Protective effect of eicosapentaenoic acid on palmitate-induced apoptosis in neonatal cardiomyocytes

Biochimica et Biophysica Acta 1781 (2008) 685–693

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Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b b a l i p

Protective effect of eicosapentaenoic acid on palmitate-induced apoptosis in neonatal cardiomyocytes Christine Leroy ⁎, Sabine Tricot, Bernard Lacour, Alain Grynberg UMR1154 INRA-Univ Paris-Sud, Faculté de Pharmacie, 5 avenue J-B. Clément, Châtenay-Malabry, F-92296, France

a r t i c l e

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Article history: Received 10 April 2008 Received in revised form 7 July 2008 Accepted 23 July 2008 Available online 8 August 2008 Keywords: Polyunsaturated fatty acid Cardiomyocyte Apoptosis Palmitate Cardiolipin Mitochondria

a b s t r a c t Long chain polyunsaturated fatty acids (PUFAs) play an important role in cardioprotection. These effects have been largely attributed to membrane docosahexaenoic acid. Conversely, saturated fatty acids trigger apoptosis in cardiomyocytes, with modifications of mitochondrial properties including cardiolipin loss, cytochrome c release and caspase-3 activation. The purpose of this study was to investigate the chronic effect of eicosapentaenoic acid (EPA) on mitochondrial apoptosis induced by palmitate treatment and the associated signalling pathways. Confluent cultures of rat neonatal cardiomyocytes were treated for 2 days in media enriched with either EPA or arachidonic acid (AA) and then exposed to palmitate (0.5 mM) to induce apoptosis, in the absence of PUFA supplements. The EPA treatment resulted in significant membrane enrichment in n − 3 PUFAs, especially in docosapentaenoic acid (DPA), and a large decrease in AA. Both AA and EPA treatments prevented caspase-3 activation, translocation of Bax to the mitochondria and release of cytochrome c induced by palmitate treatment. Furthermore, EPA, but not AA prevented the loss of mitochondrial cardiolipin due to apoptosis. These results suggest that EPA supplementation is able to protect cardiomyocytes against palmitate-induced apoptosis via an implication of different mitochondrial elements, possibly through its elongation to DPA, which is very efficient in cardiomyocytes. © 2008 Elsevier B.V. All rights reserved.

1. Introduction High levels of circulating saturated fatty acids are associated with diabetes, obesity and hyperlipidemia. In the heart, the accumulation of saturated fatty acids has been proposed to play a role in the development of heart failure and diabetic cardiomyopathy [1,2] as well as ischemia–reperfusion [3]. The two major circulating fatty acids are the saturated palmitate (C16:0) and the monounsaturated oleate (C18:1). Palmitate, but not oleate, has been implicated in the induction of apoptosis in a large variety of cell types [4–9]. The mechanism of palmitate-induced apoptosis in cardiomyocytes isolated from neonatal rats involves various markers of programmed cell death including phosphatidylserine translocation, DNA laddering and caspase-3 activation [10,11]. A mitochondrial death pathway has been proposed based on the loss of mitochondrial membrane potential [11] and the release of cytochrome c from the mitochondrial intermembrane space to the cytosol. Once released, cytochrome c leads to activation of the effector caspase-9, which is a part of the apoptosome complex, which in turn activates caspase-3, a cysteine protease involved in later apoptotic events. The proteins of the Bcl-2 family play a key regulator role in apoptosis, involving interactions with mitochondria that alter the distribution of cytochrome c. The Bcl-2 family is composed by both anti-apoptotic (e.g. Bcl-2, Bcl-XL) and pro-apoptotic proteins (e.g. Bax, ⁎ Corresponding author. Tel.: +33 1 46 83 55 34; fax: +33 1 46 83 53 71. E-mail address: [email protected] (C. Leroy). 1388-1981/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.bbalip.2008.07.009

Bad). They can form ion channels in lipid bilayers and contain a lipophilic domain, which allows their targeting to the outer mitochondrial membrane (OMM) and their contribution to permeabilization [12]. Bax was proposed as the promoter of cytochrome c release by forming oligomers or in association with PTP (permeability transition pore) elements such as VDAC (voltage-dependent anion channel) and/or ANT (adenine nucleotide transporter) [13]. The release of cytochrome c into the cytosol is triggered by its release from the inner mitochondrial membrane. Cardiolipin (1,3-bis 1′, 2′diacyl-3′-phosphoryl-sn-glycerol)-sn-glycerol) (CL), a mitochondrial anionic phospholipid, is essential for cytochrome c insertion, retention, stability and function [14,15]. A decrease of CL content was reported when cardiomyocytes were treated with palmitate and was directly correlated with cytochrome c release [16,17]. The beneficial effects of (n − 3) polyunsaturated fatty acids (PUFAs) on cardiac myocytes have been thoroughly investigated and reviewed, but few studies have investigated their effect on cardiac cell death. In neonatal cardiomyocytes, eicosapentaenoic acid (EPA) was shown to affect sodium channels [18] and to prevent arrhythmia caused by high extracellular calcium, ouabain, isoproterenol or lysophosphatidylcholine [19]. Docosahexaenoic acid (DHA) and/or EPA were reported to influence several signal transduction pathways in various cell types [20,21]. However, unlike DHA, EPA does not significantly enter in cardiac membranes in vivo [22] and its beneficial effects have been often attributed to an acute effect [18]. EPA intake induces in vivo a significant increase in cardiac membrane DPA, balanced by a large

