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(The FASEB Journal. 1999;13:1501-1510.)
© 1999 FASEB

L-carnitine prevents doxorubicin-induced apoptosis of cardiac myocytes: role of inhibition of ceramide generation

NATHALIE ANDRIEU-ABADIE1, JEAN-PIERRE JAFFRÉZOU*, STÉPHANE HATEM, GUY LAURENT*, THIERRY LEVADE{dagger} and JEAN-JACQUES MERCADIER

INSERM Unit 460, UFR de Médecine X. Bichat, Paris, France;
* CJF INSERM 9503, Institut Claudius Régaud, Toulouse, France; and
{dagger} INSERM Unit 466, Laboratoire de Biochimie Médicale, Centre Hospitalier Universitaire de Rangueil, Toulouse, France

1Correspondence: INSERM U.460, UFR de Médecine X. Bichat, 16 rue Henri Huchard, 75870 PARIS Cedex 18 France. E-mail: andrieu{at}bichat.inserm.fr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Besides the well-documented effect of the chemotherapeutic drug doxorubicin on free radical generation, the exact signaling mechanisms by which it causes cardiac damage remain largely unknown and are of fundamental importance in understanding anthracycline cardiotoxicity. In this study, we describe that a 1 h treatment of isolated adult rat cardiac myocytes with doxorubicin (0.5 µM) induced DNA fragmentation associated with the classical morphological features of apoptosis observed after 7 days of culture. The doxorubicin toxicity was preceded by an increase in intracellular ceramide levels with a concurrent decrease in sphingomyelin. Anthracycline-induced ceramide accumulation resulted from the activation of a sphingomyelinase assayed under acidic conditions, an effect related to an increase in Vmax. Pretreatment of cardiac myocytes with L-carnitine (200 µg/ml), a compound known for its protective effect on cardiac metabolic injuries, was found to dose-dependently inhibit the doxorubicin-induced sphingomyelin hydrolysis and ceramide generation as well as subsequent cell death. However, L-carnitine did not protect cardiac myocytes from apoptosis induced by exogenous cell-permeant ceramide. L-carnitine pretreatment did not affect the sphingomyelinase basal activity but abolished the doxorubicin-induced increase in Vmax. Moreover, in vitro studies conducted on cell extracts or with purified acid sphingomyelinase demonstrated that L-carnitine exerted a dose-dependent, sphingomyelinase inhibitory effect (through Vmax reduction). Taken together, these findings show that by inhibiting a (perhaps novel) drug-activated acid sphingomyelinase and ceramide generation, L-carnitine can prevent doxorubicin-induced apoptosis of cardiac myocytes.—Andrieu-Abadie, N., Jaffrézou, J.-P., Hatem, S., Laurent, G., Levade, T., Mercadier, J.-J. L-carnitine prevents doxorubicin-induced apoptosis of cardiac myocytes: role of inhibition of ceramide generation.


Key Words: staurosporine • cardiotoxicity • oxidative stress • tumor necrosis factor


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
DOXORUBICIN IS AN anthracycline antibiotic effective in the treatment of acute leukemias and malignant lymphomas as well as a number of solid tumors (1 , 2) . Unfortunately, the cardiotoxicity of doxorubicin limits its clinical use and threatens the cardiac function of many patients with cancer (3) . The exact mechanisms that underlie doxorubicin-induced cardiac damage, even though not yet fully understood, are certainly connected with the ability of the drug to induce membrane alterations through lipid peroxidation (4) , to generate free radicals (5) , to rise myocardial concentration of sodium and calcium (6) , and to impair myocardial DNA and RNA synthesis (7) . Recently, doxorubicin has been reported to induce apoptosis in cardiac myocytes (8 , 9) , as was shown for ischemia/reperfusion (10) , tumor necrosis factor {alpha} (TNF-{alpha})2 (11) , and staurosporine (12) . Since the molecular mechanisms leading to cardiac cell apoptosis are not known, understanding this form of cardiac cell death and its regulation may help elucidate many common cardiac pathologies.

