek chambers (NUNC) at a density of 90 000 myocytes/well or on laminin pre-coated culture flasks (Falcon) at a density of 600 000 myocytes/25 cm2. After an adhesion period of 1 h, myocytes were rinsed twice with culture medium to eliminate non adherent cells, dead cells and debris. Cytosine β-D-arabino-furanoside (10 µM) (Sigma–Aldrich) was added to the medium to inhibit the proliferation of any non muscle cells remaining in the culture dish. The culture medium was renewed on day 1 and every other day thereafter. Myocytes were grown until day 7.

2.2 Pharmacological treatments
All pharmacological agents were added on the day of myocyte isolation (day 0), at the time of medium renewal following the 1-h adhesion period. Doxorubicin (0.5 µM) was added to the culture medium for 1 h and then washed out. The membrane-permeant ceramide analog C2-ceramide (10, 25 and 50 µM) was added on day 0 for 24 h, and so was the inactive analog C2-dihydroceramide. The ceramide glucosyltransferase inhibitor PDMP (D,L-threo-1-pheny-2-decanoylamino-3-morpholino-1-propanol, 10 µM) was added on day 0 and maintained in the culture medium until day 7. Doxorubicin was from Sigma–Aldrich and was dissolved in culture medium. C2-ceramide (N-acetyl-D-sphingosine) was from Sigma–Aldrich and was dissolved in dimethylsulfoxide. C2-dihydroceramide (N-acetyl-D-dihydrosphingosine) was from Calbiochem–Novabiochem Corporation and was dissolved in ethanol. Final concentrations of dimethylsulfoxide and ethanol were 0.1%. The same percentage of solvents was used in control cultures. PDMP was from Alexis Corporation and was dissolved in sterile water.

2.3 Eosin–hematoxylin staining and in situ detection of DNA fragmentation
Cultured myocytes were stained with eosin and hematoxylin to assess morphological changes during treatment with doxorubicin or permeant ceramides. Methanol-fixed myocytes (–20°C for 10 min) were first incubated in a coplin jar containing 1% eosin (Merck) for 20 s and then washed in water before incubation in a second coplin jar containing Harry’s hematoxylin (Diagnostica Merck) for 10 s. After several washes in water, the slides were dehydrated and mounted (Eukitt, Kindler GmbH).

DNA fragmentation was detected in situ by means of the TUNEL method (in situ cell death detection kit, Boehringer Mannheim). Cultured myocytes were fixed with methanol at –20°C for 10 min and permeabilized with 0.1% Triton X100 (Sigma–Aldrich) at 4°C for 2 min. After two 10-min washes in PBS, cells were incubated with terminal deoxynucleotidyl transferase and FITC-conjugated-dUTP at 37°C for 1 h. After two washes in PBS, coverslips were mounted with Fluoprep (bioMérieux). Positive control cells were myocytes treated with DNase I (1 mg/ml) at 37°C for 10 min. Negative controls omitted the terminal deoxynucleotidyl transferase during the TUNEL reaction. Three to five hundred cells were counted in each experimental condition, using a microscope fitted with an eyepiece reticule grid (400x magnification); the proportion of positive myocytes was expressed as a percentage of the total number of cells counted.

2.4 DNA extraction and electrophoresis
Cultured myocytes were scraped off the flask and centrifuged (1000xg, 5 min) in PBS at 4°C. The pellet was resuspended in 0.5 ml lysis buffer comprising 75 mM NaCl, 0.5% SDS, 10 mM Tris–HCl, 10 mM EDTA, pH 8.0, and 0.15 mg/ml proteinase K (Sigma–Aldrich), and incubated at 50°C for 3 h [17]. The lysate was then incubated with 200 µg/ml RNase A (Sigma–Aldrich) at 37°C for 1 h. After extraction with phenol and chloroform, DNA was precipitated with 2 vols of ethanol and centrifuged; the pellet was washed with 70% ethanol and resolubilized in an appropriate volume of TE (10 mM Tris–HCl, 1 mM EDTA, pH 8.0). DNA was quantified by means of spectrophotometry. Equal amounts of each sample (10 µg) were electrophoresed on 1.5% agarose gels containing 0.5 mg/ml ethidium bromide, alongside 2 µg of molecular weight marker (123 bp DNA ladder, Sigma–Aldrich). DNA laddering was visualized under UV light.