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decrease in AA [22]. Recent findings demonstrated that high AA content in the diabetic heart favours cardiolipin breakdown and resulted in various mitochondrial functional damages [23]. The present study was designed to evaluate the chronic effect of EPA on cardiomyocytes apoptosis. We used neonatal rat cardiomyocytes cultures exposed to palmitate as a model of apoptosis to investigate the possible protective effect of the presence of EPA. The protective effect was evaluated on several apoptosis markers including caspase-3 activity, cytochrome c release, cardiolipin content and Bax and Bcl-2 expression. The major finding of this study showed that the replacement of AA by EPA in cell phospholipids was able to protect cardiomyocytes against palmitate-induced apoptosis by influencing the mitochondrial apoptosis pathway. 2. Materials and methods The investigations were carried out in agreement with the National Institute of Health guidelines for the Care and Use of Laboratory Animals (NIH Pub. No 85-23, Revised 1996) in animals quarters in agreement with national regulations. 2.1. Cardiomyocyte culture These investigations were performed on neonatal rat cardiac myocytes in culture. The hearts were removed from 2 to 4-day-old Wistar rats and the myocytes were isolated and cultured according to previously published procedures [24]. The ventricles were collected in sterile conditions, cut in small pieces, washed three times and minced in calcium-free saline at 30 °C. The tissue was then submitted to a seven steps proteolytic dissociation (10 min, 30 °C) with trypsin (0.3%, DIFCO, Paris, France). The first supernatant was discarded and the next six supernatants were pooled and centrifuged (1000 g, 15 min) and the cell pellet was resuspended in culture medium. The myocyte proportion was increased through a two-step selective adhesion procedure [25]. The isolated myocytes were resuspended in the culture medium and seeded in 60 mm plastic dishes (Falcon Primaria, Becton Dickinson, Pont de claix, France) at a density of 2 × 106 cells per dish. The culture was grown in Ham F10 basal medium supplemented with 10% fetal calf serum (FCS, Cambrex, Emerainville, France), 10% human serum (EFS, Rungis, France), penicillin and streptomycin (Sigma, Lyon, France). The free calcium concentration in the medium was standardised at 1.2 mM. The medium was renewed 24 h after seeding and every 2 days thereafter. The cells were incubated at 37 °C in a humidified atmosphere (with 5% CO2). Confluence was reached within 2 days and all the experiments were conducted on spontaneously beating 5-day-old cultures.

free bovine serum albumin (BSA, Sigma-Aldrich, Saint Quentin Fallavier Cedex, France) for a final palmitic acid concentration 0.5 mM and a palmitic acid/BSA molar ratio of 6/1. The medium was shaken at 37 °C until achievement of a limpid solution. FCS was then added to the medium at 20% final concentration before filter sterilization (0.2 μm). A similar process was used to prepare the control medium using oleic acid (OA) instead of palmitic acid. For control experiments, BSA was added as described above, but in the absence of palmitic acid. 2.4. Experimental protocol After five days of culture, the cells were incubated for 48 h in different medium containing either BSA alone (Ccells) or experimental media (AA or EPA supplemented cells) in order to modify the lipid membrane composition of the cardiomyocytes. Then, the experimental media were removed and replaced by the palmitic acid medium (Palm) or control medium (Ctrl) to induce apoptosis. After 16 or 24 h, the cells were washed twice with ice-cold PBS and harvested by scraping in 2 ml/dish of PBS. The cell pellet was obtained by centrifugation at 200 g, 5 min at 4 °C and stored at −80 °C. 2.5. Cell viability Two different methods were used to evaluate the cell viability in our experimental protocol: the MTT test (in vitro toxicology assay kit MTT based, Sigma-Aldrich, Saint Quentin Fallavier Cedex, France) and the determination of lactate dehydrogenase (LDH) release in the medium (in vitro toxicology assay kit lactate dehydrogenase based, Sigma-Aldrich, Saint Quentin Fallavier Cedex, France). The MTT assay used as an index of cell viability and growth, is based on the ability of viable cells to reduce MTT from a yellow water-soluble dye to a dark blue insoluble formazan product [27]. The activity of LDH released in the culture media was measured spectrophotometrically as an index of necrotic cell death [28]. 2.6. Quantification of apoptotic cardiomyocytes To quantify myocytes undergoing apoptosis, cells monolayers were stained with Hoechst 33342, 10 μg/ml (Sigma-Aldrich, Saint Quentin Fallavier Cedex, France). The morphological features of apoptosis (chromatin condensation and fragmentation) were monitored by fluorescence microscopy (Leica). At least 300 cells from ten randomly selected fields were counted in each experiment. Results were expressed as % of apoptotic nuclei and then normalised to Ccells using 1 as an arbitrary value.

2.2. PUFA-enriched media 2.7. Caspase-3 and caspase-8 activity assay To investigate the effect of membrane PUFA enrichment, the cells were incubated 48 h with a culture medium containing either EPA or AA (Cayman Chemical Company) linked to albumin (fraction V, Sigma) with a final molar ratio fatty acids/BSA closed to 2. The final concentration of added PUFAs was 80 μM [26]. The AA supplemented group was designed to create a control group receiving a PUFA supplement quantitatively similar to the EPA group, and to avoid large alterations in the polyunsaturated/saturated fatty acid ratio of the culture medium, since FCS displays a low PUFA level. 2.3. Apoptosis induction The cardiomyocytes were incubated 16 or 24 h in a 20% FCSHamF10 medium containing 0.5 mM palmitic acid. Palmitic acid C16:0 (Sigma-Aldrich, Saint Quentin Fallavier Cedex, France) was first dissolved in ethanol (195 mM palmitic acid stock solution). This preparation was added to the HamF10 supplemented with fatty acid-

Briefly, the cell pellets were lysed in 50 mM Hepes pH 7.5, 150 mM NaCl, 15 mM MgCl2, 1 mM EDTA, 2.5 mM EGTA, 0.1% Tween 20, 0.1 mM sodium orthovanadate, 1 mM NaF, 10 mM b-glycerophosphate, 2% of a stock solution of protease inhibitor cocktail (Complete, Roche), and 1 mM dithiothreitol. Protein concentrations were determined with bicinchroninic acid protein assay (Sigma-Aldrich, Saint Quentin Fallavier Cedex, France) and 100 μg of proteins was loaded in microplates. Caspase-3 and caspase-8-like activity was determined in a 100 μl reaction mixture using synthetic fluorogenic substrates AcDEVD-AFC N-acetyl-Asp-Glu-Val-Asp-AFC (7-amino-4-trifluoromethyl coumarin, Biomol International ®) and Ac-IETD-AFC Nacetyl-Ile-Glu-Thr-Asp-AFC (7-amino-4-trifluoromethyl coumarin, Biomol International ®) respectively. The reaction was done in optimal activity buffer (20 mM PIPES pH 7.2, 100 mM NaCl, 1 mM EDTA, 0.1% CHAPS, 10% sucrose, 10 mM of dithiothreitol). The release of AFC from the substrate was measured after 2 h as emission at 505 nm

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upon excitation at 400 nm using a microplate fluorescence reader (TECAN, Genios). In some experiments, Ccells were treated with TNFα (Sigma-Aldrich, Saint Quentin Fallavier Cedex, France) for 24 h at the final concentration of 200 ng/ml in order to induce the caspase-8 activity (positive control).

graph (Agilent Technologies Massy, France) on a PVA-Sil column (PVASil 120A, S-5 μm, YMC, USA) at 35 °C, and the peaks were quantified by light scattering detector (Eurosep, Cergy Pontoise, France) [32].