One strategy for the prevention of anthracycline-induced cardiotoxicity is based on the use of L-carnitine (ß-hydroxy-{gamma}-trimethylaminobutyric acid), a compound known for its function on the transport of long-chain fatty acids into the mitochondrial matrix (where the fatty acyl group is metabolized) (13) , but also for its anti-apoptotic activity (14 15 16) . Besides its recognized protective effects on cardiac metabolic injury induced by doxorubicin in different animal models (17 18 19 20 21 22 23) , in vivo (17 , 18) or in vitro (19 20 21) , L-carnitine and some of its analogs have been shown to influence apoptosis induced by many agents on different cell types. Addition of L-carnitine reduced apoptotic cell death in hepatocyte growth factor-deprived murine C2.8 hepatocytic cells (15) or in Fas-sensitive cell lines (16 , 24) . Moreover, acetyl-L-carnitine has been reported to inhibit apoptosis triggered by serum deprivation in a teratocarcinoma cell line (14) .

Due to its protective effect on cardiac alterations and its anti-apoptotic properties, it seemed interesting to study the effects of L-carnitine on doxorubicin signaling. Since the sphingomyelin (SM) –ceramide pathway has been shown to be activated during anthracycline-induced apoptosis in different cell types (25 26 27 28) as well as in cardiac myocytes stimulated by TNF-{alpha} (11) or ischemia and reperfusion (10) , we hypothesized that the cardioprotective effects of L-carnitine on doxorubicin treatment could be related to interference with ceramide accumulation. Ceramide is a sphingosine-based lipid signaling molecule produced by sphingomyelinase (SMase) -catalyzed hydrolysis of SM, one of the most abundant sphingolipid species in cell membranes. To date, production of ceramide has been ascribed to at least two distinct SMases: the acid lysosomal SMase, which is deficient in patients affected with Niemann-Pick disease, an inborn lysosomal SM storage disorder; and the neutral, magnesium-dependent SMase, probably located in the plasma membrane. Both enzymes have been reported to be activated in several cell types exposed to cytokines, antibodies, growth factors, and anticancer drugs (29 , 30) .

In this study, we provide evidence that L-carnitine is able to prevent doxorubicin-induced apoptosis of cultured adult rat cardiac myocytes, likely by inhibiting SM degradation and concomitant ceramide generation. Anthracycline-induced ceramide accumulation in the cardiac myocytes resulted from the activation of a SMase (assayed under acidic pH), which was abolished by L-carnitine treatment. Finally, our observations open the possibility that in this cellular model, ceramide generation is linked to mitochondrial metabolism and perhaps regulated by novel factors.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Isolation of adult rat ventricular myocytes
Cardiac myocytes were cultured after dissociation from adult male Wistar rat hearts. Briefly, hearts were quickly excised and perfused retrogradely (8 ml/min) using a Langendorff apparatus with Krebs buffer (10 mM HEPES pH 7.4, 4.75 mM KCl, 1.2 mM KH2PO4, 35 mM NaCl, 16 mM Na2HPO4, 25 mM NaHCO3, 10 mM glucose, 134 mM sucrose) containing collagenase (0.62 IU/ml, from Boehringer Mannheim) and hyaluronidase (147 IU/ml, from Sigma, St. Louis, Mo.) at 37°C for 15 min. After several low-speed centrifugation (100 x g) and sedimentation steps, cardiac myocytes were resuspended in DMEM supplemented with 10% fetal bovine serum, 4% nonessential amino acids, 1 mM insulin, 10 µM cytosine arabinose, and antibiotics. Cells were plated on laminin-coated (10 µg/ml) 2-well chambers at a density of 100,000 viable myocytes/well or on laminin-coated culture 25 cm2 flasks at a density of 600,000 viable cardiac myocytes/flasks and allowed to adhere for 1 h in a humidified 5% CO2 atmosphere at 37°C.