2.5 Intracellular ceramide assay
Myocytes grown in culture flasks were scraped off and centrifuged (1000xg, 5 min) in PBS at 4°C. Cell pellets were suspended in 0.7 ml of distilled water and cells were further disrupted by sonication at 4°C for 10 s using a Soniprep MSE sonicator probe. An aliquot was taken for protein determination and the homogenate was extracted with 2.5 ml of chloroform/methanol (2:1, v/v), vortex-mixed and centrifuged at 1000xg for 15 min. The organic phase was evaporated under nitrogen. Ceramide mass was measured as described elsewhere [18] by using Escherichia coli diacylglycerol kinase and [{gamma}-32P]ATP. Radioactive ceramide-1-phosphate was isolated by thin-layer chromatography with chloroform/acetone/methanol/acetic acid/water (50:20:15:10:5, v/v) as the developing solvent.

2.6 Doxorubicin administration to rats in vivo
Six male Wistar rats weighing 220–240 g were injected intraperitonally every other day with 2 mg/kg doxorubicin over a period of 2 weeks, resulting in a total cumulative dose of 14 mg/kg per animal, a protocol which has been shown to induce various degrees of heart failure 3–6 weeks later [19]. Six age-matched rats injected with vehicle only were used as controls. All rats received food and tap water ad libitum. Two weeks after the last injection, rats were weighed and anesthetized as described above. Hearts were rapidly excised and rinsed in cold saline. Ventricles were dissected from the atria and large vessels, weighed, frozen in liquid nitrogen and stored at –80°C until use. For ceramide assay, ventricles were pulverized in liquid nitrogen and the resulting powder was suspended in 2 ml of distilled water. An aliquot was taken for protein determination. The extraction and assay of ceramide mass were carried out as described above using 0.5 ml of ventricular homogenate.

2.7 Statistical analysis
Data are expressed as means±S.E.M. The number of cultures performed for each experiment is indicated throughout the text, each culture arising from distinct myocyte isolations. All values were compared using the Student’s unpaired t-test. For the results of TUNEL reaction, the percentages of positive cells in the various experimental conditions were compared after arcsinus transformation. Differences were considered significant when p values were below 0.05.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Doxorubicin induces delayed apoptosis in cultured cardiac myocytes
In keeping with the rapid apoptotic effect of daunorubicin on leukemic cell lines [11,12], in a first set of experiments cardiomyocytes were exposed to 0.5 µM doxorubicin for 1 h and examined during the first 24 hours of culture for morphological changes and survival. As no significant changes were observed during this early period of culture, myocytes were maintained in long-term culture to examine whether doxorubicin induced late toxicity during serum-induced myocyte growth. In control conditions, myocytes kept their rod shape during the first 2 days in culture but started to become rounded and to spread out around day 3 (not shown); thereafter they continued to increase in size and began to establish cell–cell contacts on day 7 (Fig. 1A). Myocytes exposed to 0.5 µM doxorubicin for 1 h on day 0 showed no morphological alterations compared with control myocytes until days 5–6, when their spreading appeared to slow. On day 7 they were markedly shrunken, exhibited thin cytoplasmic extensions around the cell body, and had irregular and condensed nuclei (Fig. 1B). Higher concentrations of doxorubicin (1–10 µM) inhibited cell spreading after day 3 or had a marked toxicity, as indicated by the rapid decrease in cell density (not shown).


Figure 1
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  		Fig. 1 Effect of 0.5 µM doxorubicin on the morphology of adult rat ventricular myocytes after 7 days of culture in the presence of 10% FCS. Myocytes were treated (B) or untreated (A) with doxorubicin on day 0 for 1 h. Methanol-fixed cells were stained with eosin and hematoxylin as described in Methods. Bar: 25 µm.

 
To determine whether the above morphological changes were related to apoptosis, nuclear DNA fragmentation was assessed using the TUNEL method and DNA gel electrophoresis. Following doxorubicin treatment, the proportion of cells showing DNA fragmentation on day 3 averaged 11±1%, a value not different from that in control cultures (Fig 2A). In sharp contrast, on day 7, doxorubicin-treated cultures showed a marked increase in the number of TUNEL-positive myocytes, compared both to control cultures (82±3 vs. 12±1%, n=7; p<0.001) and to doxorubicin-treated cultures on day 3 (11±1, n=6; p<0.001). Fig. 3 gives a typical example of DNA fragmentation visualized in situ on day 7 by using the TUNEL method in doxorubicin-treated and control cultures. Internucleosomal DNA fragmentation in doxorubicin-treated cells on day 7 was confirmed by the consistent observation of characteristic DNA ladders on agarose gels (Fig. 4). Taken together, these results indicated that exposure of freshly isolated adult rat ventricular myocytes to 0.5 µM doxorubicin for 1 h was associated with a delayed apoptotic process.