2.8. Separation of cytosolic and mitochondrial fractions

The data are expressed as mean ± SEM. The data were submitted to a one-way analysis of variance (ANOVA). A p value less than 0.05 was considered to be statistically significant. When statistically different, the means were compared by the Newman–Keuls test. The statistical analysis was done with the NCSS60 software.

The cells were plated in 60-mm culture dishes (2 × 106 cells) and incubated in PUFA-enriched media as described above. After 16 h of palmitate treatment, the medium was removed and the cells were washed three times with ice-cold PBS and harvested. The plasma membrane was then permeabilized using digitonin (30 μM) in a permeabilization buffer (250 mM sucrose, 20 mM Hepes pH 7.4, 10 mM KCl, 1 mM MgCl2, 1 mM EGTA, 1 mM EDTA supplemented with 1 mM pmsf, 1 mM DTT, 1 mM sodium orthovanadate and protease inhibitor cocktail). After 10 min, the cells were centrifuged 2 min at 15700 g at 4 °C. The supernatant was removed and centrifuged at 100 000 g for 60 min at 4 °C to obtain the cytosolic fraction. The pellet was resuspended in mitochondrial lysis buffer (RIPA) and disrupted by 10 passages through a 25G needle. The homogenate was centrifuged at 750 g for 10 min at 4 °C and the supernatant was then centrifuged at 12 000 g for 10 min at 4 °C to obtain the mitochondria pellet. Proteins were determined using the bicinchroninic acid protein assay kit. Immunodetection of cyclooxygenase II (COX II) was used as a loading control of mitochondria fraction and to check the absence of mitochondrial material contamination in the cytosolic fraction. 2.9. Western blot

2.12. Statistical analysis

3. Results 3.1. Validation of our experimental conditions Most of palmitate-induced apoptosis studies on cultured cardiomyocytes were performed using a 2/1 fatty acid/BSA molar ratio. However, for a palmitate concentration of 0.5 mM, the albumin concentration in the medium exceeds 15 g/l. High level of albumin induces a diminution of free calcium in the medium and all electrophysiological studies on cardiomyocytes have been done for a 6/1 molar ratio rather than 2/1 ratio. In order to evaluate the importance of palmitate/BSA molar ratio on apoptosis induced by palmitate, we have compared caspase-3 activity for 2/1 and 6/1 molar ratio respectively. As shown in Fig. 1A, palmitate was able to induce caspase-3 activity similarly with 2/1 and 6/1 ratio. In our model, apoptotic nuclei were quantified following treatment with 0.5 mM palmitate or oleate, a mono-saturated fatty acid, described

Fractions were separated on NuPAGE 4–12% Bis–Tris gels (Invitrogen, Cergy-Pontoise, France) and transferred onto a nitrocellulose membrane. The membranes were blocked with 5% non-fat dry milk and incubated overnight at 4 °C with primary antibodies: mouse monoclonal anti-cytochrome c (Biosource, Clinisciences, Montrouge, France), purified polyclonal anti-Bax (Biolegend, Ozyme, SaintQuentin en Yvelines, France), monoclonal anti-Bcl-2 (Santa Cruz Biotechnology Inc., California, USA), mitochondrial oxphos complexes (Mitosciences, Eugene, Oregon, USA) and monoclonal anti-β actin (Sigma-Aldrich, Saint Quentin Fallavier Cedex, France). As secondary antibodies, we used horseradish-peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG (Cell Signaling, Ozyme, Saint-Quentin en Yvelines, France) for 1 h at room temperature. Bound antibodies were detected using the enhanced chemiluminescent system ECL (Amersham Bioscience, Saclay, France) with Kodak Biomax light films. Gel band intensity was quantified using Scion Corporation software. 2.10. Fatty acids analysis The cells were harvested in 1 ml distilled water and lipids were extracted in a 2:1 chloroform–methanol mixture [29]. Phospholipids (PL) were separated from nonphosphorous lipids (NL) on silica acid cartridges [30], and the fatty acids were trans-methyled with BF3methanol. The methyl esters were analysed by gas chromatography on an econocap EC-WAX capillary columns (0.32 × 30 m, Alltech Associates) using heptadecanoic acid (C17:0) as internal standard [22]. 2.11. Analysis of phospholipids The phospholipids were extracted from cardiomyocytes by the Bligh and Dyer method [31]. Briefly, a sample of 250 μl of cell lysate was shaken with 1100 μl of chloroform/methanol (1:1, v/v) and 250 μl of water. The mixture was sonicated 30 s in ice. After 6 h, the mixture was centrifuged for 10 min at 3500 g. The lower layer of the lipid extract was collected and dried in a nitrogen stream at 20 °C. The phospholipids were separated by high performance liquid chromato-

Fig. 1. Validation of the experimental conditions. Two different acid palmitic/BSA molar ratio (2/1 and 6/1) were tested on caspase-3 activity assay (A). Cells were treated (Palm) or not (Ctrl) with palmitate (0.5 mM) for 24 h before caspase-3 activity measurement. Fluorescence values expressed as arbitrary unit (AU) were normalised to protein content. Each column represents mean ± SEM, n = 3 independent experiments. ⁎⁎⁎p b 0.0001. Identification of apoptotic morphological changes using Hoechst 33342 compound (B). Cells were incubated for 24 h with palmitic acid (Palm, 0.5 mM), oleic acid (OA, 0.5 mM) or BSA alone (Ctrl). Results were expressed as % of apoptotic nuclei and then normalised to Ccells using 1 as an arbitrary value. Each column represents mean ± SEM, n = 3 independent experiments. ⁎⁎⁎p b 0.0001.