Treatment of cardiac myocytes with drugs
After the 1 h incubation period on day 0, cells were washed with fresh medium before treatment with doxorubicin (0.5 µM; Sigma) for 1 h or with C2-ceramide (50 µM; Sigma) for 24 h in the presence or absence of varying of concentrations of L-carnitine (2–200 µg/ml; Sigma) added 1 h before the cytotoxic drugs. Doxorubicin-treated cells were then washed twice with culture medium and further incubated for 3 or 7 days in anthracycline-free media with or without L-carnitine. In some experiments, a radiolabeled doxorubicin ([14-14C]doxorubicin hydrochloride, 55 mCi/mmol; Amersham) was used at the concentration of 0.5 µM as described previously (31) .

Cytochemical stainings
Morphological nuclear alterations were evaluated on fixed cells by fluorescence microscopy using DAPI (4',6'-diamidino 2-phenylindole) (32) or by light microscopy using eosin-hematoxylin staining [1% eosin (Merck, Rahway, N.J.) for 20 s and Harry's hematoxylin (Diagnostica Merck) for 10 s].

DNA extraction and analyses
After treatment, the adherent cells were harvested by scraping, then centrifuged and incubated in 0.5 ml of 10 mM Tris-HCl pH 8.0 containing 10 mM EDTA, 75 mM NaCl, 0.5% sodium dodecyl sulfate and 0.15 mg/ml proteinase K at 50°C for 3 h. RNase A (200 µg/ml) was then added and the lysate was incubated at 37°C for an additional hour (33) . After phenol-chloroform extraction, the DNA was ethanol precipitated and resuspended in TE (10 mM Tris-HCl pH 8.0 and EDTA 1 mM). DNA laddering was resolved on a 1.8% agarose gel and visualized with ethidium bromide as described previously (34) .

Quantitative DNA fragmentation was assessed by the spectrofluorometric DAPI procedure as previously reported (32 , 35) .

Cell viability
Since cardiac myocytes are nondividing cells, viability was estimated by measuring the decrease in the number of adherent cells, using a microscope fitted with an eyepiece reticule grid. The percentage of necrotic myocytes was determined by counting trypan blue-positive cells.

Sphingolipid extraction and analysis
Treated cardiac myocytes were harvested and sedimented by low-speed centrifugation. Cell pellets were suspended in 0.6 ml distilled water and cells were disrupted at 4°C by brief sonication. An aliquot was taken for protein determination (36) . Lipids were extracted from the remaining cell homogenate via the Folch method (37) . Cellular SM levels were quantified by measuring the lipid phosphorus content (38 , 39) .

Ceramide mass was determined using the DAG kinase as described previously (40) . Briefly, the lipid extract was incubated with Escherichia coli diacylglycerol kinase (Calbiochem, Meudon, France) and [{gamma}32P]-ATP. Radioactive ceramide-1-phosphate was isolated by thin-layer chromatography using chloroform/acetone/methanol/acetic acid/water (10:4:3:2:1) as developing solvent, located by autoradiography, scraped, and quantitated by liquid scintillation counting. Quantitative results for ceramide production were obtained using an external standard (ceramide type III from Sigma) and are expressed as pmol/mg protein.

Alternatively, intracellular SM and ceramide levels were quantified after thin-layer chromatography separation using a previously described procedure based on the sphingoid base determination (41 , 42) .

In vitro SMase assays
Cellular SMase activities were assessed, as previously reported (43) , using a mixed micellar assay system. For determining neutral SMase activity, cell extracts were mixed and incubated for 2 h at 37°C with [choline-methyl-14C]SM (100,000 dpm/assay) and 0.2% Triton X-100 in a HEPES pH 7.4 buffer. Phosphocholine was extracted using chloroform/methanol (2/1 v/v). Radioactive phosphocholine generated from [choline-methyl-14C]SM was quantitated in the aqueous phase by scintillation counting. Acid SMase activity was measured as above except that a sodium acetate pH 5.0 buffer was used instead of the HEPES buffer.

The in vitro effects of L-carnitine on purified SMases were tested as above by incubating L-carnitine with a neutral SMase from Staphylococcus aureus or with an acid SMase preparation from human placenta (both enzymes were purchased from Sigma).

