Contrasting effects of arachidonic acid and docosahexaenoic acid membrane incorporation into cardiomyocytes on free cholesterol turnover

Biochimica et Biophysica Acta 1841 (2014) 1413–1421

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Contrasting effects of arachidonic acid and docosahexaenoic acid membrane incorporation into cardiomyocytes on free cholesterol turnover Aline Doublet a, Véronique Robert b,1, Benoît Vedie c, Delphine Rousseau-Ralliard d,e,1, Anne Reboulleau a, Alain Grynberg f,1, Jean-Louis Paul a,c, Natalie Fournier a,c,⁎ a

Univ Paris-Sud, EA 4529, UFR de Pharmacie, 92296 Châtenay-Malabry, France INRA, UMR 1319 MICALIS, Commensal and Probiotics–Host Interactions Laboratory, 78350 Jouy-en-Josas, France c AP-HP (Assistance Publique-Hôpitaux de Paris), Hôpital européen Georges Pompidou, Service de Biochimie, 75015 Paris, France d INRA, UMR 1198 Biologie du Développement et Reproduction, 78352 Jouy-en-Josas, France e ENVA, 94700 Maisons Alfort, France f CRNH-IdF, SMBH Université Paris 13, 93000 Bobigny, France b

a r t i c l e

i n f o

Article history: Received 20 January 2014 Received in revised form 29 June 2014 Accepted 3 July 2014 Available online 11 July 2014 Keywords: Cardiomyocytes Arachidonic ω6 polyunsaturated fatty acid Docosahexaenoic ω3 polyunsaturated fatty acid Cholesterol efflux ATP binding cassette transporter A1 Cholesterol homeostasis

a b s t r a c t The preservation of a constant pool of free cholesterol (FC) is critical to ensure several functions of cardiomyocytes. We investigated the impact of the membrane incorporation of arachidonic acid (C20:4 ω6, AA) or docosahexaenoic acid (C22:6 ω3, DHA) as ω6 or ω3 polyunsaturated fatty acids (PUFAs) on cholesterol homeostasis in primary cultures of neonatal rat cardiac myocytes. We measured significant alterations to the phospholipid FA profiles, which had markedly different ω6/ω3 ratios between the AA and DHA cells (13 vs. 1). The AA cells showed a 2.7-fold lower cholesterol biosynthesis than the DHA cells. Overall, the AA cells showed 2-fold lower FC masses and 2-fold higher cholesteryl ester masses than the DHA cells. The AA cells had a lower FC to phospholipid ratio and higher triglyceride levels than the DHA cells. Moreover, the AA cells showed a 40% decrease in ATP binding cassette transporter A1 (ABCA1)-mediated and a 19% decrease in ABCG1mediated cholesterol efflux than the DHA cells. The differences in cholesterol efflux pathways induced by AA or DHA incorporation were not caused by variations in ABCs transporter expression and were reduced when ABC transporters were overexpressed by exposure to LXR/RXR agonists. These results show that AA incorporation into cardiomyocyte membranes decreased the FC turnover by markedly decreasing the endogenous cholesterol synthesis and by decreasing the ABCA1- and ABCG1-cholesterol efflux pathways, whereas DHA had the opposite effects. We propose that these observations may partially contribute to the beneficial effects on the heart of a diet containing a high ω3/ω6 PUFA ratio. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Dietary changes weakly affect the saturated and monounsaturated fatty acid content and composition in the membranes of cardiac ventricular myocytes. However, the omega-3/omega-6 dietary balance Abbreviations: AA, arachidonic acid; ABCA1, ATP binding cassette transporter A1; ABCG1, ATP binding cassette transporter G1; apo, apolipoprotein; CE, cholesteryl esters; DHA, docosahexaenoic acid; FA, fatty acids; FC, free cholesterol; LXR/RXR, liver X receptor/retinoid X receptor; MUFA, monounsaturated fatty acid; PL, phospholipids; PUFA, polyunsaturated fatty acid; SFA, saturated fatty acid; SREBP, sterol regulatory element binding proteins; TG, triglycerides ⁎ Corresponding author at: Laboratoire de Biochimie Appliquée, EA 4529, UFR de Pharmacie, 5 rue Jean Baptiste Clément, 92290 Châtenay-Malabry, France. Tel.: +33 1 46 83 57 23; fax: +33 1 46 83 53 71. E-mail address: [email protected] (N. Fournier). 1 ex-UMR 1154 INRA/Univ Paris-Sud, UFR de Pharmacie, 92296 Châtenay-Malabry, France.