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Table 1 Fatty acid composition of cardiomyocytes membrane phospholipids after 48 h of cell incubation in various fatty acid containing media Fatty acid in phospholipids C 14:0 C 16:0 C 18:0 C 20:0 C 22:0 C 24:0 ΣSFA Σ C 16:1 Σ C 18:1 Σ C 20:1 Σ MUFA C 18:3 (n − 3) C 18:4 (n − 3) C 20:4 (n − 3) C 20:5 (n − 3) EPA C 22:5 (n − 3) DPA C 22:6 (n − 3) DHA Σ (n − 3) PUFAs C 18:2 (n − 6) C 18:3 (n − 6) C 20:2 (n − 6) C 20:3 (n − 6) C 20:4 (n − 6) AA C 22:2 (n − 6) C 22:4 (n − 6) C 22:5 (n − 6) Σ (n − 6) PUFAs Σ PUFAs Ratio (n − 6) / (n − 3)

% of total fatty acids Ccells

AAcells

EPAcells

ANOVA

0.50 ± 0.70a 15.81 ± 1.60 25.63 ± 4.66 0.83 ± 0.10a 0.87 ± 0.22 1.17 ± 0.13 45.18 ± 3.49 1.47 ± 0.15 18.88 ± 1.12a 0.88 ± 0.10a 21.22 ± 1.19a 0.28 ± 0.07a 0.28 ± 0.07 0.00 ± 0.00a 0.36 ± 0.01a 1.98 ± 0.16a 2.63 ± 0.38 5.52 ± 0.40a 8.54 ± 0.88a 0.00 ± 0.00a 0.79 ± 0.05 1.74 ± 0.11a 13.49 ± 0.72a 0.62 ± 0.17 2.44 ± 0.37a 0.48 ± 0.05a 28.09 ± 1.95a 33.61 ± 2.33 5.10 ± 0.10a

0.74 ± 1.60a 17.86 ± 0.74 25.21 ± 3.12 0.50 ± 0.08b 0.66 ± 0.22 0.69 ± 0.16 45.96 ± 2.46 1.38 ± 0.07 12.95 ± 0.66b 0.38 ± 0.09 14.70 ± 0.67b 0.08 ± 0.03b 0.31 ± 0.12 0.23 ± 0.08b 0.00 ± 0.00a 1.31 ± 0.31a 1.81 ± 0.15 3.84 ± 0.12a 3.18 ± 0.16a 0.12 ± 0.02b 0.75 ± 0.09 0.69 ± 0.05b 22.03 ± 1.60b 1.09 ± 0.61 7.26 ± 0.55b 0.34 ± 0.07a 35.47 ± 1.72b 39.31 ± 2.12 9.23 ± 0.19b

0.63 ± 0.03ab 18.30 ± 0.41 24.41 ± 2.45 0.52 ± 0.05 0.54 ± 0.6 0.80 ± 0.09 45.59 ± 2.88 1.48 ± 0.11 15.62 ± 0.89b 0.53 ± 0.03b 17.60 ± 0.99b 0.10 ± 0.03b 0.21 ± 0.08 0.26 ± 0.05b 5.56 ± 0.67b 9.58 ± 0.56b 2.05 ± 0.23 17.76 ± 1.12b 5.37 ± 0.36c 0.12 ± 0.02b 0.55 ± 0.08 0.99 ± 0.07c 10.48 ± 0.77a 0.36 ± 0.13 1.28 ± 0.07a 0.17 ± 0.01b 19.31 ± 1.34c 37.07 ± 2.06 1.09 ± 0.08c

⁎ ns ns ⁎ ns ns ns ns ⁎⁎ ⁎⁎ ⁎⁎ ⁎ ns ⁎ ⁎⁎⁎ ⁎⁎⁎ ns ⁎⁎⁎ ⁎⁎⁎ ⁎⁎ ns ⁎⁎⁎ ⁎⁎⁎ ns ⁎⁎⁎ ⁎⁎ ⁎⁎⁎ ns ⁎⁎⁎

SFA: saturated fatty acid, MUFA: monounsaturated fatty acid, PUFA: polyunsaturated fatty acid. Cells were incubated with AA or EPA (80 μM) or BSA alone (Ccells) for 48 h. The results are expressed as mean ± SEM as percent of total fatty acids from four separate experiments. Fatty acids are expressed as the number of carbon atoms: number of double bonds. Fatty acids of interest were written in bold characters. Results were expressed as mean ± SEM and the groups that affected different letters were significantly different. ⁎p b 0.05; ⁎⁎p b 0.01; ⁎⁎⁎p b 0.001. ns means not significant.

in the literature as a control. A significant increase of apoptotic nuclei in palmitate condition was observed (Fig. 1B) in comparison to oleate treatment or BSA alone. In order to rule out a possible cytotoxic effect of PUFAs, two different cell viability tests were done. PUFAs supply in basal conditions did not change the viability of the cells, since the number of viable cells as determined by MTT test was always higher than 95%, and the release of LDH never exceeded 2% of the total LDH cell content (data not shown). 3.2. Fatty acids incorporation in total and mitochondrial membranes Incorporation of PUFAs in phospholipids of total membranes was measured after 48 h of incubation (Table 1). The fatty acids added to the medium were readily incorporated into the cellular phospholipids and altered the overall fatty acid composition of cardiomyocyte membranes. In control cells (Ccells), saturated fatty acids represented 45% of membrane fatty acids. This content was not modified in cells supplemented with the PUFAs, either AA (AAcells), or EPA (EPAcells). The rough composition of Ccells was 21% in MUFAs and 33.6% in PUFAs (28% as (n − 6) and 5.5% as (n − 3)), with (n − 6) / (n − 3) ratio and AA/DHA ratio both close to 5. The total AA content in Ccells was 13.5% (balanced by a high MUFA content), which is a rather low content for cardiac material, and can be attributed to the low PUFA content in the FCS-medium. AAcells were characterized by a lowered MUFA content (15%) and a significant increase in AA (22% vs 13.5% in Ccells). In these cardiomyocytes, both the (n − 6) / (n − 3) ratio and the AA/DHA ratio were raised to 10. Interestingly, these cells displayed a lower