Statistical analysis
Student's t test was used for statistical analysis.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of L-carnitine on doxorubicin-triggered apoptosis in cardiac myocytes
Since L-carnitine treatment has been reported to prolong the survival of rats with anthracycline-induced heart failure (17 , 18) , we investigated the effects of L-carnitine on an in vitro model of doxorubicin cardiotoxicity. Cultured adult rat cardiac myocytes were preincubated with 200 µg/ml L-carnitine for 1 h, after which 0.5 µM doxorubicin [a concentration that closely reflects the in vivo pharmacokinetics of the drug (26 , 44) ] was added for 1 h. Cells were then washed and resuspended in fresh medium containing L-carnitine. At various time points, cells were analyzed for apoptotic features. Surprisingly, no significant morphological changes were observed (as compared with untreated cells) during the early stage until 5 or 6 days of culture (data not shown). On day 7, doxorubicin treatment led to a considerable reduction in cell number (Fig. 1 A), which was associated with nuclear condensation and cell shrinkage as observed by microscopy (eosin-hematoxylin and DAPI staining) (Fig. 1B ) but without plasma membrane alterations, as indicated by trypan blue dye exclusion (Fig. 1A , inset), suggesting that doxorubicin induced apoptosis but not necrosis of adult rat cardiac myocytes. DNA quantitative analysis (Table 1 ) and electrophoresis (Fig. 1C ) revealed that doxorubicin induced ~40% of DNA fragmentation with a pattern characteristic of internucleosomal degradation. Addition of L-carnitine potently inhibited cell death (~90%) (Fig. 1A ) as well as DNA laddering (Fig. 1C ), DNA fragmentation (Table 1) , and the morphological features of doxorubicin-triggered apoptosis (Fig. 1B ). Control experiments with 0.5 µM [14-14C]doxorubicin indicated that L-carnitine did not affect anthracycline uptake (data not shown).



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  		Figure 1. Effect of L-carnitine on doxorubicin-triggered apoptosis in rat cardiac myocytes. Cardiac myocytes were preincubated in the absence or presence of L-carnitine (200 µg/ml) for 1 h, followed by a 1 h incubation with or without 0.5 µM doxorubicin. Culture medium was removed and cells were further incubated for 7 days in anthracycline-free medium with or without L-carnitine. A) Protective effect on cardiomyocyte viability. Adherent cells were treated as described above and counted. Inset: Membrane permeability was assessed with the trypan blue dye test. Results are representative of at least three independent experiments (***P<0.001). B) Morphological alterations of nuclei were evaluated either by eosin-hematoxylin staining (a–c) or by DAPI staining (d–f); a, d, control cells; b, e; doxorubicin alone; c, f, doxorubicin + L-carnitine. C) DNA fragmentation in cardiac myocytes treated with doxorubicin. DNA was extracted from treated cells, electrophoresed, stained with ethidium bromide, and photographed as described in Materials and Methods. Results for panels B and C are representative of at least three independent experiments.


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  		Table 1. Effect of L-carnitine on DNA fragmentation triggered by doxorubicin in rat cardiac myocytesa

L-carnitine did not abrogate apoptosis induced by myocyte treatment with exogenous cell-permeant ceramide for 24 h as shown by DAPI staining (Fig. 2 A), DNA electrophoresis (Fig. 2B ), and quantitative analysis (Table 1) .



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  		Figure 2. L-carnitine does not influence C2-ceramide-induced apoptosis in rat cardiac myocytes. Cardiac myocytes were treated with 50 µM C2-ceramide for 24 h in the presence or absence of L-carnitine (200 µg/ml) added to cell culture 1 h prior to the addition of ceramide. A) Morphological alterations of nuclei were evaluated using DAPI; a, C2-ceramide; b, C2-ceramide + L-carnitine. B) Agarose gel electrophoresis of DNA extracted from C2-ceramide (C2-cer) -treated cells.

Effect of L-carnitine on doxorubicin-induced ceramide generation in cardiac myocytes
Since the SM–ceramide pathway has been shown to be activated in anthracycline-induced apoptosis (25 26 27 28) , we measured ceramide levels in cardiac myocytes treated by doxorubicin in the presence or absence of L-carnitine. The amount of ceramide in doxorubicin-treated cells began to increase after 3 days, i.e., before the onset of apoptosis, and reached maximal levels at the seventh day of culture (216% of the control values; see Fig. 3 A). L-carnitine treatment (200 µg/ml) of cells led to a significant inhibition (~70%) of doxorubicin-induced ceramide generation on day 7 (Fig. 3A ). A complete inhibition of ceramide increase was observed on day 3.