Figure 2
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  		Fig. 2 Effect of 0.5 µM doxorubicin on DNA fragmentation (TUNEL staining) (A) and ceramide levels (B) in adult rat ventricular myocytes after 3 and 7 days of culture in the presence of 10% FCS. Myocytes were treated (black bars) or untreated (white bars) with doxorubicin on day 0 for 1 h. Results are means±S.E.M. ** p<0.01 and *** p<0.001 vs. control values; ++ p<0.01 vs. corresponding values at day 3.

 

Figure 3
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  		Fig. 3 In situ detection of DNA fragments using the TUNEL method in adult rat ventricular myocytes after 7 days of culture in the presence of 10% FCS. (A) Control culture; (B) culture treated on day 0 with 0.5 µM doxorubicin for 1 h. Bar: 25 µm.

 

Figure 4
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  		Fig. 4 Electrophoretic pattern (1.5% agarose gel) of DNA extracted from myocytes treated or not with 0.5 µM doxorubicin on day 0 for 1 h and grown for 7 days in the presence of 10% FCS. (A) DNA molecular weight marker (multiples of 123 bp); (B) control myocytes; (C) doxorubicin-treated myocytes.

 
3.2 Doxorubicin induces ceramide generation in cultured cardiac myocytes
As anthracyclines activate sphingomyelin hydrolysis, generate intracellular ceramide and induce apoptosis [12], we measured ceramide concentrations in cardiac myocytes in control conditions and following doxorubicin exposure (Fig. 2B). The ceramide concentration in control myocytes was 259±37 pmol/mg protein after 3 days of culture, and raised to 785±99 pmol/mg protein after 7 days of culture (n=5, p<0.01). Myocyte exposure to 0.5 µM doxorubicin for 1 h on day 0 induced a further increase in the ceramide concentration, both on day 3 (731±59 pmol/mg protein, n=5; p<0.001 vs. control) and on day 7 (1715±243 pmol/mg protein, n=5; p<0.01 vs. control; and n=5, p<0.01 vs. value on day 3). Therefore, ceramide accumulation in cultured cardiac myocytes was increased by brief doxorubicin exposure, preceding myocyte apoptosis.

3.3 Ceramide induces apoptosis in cultured cardiac myocytes
To confirm the possibility that ceramide mediates doxorubicin-induced myocyte apoptosis in vitro, myocytes were exposed to various concentrations of the membrane-permeant ceramide analog C2-ceramide. Addition of 50 µM C2-ceramide to the culture medium of freshly isolated myocytes induced, 24 h later, marked morphological changes characterized by cell shrinkage and membrane protrusions (Fig. 5B). In contrast, myocyte exposure to 50 µM C2-dihydroceramide, the inactive analog of C2-ceramide, had no effect (Fig. 5C). Morphological changes observed with C2-ceramide were associated with DNA fragmentation as shown by the TUNEL method (45±3 vs. 10±1%, n=6, p<0.001, Fig. 6). A lower C2-ceramide concentration (25 µM) induced both lesser morphological alterations (not shown) and a smaller proportion of TUNEL-positive myocytes (29±2% TUNEL-positive myocytes, n=5, p<0.001 and n=5, p<0.01 vs. control and vs. 50 µM C2-ceramide, respectively). In contrast, higher C2-ceramide concentrations (75 µM) were associated with major cytotoxicity, as evidenced by a marked decrease in cell density (not shown). These results indicated that exogenous ceramide induced rapid apoptosis of adult rat ventricular myocytes in vitro.


Figure 5
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  		Fig. 5 Effect of C2-ceramide on the morphology of rat ventricular myocytes after 24 h of culture in the presence of 10% FCS. Methanol-fixed cells were stained with eosin and hematoxylin as described in Methods. (A) Control culture; (B) culture in the presence of 50 µM C2-ceramide; (C) culture in the presence of 50 µM C2-dihydroceramide, the inactive C2-ceramide analog. Bar: 25 µm.