content in linoleic acid (18:2, n − 6) and a higher content in docosatetraenoic acid (22:4, n − 6). When the cardiomyocytes were cultured in EPA-enriched medium, the total amount of phospholipid (n − 3) PUFAs was enhanced to 17% (vs 5.5% in Ccells and 3.8% in AAcells), whereas the AA content decreased to 10% and the total (n − 6) PUFA level to 20%. The cardiomyocyte is known to be deficient in Δ4 desaturation capacity [33] and then, EPAcells were unable to produce DHA from EPA. Incubation with EPA induced an increase in EPA (5.6% of membrane fatty acids) and more significantly in DPA (9.6%) with a significant decrease in AA (3.5%). EPAcells were characterized by a (n − 6) / (n − 3) ratio of 1 and an AA/ DHA ratio close to 2. Following PUFA incorporation into total membranes, the fatty acids composition remained unchanged for 24 h after removing PUFA supply, which guaranteed the stability of the composition during the palmitate treatment (data not shown). The fatty acids composition of the mitochondrial membranes was also investigated in order to assess the specific consequences of PUFA enrichment on these membranes. The results (Table 2) roughly show the same qualitative differences as the total membrane fraction, with an increase in AA in the AAcells and an increase in both EPA and DPA in EPAcells also associated to a significant decrease in AA. However, the amplitude of the differences either on individual PUFAs or in the PUFA ratios was largely higher than in the total phospholipid fraction, especially for EPA. These results show, in accordance with our previous reports that increasing either AA or EPA in culture media of cardiomyocytes leads to membrane modifications very similar to those observed in vivo in the heart after feeding a (n − 6) and a (n − 3) diet respectively [22,26,33,34].

Table 2 Fatty acid composition of cardiomyocyte mitochondrial membranes phospholipids after 48 h of cell incubation in various fatty acid containing media Fatty acid in phospholipids C 14:0 C 16:0 C 18:0 C 20:0 C 22:0 C 24:0 Σ SFA Σ C 16:1 Σ C 18:1 Σ C 20:1 Σ MUFA C 18:3 (n − 3) C 18:4 (n − 3) C 20:4 (n − 3) C 20:5 (n − 3) EPA C 22:5 (n − 3) DPA C 22:6 (n − 3) DHA Σ (n − 3) PUFAs C 18:2 (n − 6) C 18:3 (n − 6) C 20:2 (n − 6) C 20:3 (n − 6) C 20:4 (n − 6) AA C 22:2 (n − 6) C 22:4 (n − 6) C 22:5 (n − 6) Σ (n − 6) PUFAs Σ PUFAs Ratio (n − 6) / (n − 3)

% of total fatty acids Ccells

AAcells

EPAcells

ANOVA

0.58 ± 0.06 22.18 ± 1.17a 25.95 ± 1.11a 0.94 ± 0.17 0.47 ± 0.18 1.06 ± 0.25 51.19 ± 2.94a 1.43 ± 0.07 14.98 ± 0.57a 0.42 ± 0.04a 68.02 ± 3.62a 0.45 ± 0.14 0.00 ± 0.00 0.08 ± 0.08a 0.51 ± 0.20a 1.54 ± 0.39a 2.90 ± 0.92 5.48 ± 1.73a 5.91 ± 0.45 0.13 ± 0.06 0.45 ± 0.01 1.36 ± 0.09a 12.91 ± 1.11a 0.46 ± 0.23 2.43 ± 0.40a 0.39 ± 0.07 24.04 ± 2.42a 29.52 ± 4.15a 4.39 ± 1.40a

0.88 ± 0.26 14.76 ± 1.28b 15.86 ± 0.67 0.86 ± 0.64 0.38 ± 0.12 0.89 ± 0.41 33.62 ± 3.08b 1.52 ± 0.59 10.07 ± 1.36b 0.31 ± 0.04 45.52 ± 5.07b 0.26 ± 0.10 0.00 ± 0.00 0.00 ± 0.00a 0.09 ± 0.04a 1.11 ± 0.12a 1.49 ± 0.11 2.96 ± 0.37a 3.87 ± 0.42 0.24 ± 0.01 0.42 ± 0.08 2.04 ± 0.28b 28.83 ± 3.44b 0.49 ± 0.16 12.62 ± 2.09b 0.29 ± 0.05 48.80 ± 6.62b 51.75 ± 6.99b 16.51 ± 17.89b

1.07 ± 0.19 19.88 ± 1.10a 20.15 ± 0.65c 0.74 ± 0.16 0.56 ± 0.20 0.74 ± 0.19 43.13 ± 2.49c 1.48 ± 0.47 11.19 ± 1.67 0.26 ± 0.02b 56.06 ± 4.65b 0.32 ± 0.17 0.00 ± 0.00 0.43 ± 0.11b 9.50 ± 2.22b 14.59 ± 1.81b 1.43 ± 2.25 26.26 ± 4.56b 4.31 ± 0.64 0.14 ± 0.03 0.29 ± 0.04 0.96 ± 0.03a 7.32 ± 0.45a 0.79 ± 0.38 1.70 ± 0.15a 0.17 ± 0.03 15.66 ± 1.74c 41.92 ± 6.31c 0.60 ± 0.38c

ns ⁎ ⁎⁎⁎ ns ns ns ⁎⁎⁎ ns ns ⁎ ⁎⁎ ns ns ⁎ ⁎⁎ ⁎⁎⁎ ns ⁎⁎⁎ ns ns ns ⁎ ⁎⁎⁎ ns ⁎⁎ ns ⁎⁎⁎ ⁎⁎⁎ ⁎⁎⁎

SFA: saturated fatty acid, MUFA: monounsaturated fatty acid, PUFA: polyunsaturated fatty acid. Cells were incubated with AA or EPA (80 μM) or BSA alone (Ccells) for 48 h. The results are expressed as mean ± SEM as percent of mitochondrial fatty acids from three separate experiments. Fatty acids are expressed as the number of carbon atoms: number of double bonds. Fatty acids of interest were written in bold characters. Results were expressed as mean ± SEM and the groups that affected different letters were significantly different. ⁎p b 0.05; ⁎⁎p b 0.01; ⁎⁎⁎p b 0.001. ns means not significant.