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  		Figure 3. Effect of L-carnitine on doxorubicin-triggered activation of the sphingomyelin-ceramide pathway. Cardiac myocytes were preincubated in the absence or presence of L-carnitine (200 µg/ml) for 1 h, followed by a 1 h incubation with or without 0.5 µM doxorubicin. The cells were washed and further incubated for 3 or 7 days in anthracycline-free medium with or without L-carnitine. A) Inhibition by L-carnitine of ceramide generation in cardiac myocytes treated with doxorubicin. Lipids were extracted from adherent cells, and ceramide was quantitated using the DAG kinase assay. Results (pmol/mg of protein) are expressed as % of values found in cells incubated for the same period of time in the absence of drugs; they correspond to the mean ± SE of three to four independent determinations. B) Inhibition by L-carnitine of SM hydrolysis in cardiac myocytes treated with doxorubicin. SM mass was evaluated by measuring its phosphate content as described in Materials and Methods. The values correspond to the mean ± SE of three to six independent experiments. C) Inhibition by L-carnitine of SMase activities in cardiac myocytes treated with doxorubicin. The activities of neutral magnesium-dependent and acid SMases were determined with a mixed micelar assay system using [N-methyl-14C]SM at pH 7.5 and 5 as described in Materials and Methods. Data are means ± SE of 3 to 11 independent experiments. *P<0.05; **P<0.01; ***P<0.001.

Effect of L-carnitine on doxorubicin-induced sphingomyelin hydrolysis
Ceramide may be generated either through activation of ceramide synthase (25 , 45) or by SM hydrolysis catalyzed by SMase (26) . To determine the source of ceramide generated by doxorubicin treatment in cardiac myocytes, we investigated the effect of the drug on SM degradation. Doxorubicin (0.5 µM) applied for 1 h on adult rat cardiac myocytes resulted in an SM decrease after 3 days that was maximal after 7 days of culture (31% of the control values) (Fig. 3B ). Quantitative analyses of intracellular sphingolipid levels using independent methods (two for SM and two for ceramide; see Materials and Methods) indicated that whereas 1600–1750 pmol of SM/mg were hydrolyzed on day 7 of doxorubicin treatment, ceramide levels increased by 1000–1850 pmol/mg, suggesting that SM was the source of ceramide. Consistent with this view, the ceramide synthase inhibitor fumonisin B1 (25 µM) did not affect doxorubicin-induced elevation of ceramide (data not shown). In agreement with the inhibitory effect of L-carnitine on ceramide generation, L-carnitine (200 µg/ml) also inhibited SM hydrolysis (by ~90%).

Effect of L-carnitine on acid and neutral sphingomyelinase activities
Because SM hydrolysis and concomitant ceramide generation suggested that doxorubicin triggered the activation of a SMase, we tried to measure this enzyme activity in doxorubicin-treated cardiac myocytes. As shown in Fig. 3C , treatment of cardiac myocytes with 0.5 µM doxorubicin for 7 days led to an increase in a SMase active at acidic pH (34% of the control values). No significant changes were observed in the neutral magnesium-dependent SMase activity in the presence of doxorubicin alone or with L-carnitine. However, when cells were preincubated for 60 min with 200 µg/ml L-carnitine, the doxorubicin-induced increase in SMase activity was completely abolished. In contrast, addition of L-carnitine to the cells 5 days after doxorubicin treatment did not result in inhibition of the SMase (data not shown).

Dose-effect of L-carnitine on doxorubicin-triggered ceramide generation and DNA fragmentation
Figure 4 shows the dose-response of L-carnitine on both ceramide (Fig. 4A ) production and DNA fragmentation (Fig. 4B ) induced by doxorubicin stimulation on cardiac myocytes. For both indexes, a comparable dose response was seen: a significant inhibitory effect, observed at 20 µg/ml, became maximal at 200 µg/ml.