 

Figure 6
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  		Fig. 6 Effect of C2-ceramide on DNA fragmentation (TUNEL staining) in rat ventricular myocytes grown for 24 h in the presence of 10% FCS. Results are means±S.E.M. * p<0.05 and *** p<0.001 vs. control conditions; ++ p<0.01 vs. lower C2-ceramide concentration.

 
We then examined the effect of slow accumulation of endogenous ceramide by using the ceramide glucosyltransferase inhibitor PDMP [20], which inhibits ceramide conversion into glucosylceramide [21]. After 3 days of culture with 10 µM PDMP, the myocyte ceramide concentration was not different from that in control conditions. However, on day 7 it rose to 1000±108 pmol/mg protein, compared to 574±36 pmol/mg protein in control conditions (n=5, p<0.01; Table 1). As following the brief doxorubicin exposure of myocytes at day 0, myocytes grown in the presence of 10 µM PDMP also exhibited DNA fragmentation on day 7 (62±6 vs. 11±3% TUNEL-positive myocytes in PDMP-treated and control myocytes, respectively; n=5, p<0.001; Table 1). These results indicated that slow ceramide accumulation resulting from inhibition of ceramide glucosyltransferase was associated with delayed myocyte apoptosis.


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  		Table 1 Ceramide concentration and DNA fragmentation (TUNEL staining) in rat ventricular myocytes at day 7 of culture in the presence of 10 µM PDMP and in control conditions (n=5 cultures)

 
3.4 Doxorubicin induces ceramide accumulation in rat ventricles
Intraperitoneal injection of a cumulative dose of 14 mg/kg doxorubicin to adult rats over a period of 2 weeks was associated, 2 weeks later, with a decrease in body weight (284±13 vs. 378±8, n=6, p<0.001; Fig. 7), while the ventricular weight to body weight ratio did not change (3.2±0.1 vs. 2.9±0.1 mg/g, n=6, ns), in good agreement with previous studies [19,22]. The ventricular ceramide concentration increased in doxorubicin-treated rats to twice the control value (11.01±0.64 vs. 5.24±0.88 pmol/mg protein, respectively; n=6, p<0.001).


Figure 7
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  		Fig. 7 Body weights (left), ventricular weight to body weight ratios (middle), and ventricular ceramide concentrations (right) in control and doxorubicin (Doxo)-treated rats. Doxorubicin (14 mg/kg) was administered over 2 weeks, as described in Methods. Animals were killed 2 weeks later. Values are means±S.E.M for six rats in each group. *** p<0.001.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
To our knowledge this is the first report that brief exposure of freshly isolated adult rat ventricular myocytes to an anthracycline drug: (i) induces delayed myocyte apoptosis, as indicated by typical cellular and nuclear morphological changes and DNA fragmentation, (ii) preceded by intracellular accumulation of ceramide, a mediator of apoptosis in a number of cell types. Taken together, these data confirm the existence of a functional ceramide pathway linked to the induction of apoptosis in adult cardiac myocytes, as recently demonstrated during ischemia/reperfusion-induced myocardial cell death [10] and TNF-{alpha}-induced apoptosis of cardiac myocytes in vitro [9]. Our data also extend to the adult cardiac myocyte, a terminally differentiated cell type, the observation that anthracyclin-induced apoptosis occurs, at least in part, through activation of a metabolic pathway rather than through the crippling of cell metabolism. In addition, our demonstration that doxorubicin induces myocardial ceramide accumulation in vivo suggests that activation of the pathway may also be involved in the pathophysiology of anthracycline-induced cardiomyopathy.

We used a doxorubicin concentration of 0.5 µM and a 1-h myocyte exposure time both to mimic conditions of in vivo delivery and because 0.5–1 µM daunorubicin optimally induces apoptosis and ceramide accumulation in leukemia cell lines [12]. Higher anthracycline concentrations are considered pharmacologically irrelevant and clinically unachievable [23]. Regarding the exposure time, 1-h drug treatment followed by drug-free incubation is considered to reflect in vivo pharmacokinetics more closely than longer exposure [23]. In pilot experiments with doxorubicin we found that concentrations below 0.5 µM were inefficient in inducing myocyte apoptosis, while concentrations above 1 µM were toxic for myocytes. Prolonging the exposure time beyond 1 h had no additional effect.