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3.3. Effect of membrane PUFAs on caspase-3 activity Fig. 2A shows that palmitate induced a two-fold caspase-3 activation in Ccells. The PUFA enrichment in AAcells and EPAcells did not affect the basal caspase-3 activity, but prevented the caspase-3 activation due to palmitate. 3.4. Effect of membrane PUFAs on caspase-8 activity Fig. 2B shows that palmitate was unable to induce caspase-8 activation in Ccells. By contrast, exposure of Ccells to TNF-α (200 ng/ ml for 24 h), a concentration known to increase caspase-8 activity in neonatal rat cardiomyocytes [35], induced a 4-fold stimulation of caspase-8 activity. Moreover, modifications of membrane composition by PUFAs supply had no effect on caspase-8 activities both in basal and palmitate conditions. These results suggest that activation of caspase8 is not involved in palmitate-induced apoptosis. 3.5. Effect of membrane PUFAs on palmitate-induced cytochrome c release The influence of membrane PUFA modifications was investigated on the release of cytochrome c from mitochondria to cytosol in response to palmitate treatment. A representative western blot of three independent experiments is presented in Fig. 3A. In our experimental conditions, we observed in Ccells a two-fold increase of the cytosolic/mitochondrial cytochrome c ratio, which express the

Fig. 3. Effect of membrane PUFAs on cytochrome c release. Cardiomyocytes were incubated in various PUFA-enriched media (AA or EPAcells) or in control medium without enrichment (Ccells) and then treated with acid palmitic (Palm) or with BSA alone (Ctrl) for 16 h. After digitonin permeabilization (30 μM), cytosolic and mitochondrial fractions were isolated and analysed by western blotting. Results are expressed as ratio of cytochrome c cytosolic/cytochrome c mitochondrial. (A) A representative western blot of three independent experiments. (B) Densitometric and statistic analysis of PUFA-supplemented cells western blot experiments (n = 3). Note that equal protein loading and transfer for each sample have been confirmed after Ponceau S red staining. The differences between Palm and Ctrl in each group were represented as ⁎p b 0.05 and ns = not significant. Letters represented the differences due to fatty acid composition in basal condition. The groups that affected different letters were significantly different (p b 0.05).

cytochrome c released from mitochondria to cytosol, as a response to palmitate addition. In each of three experimental series, in basal conditions, the cytochrome c release was lower in EPAcells than in AAcells (Fig. 3B). As shown, palmitate treatment failed to increase the cytochrome c release in both AA and EPA supplemented cells. These results suggest that the increase in mitochondrial AA negatively

Fig. 2. Effect of membrane PUFAs on caspase-3 and caspase-8 activity. Cardiomyocytes were pre-incubated for 48 h in acid arachidonic (AAcells) or eicosapentaenoic acid (EPAcells) enriched culture medium or in a culture medium without enrichment in fatty acids (Ccells). Then, cells were treated (Palm) or not (Ctrl) with palmitate (0.5 mM) for 24 h before caspase-3 or caspase-8 activity measurement. Cardiomyocytes incubated with TNF-α (200 ng/ml for 24 h) were used as a positive control for the induction of caspase-8 activity. Fluorescence values expressed as arbitrary unit (AU) were normalised to protein content. Each column represents mean ± SEM, n = 4 independent experiments. ⁎⁎⁎p b 0.0001.

Fig. 4. Effect of membrane PUFAs on cardiolipin concentration. Cardiomyocytes incubated in fatty acid enriched media (AA or EPAcells) or in control medium without enrichment (Ccells), were treated (Palm) or not (Ctrl) with palmitic acid for 16 h. Phospholipids were extracted and analysed by high performance liquid chromatography (HPLC) system as described in materials and methods. Levels of cardiolipin were expressed in μg/μg protein. Means ± SEM (n = 10 independent experiments). ANOVA: The results of ANOVA were presented above the bars. The differences between Palm and Ctrl in each cell group were represented as ⁎p b 0.05; ⁎⁎p b 0.01 and ⁎⁎⁎p b 0.001. ns means not significant.

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palmitate induced a significant CL decrease in membranes. The same effect was recorded in AAcells with roughly similar amplitude. Conversely, the palmitate treatment failed to induce a CL decrease in EPAcells and the amount of CL remained unchanged after palmitate treatment. These results are in accordance with those on cytochrome c release (see above). The increase in AA induced a cardiolipin loss, which plays a key role in cytochrome c binding, whereas EPA + DPA did not. However, only EPA + DPA significantly prevented cardiolipin decrease in palmitate treated cells. 3.7. Bax and Bcl-2 protein expression during palmitate apoptosis treatment in Ccells Both the pro-apoptotic Bax and the anti-apoptotic Bcl-2 proteins were expressed in Cells as shown by western blot experiments on cell lysates (Fig. 5A and B). In basal conditions, Bax was mainly located in the cytosol, although a moderate expression of Bax was observed in the mitochondrial fraction (Fig. 6A). In Ccells, the palmitate treatment did not influence Bcl-2 expression but significantly decreased the expression of Bax (Fig. 5A and B) without changes in the Bax/Bcl-2 ratio (Fig. 5C). Furthermore, palmitate induced an enrichment of Bax in mitochondria of Ccells as shown in Fig. 6B.) COX II was only recovered in the mitochondrial fractions. This observation can rule out a contamination of the cytosolic fraction by mitochondria. 3.8. Effect of membrane PUFAs on both Bax and Bcl-2 total protein expression The effects of individual PUFA enrichment on both pro-apoptotic Bax and anti-apoptotic Bcl-2 total expression were also investigated.

Fig. 5. Effect of PUFAs on Bax and Bcl-2 protein expression. Cardiomyocytes incubated in fatty acid enriched media (AA or EPAcells) for 48 h or in control medium without enrichment (Ccells), were treated (Palm) or not (Ctrl) with palmitic acid for 24 h. Protein extracts were analysed by western blotting for the pro-apoptotic Bax (A) and for the anti-apoptotic Bcl-2 (B) and results were expressed following densitometric analyses of western blots. The Bax/Bcl-2 ratio expression was then calculated and illustrated on C. Means ± SEM (n = 3 independent experiments).⁎p b 0.05; ⁎⁎p b 0.01; ns means not significant.

affected the stability of cytochrome c in the membrane, whereas the increase in EPA + DPA did not. Conversely, the two modifications prevented the further release elicited by palmitate treatment. 3.6. Effect of membrane PUFAs on cardiolipin level The effects of individual (n − 3) and (n − 6) PUFA enrichment on total steady-state CL levels were investigated. The data show a significant decrease of CL level in AAcells as compared to Ccells in basal conditions (Fig. 4), but not in EPAcells. In Ccells, the treatment with