http://dx.doi.org/10.1016/j.bbalip.2014.07.003 1388-1981/© 2014 Elsevier B.V. All rights reserved.

strongly influences the docosahexaenoic acid (DHA) vs. arachidonic acid (AA) content and composition of membrane phospholipids. An increase in DHA results in an equivalent decrease in AA. These dietary alterations of polyunsaturated fatty acids (PUFAs) in cell membranes influence cardiomyocyte functions by modulating membrane physicochemical properties such as the fluidity, organization and function of membrane proteins. Moreover, PUFAs modulate cardiac eicosanoids formed from arachidonic acid (C20:4 ω6) and eicosapentaenoic acid (C20:5 ω3) [1]. PUFA balance is known to influence several signal transduction pathways in various cell types [2]. Equally important is the ability of PUFAs to modulate cholesterol metabolism, particularly cellular cholesterol homeostasis, which results from a balance between endogenous synthesis, receptor-mediated endocytosis and cholesterol efflux. However, controversial results have been reported in various cell types such as macrophage-derived foam cells, which play a key role in atherosclerosis, as well as fibroblasts and adipocytes [3].

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Several epidemiological studies have shown the beneficial effect of a diet containing a high ω3/ω6 PUFA ratio in the prevention of cardiovascular disease [4–7]. Many experimental studies have provided evidence that ω3 PUFAs have diverse actions on various cell types such as platelets, endothelial cells, vascular smooth muscle cells and macrophages. Some studies also demonstrated that PUFAs exert several cardiac effects such as the regulation of phospholipase A2 activity [8], the alteration of receptor-mediated phospholipase C activity [9], the modulation of a βadrenergic transduction mechanism through cAMP [10–12], or an influence on the mitochondrial apoptosis pathway [13]. Nevertheless, to our knowledge, the impact of membrane PUFA composition on cardiomyocyte cholesterol homeostasis has not been investigated. Recently, we reported data on the regulation of cholesterol metabolism in cardiomyocytes. For this, we developed experimental procedures to assess cholesterol synthesis, cholesterol masses, LDL-cholesterol uptake and cholesterol efflux from primary cultures of neonatal rat cardiac myocytes [14]. We demonstrated the key importance of the ABCA1(ATP binding cassette transporter A1) and ABCG1-mediated cholesterol efflux pathways in maintaining a constant free cholesterol (FC) pool size, which is critical for ensuring several major functions such as membrane domain rigidity, ion exchangers, β-adrenergic signaling [15–17], Ca2+ current regulation [18] or MG53 protein-mediated cardiac membrane repair [19]. The purpose of the present study was to evaluate the impact of the membrane incorporation of either AA or DHA as ω6 and ω3 PUFAs on cholesterol homeostasis in cardiomyocytes. Our results show that cholesterol biosynthesis, ABCA1- and ABCG1cholesterol efflux pathways, and overall cellular FC pool size are markedly decreased in AA-enriched cardiomyocytes compared with DHAenriched cardiomyocytes. These findings suggest that the potentially deleterious in vitro effects of ω6 PUFA membrane incorporation may partially contribute to the beneficial effects on the heart of a diet containing a high ω3/ω6 PUFA ratio.