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  		Figure 4. Dose effect of L-carnitine on doxorubicin-triggered ceramide generation and DNA fragmentation. Cardiac myocytes were preincubated in the absence or presence of different concentrations of L-carnitine for 1 h, followed by a 1 h incubation with or without 0.5 µM doxorubicin. The cells were then washed and further incubated for 7 days in anthracycline-free medium with or without L-carnitine. A) Ceramide levels in cardiac myocytes treated with doxorubicin. B) Quantitative DNA fragmentation in cardiac myocytes treated with doxorubicin as determined by the spectrofluorometric DAPI. Results are means ± SE of triplicate determinations (*P<0.05; **P<0.01).

Effect of L-carnitine on SMase kinetic parameters
To obtain a more detailed estimation for L-carnitine-mediated SMase inhibition, we performed enzyme kinetic analysis. Apparent Km and Vmax were determined for the substrate [choline-methyl-14C]SM by fitting initial rates of SM hydrolysis at various substrate concentrations to the Michaelis-Menten equation. As shown in Fig. 5 , doxorubicin treatment of cardiac myocytes did not significantly affect Km, but increased Vmax (1.5-fold increase over the control level). Pretreatment with L-carnitine did not affect basal activity of the SMase but completely abolished the increase in the Vmax seen with doxorubicin. In all instances, Km values remained unchanged.



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  		Figure 5. Effect of L-carnitine on the kinetic parameters of SMase in cardiac myocytes. Cardiac myocytes were preincubated in the absence or presence of L-carnitine (200 µg/ml) for 1 h, followed by a 1 h incubation with or without 0.5 µM doxorubicin. The cells were then washed and further incubated for 7 days in anthracycline-free medium with or without L-carnitine. After treatment, cell pellets were homogenized in lysis buffer; acid SMase activity was determined at pH 5.0 using different concentrations of radiolabeled [N-methyl-14C]SM. B) Results were plotted double reciprocally. C) Km and Vmax values were calculated according to Lineweaver-Burk. Each plot is representative of at least two separate experiments.

To determine whether L-carnitine was able to directly inhibit SMase activity, we performed in vitro studies by mixing purified enzyme from a bacterial source (neutral SMase) or from human placenta (acid SMase) with L-carnitine. When acid SMase was preincubated for 30 min at 4°C with L-carnitine, followed by incubation with substrate for 2 h at 37°C, a dose-dependent inhibition of acid SMase by L-carnitine was observed (Fig. 6 ). This effect seemed irreversible, as suggested by the fact that a similar reduction in sphingomyelinase activity (24%) was observed after preincubating the enzyme for 30 min with L-carnitine and then diluting it to decrease the carnitine concentration by at least threefold. Acid SMase activity was also reduced (by ~30%) by the D-enantiomer of carnitine and by the acetyl ester of L-carnitine (data not shown). Neutral SMase activity remained unchanged in the presence of L-carnitine. A kinetic analysis was also performed to learn whether L-carnitine affects the Vmax or Km of acid SMase. Double-reciprocal plots showed that L-carnitine did not significantly affect the Km but decreased the Vmax, suggesting that L-carnitine affects the enzyme activity rather than substrate accessibility.