Fig. 6. Translocation of Bax from cytosol to mitochondria induced by palmitate in Ccells (A) and effect of PUFAs on Bax mitochondrial expression (B). Cardiomyocytes incubated in a specific fatty acid enriched media (AA or EPAcells) for 48 h or in control medium without enrichment (Ccells), were treated (Palm) or not (Ctrl) with palmitic acid for 24 h. Following permeabilization with digitonin (30 μM), cardiomyocytes were fractionated in order to separate cytosolic (c) and mitochondrial (m) fractions. Bax protein expression in both cytosolic and mitochondrial fractions was analysed by western blotting. Following densitometric analysis, mitochondrial Bax expression was represented as percentage of control. Note that equal protein loading and transfer for each sample have been confirmed after Ponceau S red staining. Panel A shows a representative western blot experiment of cytosolic and mitochondrial fractions of Ccells for Bax and COX II (mitochondrial marker). Western blot experiments of each PUFA-enriched cell in parallel of Ccells were performed on three independent cell cultures and mitochondrial Bax expression was expressed as a percentage of control (B). Means ± SEM (⁎p b 0.05). ns means not significant.

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Fig. 5A showed an increase of Bax in basal conditions in AAcells and a decrease in EPAcells as compared to Ccells. However, these alterations were extremely moderate, but resulted in a statistically significant difference between AAcells and EPAcells. Moreover, both AA and EPA enrichment prevented the decrease in Bax expression associated to the palmitate treatment (Fig. 5A and B). Conversely, Bcl-2 expression was neither influenced by PUFA treatment of cardiomyocytes nor by palmitate treatment (Fig. 5B). A trend was observed in each experiment for a slight increase in Bcl-2 expression in the EPAcells, which was not confirmed statistically. Similarly, the Bax/Bcl-2 ratio was increased in AAcells and decreased in EPAcells (Fig. 5C) and the difference was significant (p b 0.05) whereas none of these 2 groups was individually different from Ccells. 3.9. Effect of membrane PUFAs on the palmitate-induced translocation of Bax to mitochondria In order to evaluate the individual effect of PUFAs on the palmitateinduced translocation of Bax to mitochondria, western blots experiments were performed in cardiomyocytes controls (Ccells) or cardiomyocytes supplemented with either AA or EPA. Palmitate treatment of Ccells induced a three-fold increase of Bax mitochondrial expression (Fig. 6A and B), whereas this effect of palmitate was totally prevented in PUFA-supplemented cardiomyocytes whatever the PUFA used (AA or EPA). 4. Discussion In this study, we used the in vitro model of palmitate-induced apoptosis in rat neonatal cardiomyocytes to investigate the influence of individual PUFA membrane composition on programmed cell death in the heart. Apoptosis was induced by 0.5 mM palmitate with a palmitate/BSA ratio of 6/1. Although some papers were obtained with this ratio, it is different from the most often used 2/1 ratio [4,10,11,16]. This model involved 2 successive treatments with fatty acids, the first one with PUFAs to modify the membrane composition, and the second one with palmitate to induce apoptosis. The first treatment featuring the dietary supply was made with a “physiological” 2/1 ratio, whereas the second one featuring an aggression was made with a “pathological” ratio 6/1. The use of 2 treatments with a 2/1 ratio would result in a very high BSA content in the medium, which would in turn severely affect the free calcium concentration and induce a significant decrease in beating rate and the occurrence of arrhythmias. Our results indicate that the 2/1 and 6/1 palmitate/BSA ratios similarly induce apoptosis in cardiomyocytes, as already reported in the same model for ROS generation [36]. The results showed that cardiomyocytes exposed to palmitate display an increased caspase-3 activity and mitochondrial alterations particularly a cardiolipin loss leading to a cytochrome c release in cytosol in agreement with the literature [11,16], without activation of the caspase-8. In the present work, we showed that this palmitate effect was associated with a translocation of Bax to the mitochondria, which confirms the central role of mitochondria in the cardiac myocyte cell death process. The various fatty acid treatments used in this study resulted in the expected modifications of fatty acid profile of membrane phospholipids as previously described in the same model [26,37,38]. In the present study, the modification of membrane PUFA profile by supplementation with either EPA (n − 3 PUFA) or AA (n − 6 PUFA) totally prevented the increase of caspase-3 activity induced by palmitate, the translocation of Bax to the mitochondria and the release of cytochrome c into the cytosol. Furthermore, the prevention on mitochondrial cardiolipin loss induced by palmitate was significantly dependent on the PUFA introduced in membrane phospholipids. Cardiolipin is an important phospholipid that contributes to the strength of mitochondrial structure and function and consequently to cell survival. A close interaction between cytochrome c and cardiolipin