μM (final albumin concentration: 4.1 g/L). The concentration of 90 μM of FA was selected because it is within the concentration range that our group and others currently use to induce marked effects on several functions of cardiomyocytes without inducing cellular toxicity (60–100 μM) [10–12]. After a 32-h incubation, the cells were further grown for 48 h in either basal medium to produce the standard cardiomyocytes (Std cells) or the PUFA-supplemented media to produce AA- or DHAenriched cardiomyocytes (AA cells and DHA cells, respectively). 2.3. General experimental design to study the impact of fatty acids The results of PUFA incorporation on endogenous cholesterol synthesis were evaluated by incubating the cardiomyocytes for 18 h in serumfree medium containing [3H]-acetate. In addition, cardiomyocytes were radiolabeled with [3H]-cholesterol, equilibrated overnight in BSAserum free medium and then subjected to cholesterol efflux measurements. Moreover, the impact on FA profiles, cholesterol masses, phospholipid and triglyceride contents, and mRNA or protein expression for ABC transporters was determined after overnight equilibration in 0.5% BSA-serum free medium. 2.4. Fatty acid analysis The cells were harvested in distilled water and lipids were extracted in a 2:1 chloroform-methanol mixture [22]. Phospholipids (PL) were separated from nonphosphorous lipids on silica acid cartridges [23] and the FA were trans-methylated with 7%. BF3-methanol The methyl esters were analyzed as described elsewhere by gas chromatography (GC 3900, Varian, Les Ulis, France) on econocap EC-WAX capillary columns (30 m × 0.32 m, Alltech Associates, Deerfield, IL, USA) coupled to a flame ionization detector [13,24]. A mixture was used to identify FA methyl esters in samples, and the results were expressed as the relative abundance of total FA using heptadecanoic acid (C17:0) as an internal standard.

2. Material and methods 2.5. Assessment of endogenous cholesterol synthesis 2.1. Cardiomyocyte primary culture Cardiac myocytes were prepared from 2- to 4-day-old Wistar rats as previously described [13,14,20]. Briefly, the ventricles were minced and the cardiac cells were dissociated during 7 proteolytic treatments in trypsin at 30 °C. The cells from the last 6 steps were resuspended in culture medium, and the preparation was enriched in myocytes by 2 successive preplating periods (30 and 120 min). The final suspension was seeded on 60 mm Petri dishes (Falcon Primaria; Becton Dickinson, Pont de claix, France; 2 × 106 cells/dish) or on 24-well plates (Falcon Primaria; 12 × 104 cells/well). The cells were grown in standard complete culture medium (Ham's F10 medium supplemented with 10% fetal calf serum, 10% human serum and antibiotics with a free calcium concentration standardized at 1.2 mM) renewed after 24 h and after 48 h thereafter. We previously reported that cardiomyocytes cultured in these conditions contain less than 5% of non-muscle cells [21]. After 96 h in the standard medium, the cells were further grown either in standard medium or in one of the experimental media for 80 h and then subjected to experimental treatments. All experiments were conducted 7–8 days after plating.

As previously described [14], de novo cholesterol synthesis was assayed by thin layer chromatography after an 18-h incubation of the cells with [3H] acetate as a radiolabeled precursor of cholesterol in serum-free medium supplemented with 0.5% BSA. Free cholesterol bands well separated from bands of diglycerides, monoglycerides or phospholipids were quantified by liquid scintillation counting, and the results were expressed as cpm FC/μg protein/18 h. 2.6. Determination of free and esterified cholesterol masses After cell lysis with 0.2 M NaOH, a sample was removed for protein determination using the BCA method, while the remaining cell lysate was used to quantify FC and cholesteryl ester (CE) contents using HPLC as previously described [14,25]. This method allows the determination of free cholesterol and 7 cholesteryl esters: linoleate, oleate, arachidonate, docosahexaenoate, palmitate, myristate and stearate. In these experimental conditions, cholesteryl eicosapentaenoate and cholesteryl docosapentaenoate, if present, comigrate with cholesteryl docosahexaenoate as a unique cholesteryl-ω3 peak. The results were expressed as nmol cholesterol/mg cell protein.

2.2. Membrane fatty acid alterations 2.7. Measurement of cellular phospholipid and triglyceride contents To investigate the effect of membrane PUFA enrichment, the cardiomyocytes were incubated 80 h with the experimental media containing either AA or DHA (Cayman Chemical Company, Ann Harbor, MI) prepared by adding FA bound to serum albumin to the standard culture medium as previously described [8]. Some cells were kept untreated in standard medium (standard cells). The final molar ratio of FA/albumin was approximately 1.5 and the final concentration of added FA was 90

The cells were scraped into ice-cold lysis buffer (10 mM Tris–HCl, 1% Triton X-100, 0.5% Nonidet-P40 with protease inhibitors), and lipids were extracted from cardiomyocytes by the Folch method [22]. Briefly, 4 mL of a 2:1 chloroform–methanol mixture was added into 200– 400 μL of cell lysate and vigorously mixed by vortex, followed by the addition of 1 mL of 0.73% NaCl solution to cause phase separation. The