Our observation that myocyte apoptotic death only occurred 7 days after a brief exposure to doxorubicin, whereas a significant increase in ceramide accumulation occurred after only 3 days, suggests that doxorubicin-induced apoptosis of adult rat ventricular myocytes in vitro is a slow process which results from ceramide accumulation. Moreover, the fact that the intracellular concentration of ceramide increased on days 3 and 7 in doxorubicin-treated and control myocytes, respectively, in the absence of any increase in the proportion of TUNEL-positive myocytes (Fig. 2), also suggests the existence of a ceramide concentration threshold required to trigger downstream apoptotic mechanisms. This is also supported by our observation that high concentrations (50 µM) of the membrane-permeant ceramide analog C2-ceramide, which mimics the proapoptotic effect of agents such as TNF-{alpha} [24], induced myocyte apoptosis within 24 h, whereas lower C2-ceramide concentrations (e.g. 10 µM) failed to increase the number of TUNEL-positive myocytes, even after several days of culture (not shown). The possibility that myocyte apoptosis occurs only when intracellular ceramide concentration reaches a threshold is also supported by our finding that slow accumulation of endogenous ceramide induced by the ceramide glucosyltransferase inhibitor PDMP caused apoptosis of ventricular myocytes with a time course similar to that following brief doxorubicin exposure. Taken together, these results strongly suggest that ceramide accumulation following doxorubicin exposure is responsible for the delayed myocyte apoptosis. Nevertheless, the causal role of ceramide in the induction of myocyte apoptosis can only be confirmed by the use of specific inhibitors, which are not available.

There are potentially a large number of mechanisms by which anthracycline-induced ceramide accumulation could induce myocyte apoptosis. One is the activation of caspase proteases. An ischemia–reperfusion sequence of the rat left ventricle in vivo is associated with myocyte apoptosis and increased CPP32 levels which co-localizes in the ischemic/reperfused region [25] and it has been shown that ceramide-signaled apoptosis in vincristine-treated ALL-697 leukemia cells occurs via the activation of a CPP32-like protease, an effect which is inhibited by Bcl-2 [26,27]. Another possibility is an increased expression of p53 as observed during stretch-induced cardiac myocyte apoptosis and the resulting release of angiotensin II [28]. It is also possible that ceramide accumulation induced the upregulation of Fas protein as observed following hypoxia of cultured neonatal rat cardiomyocytes [29] or the ischemia–reperfusion sequence of rabbit ventricle in vivo which also induced activation of stress-activated protein kinase (SAPK) [30]. Herr et al. [31] found that that ceramide generated by doxorubicin induced the expression of Fas-ligand (CD95-L), which then caused cell death by activating Fas at the cell surface through an autocrine or paracrine process, another possibility which could explain the delayed apoptosis in our study. Moreover, Verheij et al. [32] have shown that ceramide can induce apoptosis in primary cultures of bovine endothelial cells and in U937 monoblastic leukemia cells by activating the SAPK/JNK signaling pathway. Of special importance is the role of mitochondria in the effector phase of apoptosis. Indeed, numerous investigations define mitochondrial perturbation as a committed step in ceramide signaling of apoptosis and the mitochondria appear to function as a cellular sensor of ceramide which signals mitochondrial permeability transition (PT) and induces the release of apoptosis inducing factor (AIF) and cytochrome c (Cyt c), a process which is inhibited by Bcl-2 [8]. Importantly, a major cause of anthracycline cardiotoxicity is free radical generation [1,33], and recent data implicate mitochondrial hydrogen peroxide generation in ceramide-induced apoptosis [34]. It is thus possible that ceramide accumulation also participates in the free radical myocyte injury induced by doxorubicin.