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exists and a direct relationship between cardiolipin loss and cytochrome c release from mitochondria has already been identified as a key step in the pathway to apoptosis [17]. In basal conditions, the total level of cardiolipin was not influenced by membrane enrichment with EPA but was significantly reduced when the cardiomyocytes membranes were enriched with AA. A chronic n − 6 PUFA feeding was already reported to activate mitochondrial phospholipase A2 in the rat heart [34,39,40], and hence to trigger cardiolipin hydrolysis and a loss in total cardiolipin mass. Our results in AA-enriched cells confirm these observations. The drop of cardiolipin induced by palmitate treatment in Ccells could be attributed to the low affinity of cardiolipin-synthase for saturated phosphatidylglycerol [17]. Similarly, investigations showed that ischemia is able to induce a severe loss in mitochondrial cardiolipin [41]. This cardiolipin loss due to palmitate treatment was also observed in AA but not in EPAcells. This selective protective effect of EPA on cardiolipin content has, to our knowledge, not been reported in the literature. It may be related to the resistance of EPAcells to the release of mitochondrial cytochrome c into cytosol. The increased cytochrome c release into cytosol of AAcells in the absence of palmitate is the consequence of the CL loss observed in these conditions. Moreover, several nutritional studies reported that the modification of mitochondrial phospholipids by fish oil (n − 3 PUFAs) feeding prevented the loss of mitochondrial cardiolipin and enhanced the cardioprotection against ischemicinduced cardiac arrhythmias, mitochondrial calcium overload and activation of the mitochondrial permeability transition pore leading to apoptosis [42,43]. Feeding rats a diet enriched in PUFAs leads to a modification of heart mitochondrial cardiolipin fatty acid composition, which may affect mitochondrial integrity and function. A recent publication reported that increasing the unsaturation in cardiolipin protected the cytochrome c from photodynamic damage [44]. In a review on the diabetic heart, Ghosh and Rodrigues [23] suggested that acute EPA may prevent calcium overload associated with the mitochondrial pore transition opening, which leads to cytochrome c release. Moreover, several studies reported a beneficial effect of EPA on cardiomyocyte function by chronic exposure leading to membrane profile modifications [26,38,45–47]. Our results showed that the protection against palmitate-induced apoptosis due to EPA was based, at least in part, on the maintenance of cardiolipin content in mitochondria membranes. This effect may result either from a diminution of lysocardiolipin production due to cardiolipin PUFA oxidation or to an increase in cardiolipin synthesis and turnover. As reported in the literature, cardiomyocyte apoptosis induced by palmitate is not associated to an increased ROS formation [36] and cardiolipin oxidation may thus not be higher in palmitate treated cells than in control cells. Moreover, the total membrane unsaturation (and thus the sensitivity to oxidation) is higher in AAcells than in Ccells and much higher win EPAcells due to the high content in DPA with 5 double bonds. The probability to oxidize the cardiolipin is contradictory with the observed cardiolipin loss in palmitate treated cells. This protective effect of EPA/DPA on cardiolipin may rather be attributed to an increase in synthesis, which may involve their properties in PPAR activation since the synthesis of cardiac cardiolipin was reported to be stimulated by PPAR-α activation in rodent models [48]. However, this effect of EPA supply can hardly be attributed to EPA itself because this PUFA is weakly incorporated in cardiac mitochondrial phospholipids in vivo but lead to a significant increase in DPA (docosapentaenoic acid; 22:5 n − 3), the elongation product of EPA. Nevertheless, the supply of EPA to the cardiomyocytes in this study led to a total content of 33.9% of n − 3 PUFAs (as EPA + DPA + DHA) in mitochondrial membranes (vs 7.5% in control cells) balanced by a significant decrease in AA (6.7% vs 15.1% in control cells). The proteins of the Bcl-2 family, including both anti-apoptotic (e.g. Bcl-2, Bcl-XL) and pro-apoptotic members (e.g. Bax, Bad) are known to regulate apoptosis in the cardiovascular system [49]. Bax is abundant in neonatal cardiomyocytes [50,51] and is largely located in the cytosol

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in healthy cells. Under apoptosis stimulus, the translocation of Bax to mitochondria outer membrane occurs. After oligomerization, it triggers cell death through permeability transition pore opening resulting in mitochondrial dysfunction [13]. However, the mechanism by which Bax induces cytochrome c release from the intermembrane space remains controversial. Two hypotheses have been considered, either the formation of channels in the outer mitochondrial membrane by Bax itself or the interaction of Bax with an existing outer membrane protein such as VDAC [52]. Bax translocation and permeabilization of membranes are blocked by anti-apoptotic proteins like Bcl-2 [53]. Thus, an increase in Bax/Bcl-2 ratio contributes to the formation of pores in mitochondria and results in an efflux of pro-apoptotic factors, including cytochrome c [54]. In this study, we showed that palmitate-induced apoptosis involves the migration of the pro-apoptotic Bax from cytosol to mitochondria. This Bax translocation was also reported in the same model during stimulated ischemia [55] and in Rho-A activated cells [56]. Interestingly, in the present study, both (n − 3) and (n − 6) PUFA-enriched cells were resistant to mitochondrial Bax translocation in response to palmitate action, suggesting that increasing the PUFAs proportion in the mitochondrial membranes could be responsible for the decrease of Bax insertion in the mitochondria, whatever the PUFA supplied. Further investigations are required to explain these data, including the effect of PUFAs on PTP elements such as VDAC or ANT. Since Bax is located in various organelles (cytosol, nucleus, mitochondria and recently endoplasmic reticulum), western blot assays were performed on total cell lysates in order to investigate the impact of palmitate and PUFAs on total Bax, Bcl-2 expression and Bax/Bcl-2 ratio which determines the ultimate fate of the cells. In Ccells, palmitate induced a moderate but significant decrease of total Bax expression whereas the Bcl-2 expression level remained unchanged. Nevertheless, the balance between levels of Bax and those of anti-apoptotic Bcl-2 represented by the Bax/Bcl-2 ratio remained unchanged by palmitate treatment. Our results demonstrate a small increase of total Bax expression in AAenriched cells, and a decrease in EPA-enriched cells. Conversely, no modification of Bcl-2 expression could be observed in either AAcells or EPAcells. However, the increase of total Bax in (n − 3) PUFAs and the decrease in AA (EPAcells) were associated with a significant decrease of the Bax/Bcl-2 ratio suggesting that the anti-apoptotic effect of EPA chronic treatment may be associated to a modification of the apoptotic protein profile. The modification of Bax expression could be directly attributed to palmitate treatment, since (n − 3) PUFAs were reported not to influence Bax, Bcl-2 and caspase-3 gene expression [57,58]. However, several members of the Bcl-2 family undergo post-translational modifications, including phosphorylation and proteolysis, that contribute to alter their function [59]. Interestingly, the results of the present study demonstrate that palmitate was inefficient to lower Bax total expression and to increase Bax mitochondrial expression in the PUFA-enriched cells. Further experiments are necessary to elucidate the mechanisms involved in these PUFA effects. In conclusion, this study showed that palmitate acute treatment induces apoptosis in cardiomyocytes, as evidenced by apoptotic nuclei apparition, caspase-3 activation, cytochrome c release from mitochondria, mitochondrial cardiolipin loss and Bax translocation to mitochondria. Moreover, the results demonstrated a protective effect of EPA/DPA on palmitate-induced apoptosis, as evidenced by the prevention of CL decrease, cytochrome c release, relocalisation of Bax to mitochondria and finally caspase-3 activation. However, the final attribution of this anti-apoptotic effect to DPA will require in vivo experimentations in animals fed EPA, which heart phospholipids will display a high DPA content and a low EPA content. The results observed after AA enrichment are more controversial since AA prevented cytochrome c release although the spontaneous release was increased and failed to maintain CL homeostasis. Nevertheless, AA elicited the same preventive effect than EPA on Bax

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