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  		Figure 6. Effect of L-carnitine on purified acid or neutral SMases. 20 or 200 µg/ml of L-carnitine was preincubated with a neutral SMase from S. aureus (200 mU/assay) or a placental acid SMase (280 mU/assay) for 30 min before incubation for 2 h at 37°C with radiolabeled [N-methyl-14C] SM, as described in Materials and Methods. For acid SMase, various concentrations of [N-methyl-14C]SM were used. Enzyme activities were determined (A, B) and results are plotted double reciprocally (C). Km and Vmax values were calculated according to Lineweaver-Burk. Each plot is representative of at least two separate experiments.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The progressive loss of cardiac myocytes observed in numerous physiological and pathophysiological conditions regarding normal and diseased myocardium is not well understood. Recent studies showed that apoptosis could be one of the major processes that lead to the progressive deterioration of myocardial function responsible for some cardiac pathologies including heart failure, hypertrophy, and myocardial infarction (46 , 47) . However, apoptotic signaling pathways in cardiac myocytes still remain to be elucidated. One of the strategies to investigate apoptotic mechanisms induced in cardiac myocytes relies on the use of cardioprotective molecules, which can help determine the nature, function, and regulation of some intracellular targets playing an essential role in cell death signaling. For example, Wang and co-workers reported that insulin-like growth factor I, which is known to be an important survival growth factor for myocardium (48) , can inhibit cardiomyocyte apoptosis induced by doxorubicin by attenuating Bax induction and caspase 3 activation (9) . Similarly, carvedilol, a cardioprotective, vasodilating ß-adrenoreceptor antagonist, prevents myocardial ischemia/reperfusion-induced apoptosis by down-regulation of the SAPK signaling pathway, inhibition of Fas receptor expression, and by ß-adrenergic blockade (49) .

In the present study, L-carnitine, which has a well-established protective role in the ischemic myocardium (50) but also in the anthracycline-induced cardiotoxicity (17 18 19 20 , 23) , is shown to block the doxorubicin-induced apoptosis in cardiac myocytes, most likely by inhibiting the SM–ceramide pathway. Indeed, ceramide appears as an important mediator of programmed cell death in response to many cytotoxic agents including TNF-{alpha} (51) , Fas (52) , and chemotherapeutic drugs (25 26 27) in several leukemic cells. Moreover, activation of the sphingolipid pathway in cardiac myocytes in response to cardiac injury induced by ischemia/reperfusion (10) or by TNF-{alpha} (11) has just been recognized. However, the potential source of ceramide has not been shown in these studies. Regarding doxorubicin-induced cell death of cardiac myocytes, the importance of ceramide as a potential mediator is highlighted by the following observations: 1) ceramide has been reported to mediate anthracycline-induced apoptosis of leukemic cells (26) ; 2) ceramide generation preceded apoptosis onset; and 3) cardiomyocyte cell death could be reproduced by addition of exogenous cell permeant ceramide or by agents that increase intracellular ceramide concentration (53) . The present data clearly demonstrate that the doxorubicin-induced production of ceramide in cardiac myocytes did originate from the breakdown of SM by an SMase assayed under acidic conditions and that could be inhibited by L-carnitine pretreatment. Whereas the anthracycline-induced ceramide generation was not completely blocked by L-carnitine, the SM hydrolysis as well as the SMase activation were completely inhibited by the drug, perhaps suggesting that a small part of ceramide increase results from additional mechanisms. With regard to the SMase that is activated by doxorubicin treatment in rat cardiac myocytes, we observed maximal stimulation under acidic conditions. This is the first description by our group of the implication of such a SMase in apoptosis signaling. Whether this SMase corresponds to the lysosomal hydrolase previously characterized in human or murine cells that has controversially been implicated in cell signaling (29 , 30 , 54 , 55) awaits further investigation.

The cardioprotective action of L-carnitine has long been documented (17 18 19 20 21 22 23) . The effect of L-carnitine could be mediated by different intracellular targets. Indeed, the most frequently reported role of L-carnitine in heart muscle is its implication in the transport of long chain fatty acids across the mitochondrial membrane into the matrix where the fatty acyl group is metabolized to produce cell energy (13) . However, despite the amount of data in the literature, the present observations suggest a novel mechanism for L-carnitine-mediated protection against cardiac cell injuries. We show that L-carnitine can act on cell signaling by inhibiting the activation of a SMase, and therefore the generation of ceramide, an event believed to be crucial for triggering apoptosis (29 , 30) . Moreover, experiments conducted in vitro with purified bacterial or placental SMases confirmed that acid SMase can be inhibited by L-carnitine, whereas the neutral enzyme activity is not altered by the drug, supporting the results obtained in the living cell. In accordance with these observations, cell-permeant ceramide induced cardiomyocyte apoptosis, bypassing the doxorubicin-induced SMase activation. This event was not blocked by L-carnitine treatment, strongly supporting the view that L-carnitine acts upstream of ceramide generation in the apoptotic signaling cascade.