We used 10% FCS in the culture medium throughout cell culture to avoid a possible intrinsic proapoptotic effect of serum withdrawal on cardiac myocytes [5]. We cannot rule out the possibility that this affected doxorubicin induction of apoptosis in these cells. It has been shown in other cell types that a balance between SAPK/JNK and ERK signaling pathways may determine the apoptotic outcome; in addition, while ceramide initiates the apoptotic pathway it does not commit the cells to death, as ERK and PKC signaling or Bcl-2 can attenuate the process at multiple checkpoints downstream of ceramide generation (for a review, see Ref. [8]). It is thus possible that the FCS and insulin added to the culture medium had a protective effect, delaying ceramide-induced apoptosis by activating the ERK cascade [35] or by inhibiting the activity of CPP32 protease, as reported in cultured fetal cardiomyocytes exposed to doxorubicin [5]. Culture of adult cardiac myocytes in the presence of serum is also known to be associated with a degree of cell dedifferentiation [13,14], a process which may limit the relevance of our findings to the in vivo situation. However, the fact that ceramide also accumulated in the ventricles of rats injected with doxorubicin clearly indicates that the ceramide pathway can also be triggered by doxorubicin in vivo. Further studies are needed to determine whether such accumulation is also associated with the induction of myocyte apoptosis.

The source of doxorubicin-induced ceramide generation in cardiac myocytes is important with respect to possible pharmacological inhibition of anthracyclin cardiotoxicity. In leukemic cell lines treated with daunorubicin, ceramide is generated either by sphingomyelin hydrolysis through neutral sphingomyelinase activation [12] or by de novo synthesis through the activation of ceramide synthase [11] and Mansat et al. [36] reported that serine protease inhibitors which blocked the neutral sphingomyelinase activation, also prevented ceramide generation and apoptosis occurring after U937 and HL60 cell exposure to daunorubicin. Studies are under way to identify the process underlying doxorubicin-induced ceramide accumulation in cardiac myocytes.

Another important question is the contribution of apoptosis to the toxicity of anthracyclines in vivo. Zhang et al. [37] have reported that the cardiomyopathy induced by doxorubicin in spontaneously hypertensive rats is not associated with apoptosis of cardiac myocytes but rather with apoptosis of non muscle cells of interstitial tissue. However, in the Zhang et al. [37] study apoptosis was assessed one week after the last injection of a rather low dose of doxorubicin delivered at wide time intervals (1 mg/kg, once a week for 12 weeks) and it is possible that apoptosis occurred earlier or later, with no trace at the time of investigation. More likely, the degree of myocyte apoptosis induced by doxorubicin in this model may have been too low to be detected by the TUNEL method. Indeed, there seems to be now a general agreement that the percentage of apoptotic myocytes detected in vivo at any given time is very low (less than 1 per 1000 myocytes), regardless of the pathophysiological circumstances. This could be due also to the fact that in rats with genetic hypertension, ventricular myocytes undergo an hypertrophic process as in our in vitro experimental conditions, with anti-apoptotic pathways being activated and which hinders the induction of apoptosis by doxorubicin. At all events, our observation that ceramide also accumulates in rat ventricles following doxorubicin exposure in vivo warrants studies aimed at identifying a ceramide-mediated doxorubicin-induced cardiac myocyte apoptotic pathway in animal models of anthracycline-induced cardiomyopathy.

Whatever the contribution of cardiac myocyte apoptosis in the anthracycline-induced cardiomyopathy, the present observation that doxorubicin caused ceramide accumulation in cardiac myocytes also raises the question of the other effects of the sphingolipid in the pathogenesis of this cardiomyopathy. For instance, heart failure resulting from anthracycline exposure is also characterized by a myofibrillar degeneration [1,38], a process also observed in vitro [39] and Sussman et al. [40] observed that PKC{varepsilon} activation is an important event in the initiation of doxorubicin-induced myofibrillar degeneration in neonatal cardiac myocytes, a protein kinase which can be activated by ceramide [41]. In conclusion, the results of our study strongly suggest that doxorubicin-induced apoptosis of adult rat ventricular myocytes in vitro is mediated, at least in part, by slow intracellular accumulation of ceramide. The relative importance of each downstream pathway must now be determined with a view to developing new therapeutic strategies. Prevention of ceramide generation may be one such possibility if the watershed position of this sphingolipid in several pro-apoptotic pathways in cardiac myocytes is confirmed.

Time for primary review 27 days.


   Acknowledgements
 
We thank Michèle Heimburger and David Young for their help in preparing and restyling the manuscript, respectively. This work was supported in part by grants from the Institut National de la Santé et de la Recherche Médicale (INSERM), the Centre National de la Recherche Scientifique (CNRS) and the Association Française contre les Myopathies (AFM). Nathalie Andrieu was supported by a grant from the Association pour la Recherche contre le Cancer (ARC).


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

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