The inhibition of L-carnitine on acid SMase could be compared with recent findings by Hannun's group on the inhibitory effect of glutathione (GSH) on neutral SMase (56) . In that study, GSH depletion was shown to occur upstream of the neutral SMase activation in the TNF-{alpha} signaling pathway. In analogy to GSH depletion, a reduction of cellular L-carnitine levels could regulate acid SMase. Indeed, cardiac injuries induced by ischemia (57) or doxorubicin treatment (21) are known to be accompanied by reduced L-carnitine levels in cardiac myocytes. This hypothesis would agree with our results with the dose-dependent inhibition effect of L-carnitine on ceramide generation and apoptosis evoked by doxorubicin in cardiac myocytes, and also with a recent study of the dose-dependent effects of the L-carnitine in myocardial protection in normothermic ischemia (58) .

Another reported role for L-carnitine is its antioxidant properties, which were described in peripheral blood lymphocytes during acute HIV syndrome (59) or in the ischemic heart (60) . On the other hand, doxorubicin is known to injure the heart by generating reactive oxygen species (5 , 61) . However, even though the free radical hypothesis proposed to explain the doxorubicin cardiotoxicity is popular (62 , 63) , it is still controversial (5) . Indeed, it seems that 1) at cardiotoxic concentrations, anthracyclines often fail to generate free radicals and 2) free radical scavengers often fail to prevent doxorubicin cardiotoxicity (5) . Moreover, our results do not support the hypothesis that L-carnitine acts as an antioxidant. Indeed, although L-carnitine abolished doxorubicin cardiotoxicity, ceramide toxicity [which is known to implicate the mitochondrial production of free radicals (64 , 65) ] was not prevented by L-carnitine, suggesting that L-carnitine does not simply interfere with oxygen free radical formation. Finally, since depletion of L-carnitine has been associated with inhibition of carnitine palmitoyltransferase I (CPT I) in cardiac tissue after adriamycin treatment (21) , it could be speculated that the doxorubicin-induced apoptosis of cardiac myocytes is mediated by decreased CPT-dependent fatty acid oxidation. Indeed, inhibition of CPT I has recently been described to induce programmed cell death in some cell systems (66) . This phenomenon was linked to the generation of ceramide. However, although this work emphasized the role of sphingolipids in the induction of apoptosis, the increased ceramide levels reported by these authors are presumed to have derived from an enhanced de novo ceramide synthesis. This contrasts with our findings showing that ceramide originates from SM breakdown.

In summary, we have shown that in adult cardiac myocytes, apoptosis induced by doxorubicin is associated with the activation of a SMase leading to SM hydrolysis and a concomitant ceramide generation. L-carnitine treatment resulted in attenuated ceramide accumulation by interaction with the SMase, leading to the inhibition of the doxorubicin-induced cardiac apoptosis. Although the possibility that L-carnitine prevents apoptosis through its action on fatty acid metabolism cannot be ruled out, our observations suggest that the doxorubicin effects on mitochondrial homeostasis could be mediated by ceramide and inhibited by L-carnitine through impairment of ceramide production. Additional investigations will of course be required in order to define the exact contribution of this mechanism in the cardioprotective function of L-carnitine. Using cardiac myocytes from acid SMase-deficient rats may help delineate the precise contribution of acid lysosomal SMase in doxorubicin signaling.


   ACKNOWLEDGMENTS
 
The authors thank Pr. R. Salvayre (Toulouse, France) for helpful comments. The technical assistance of S. Carpentier is gratefully acknowledged. This work was supported by grants from INSERM. N.A.A. is a recipient of an Association pour la Recherche contre le Cancer fellowship.


   FOOTNOTES
 
2 Abbreviations: C2-ceramide, N-acetyl-D-sphingosine; CPT I, carnitine palmitoyltransferase I; DAPI, 4',6'-diamidino 2-phenylindole; GSH, glutathione; SAPK, stress-activated protein kinase; SM, sphingomyelin; SMase, sphingomyelinase; TNF, tumor necrosis factor.

Received for publication December 21, 1998. Revised for publication March 24, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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