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mixture was centrifuged at 4 °C for 10 min at 3000 g. The lower phase of the lipid extract was dried under nitrogen. The PL and triglyceride (TG) contents were determined enzymatically using kits from DiaSys (DiaSys, Holzheim, Germany) and Biomerieux (Biomerieux, Marcy l'Etoile, France), respectively, and expressed as μg/mg protein determined using the BCA method. 2.8. Efflux of labeled cholesterol Cardiomyocytes were subjected to cholesterol efflux measurements as previously described [14]. Briefly, the cardiomyocytes were radiolabeled for 48 h with [3H]-cholesterol in complete culture medium supplemented or not with PUFAs, equilibrated in BSA-incomplete medium for 18 h, and incubated with either 10 μg/ml apolipoprotein AI (apo AI) (Sigma-Aldrich), or 25 μg PL/ml of human HDL, or 5% normolipidemic human serum for 4 h. The percentage of FC efflux to apo AI was considered as the ABCA1-mediated efflux whereas the percentage of FC efflux to HDL was considered as primarily mediated by aqueous diffusion and ABCG1, the SR-BI being not expressed in cardiomyocytes [14]. The efflux to human serum containing several types of acceptors involved all of these pathways. In addition, the cells were incubated for 30 min with 1 mg/ml methyl-β-cyclodextrin (Sigma) to assess the aqueous diffusion pathway [26]. In some experiments, the cardiomyocytes were incubated with LXR/RXR agonists (5 μg/ml 22-hydroxy-cholesterol and 10 μM 9-cis-retinoic acid) to induce ABCA1 and ABCG1 expression [14]. Each sample was analyzed in triplicate. Fractional cholesterol efflux was calculated as the percentage of the radioactivity released from cells into the medium relative to the total radioactivity in cells and medium and the values were corrected for the small amount of [3H]cholesterol released into the medium in the absence of acceptors. 2.9. Analyses of ABCA1 and ABCG1 mRNA levels Total RNA was extracted from cultured cells using an RNeasy Mini kit (QIAGEN, Courtaboeuf, France) and real-time quantitative PCR was performed using ABI PRISM 7900 (Applied Biosystems, Darmstadt, Germany) with the primers reported previously [14]. Each sample was assayed in triplicate, and relative quantifications normalized to 18S mRNA were expressed as fold-variation over the standard condition (cardiomyocytes grown in standard medium arbitrarily set to 1). 2.10. Western blotting analysis for ABCA1 or ABCG1 The cardiomyocytes were lysed on ice [27] and the total cell lysate (30 μg) was electrophoresed under reducing conditions on 3–8% NuPAGE Tris acetate gels (Novex, Carlsbad, CA) as previously described [14]. The results were expressed as the ratio of ABCA1 or ABCG1 to βactin (loading control) using Adobe Photoshop 7.0 software (Adobe, San Jose, CA). 2.11. Statistics The data were expressed as the mean ± SEM. Comparisons between groups were evaluated by one-way ANOVA with the Bonferroni posthoc test using StatView 5.0 software (SAS Institute, Berkeley, CA). Significance was defined as P b 0.05. 3. Results 3.1. AA cells and DHA cells showed strong differences in membrane fatty acid composition We first compared the cell PL fatty acid composition between AAand DHA-enriched cardiomyocytes in 3 independent cultures. As expected, the PUFAs were incorporated into PL and strongly affected the ω6 and ω3 profiles (Fig. 1). Compared with the Std cells, enrichment

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with AA or DHA weakly affected the membrane content of saturated (SFA) and monounsaturated fatty acids (MUFA). AA cells contained a very high level of AA (C20:4 ω6) (34%) and its elongation product C22:4 ω6 (8%), which was poorly desaturated in C22:5 ω6 (b 0.5%). In contrast, DHA cells contained a high level of DHA (C22:6 ω3) (20%), associated with an increase in EPA (C20:5 ω3). Compared with the Std cells, this increase in ω3 PUFA content was associated with a decrease in all ω6 PUFAs, including AA and linoleic acid (C18:2 ω6). The ω6/ω3 ratios were markedly different between ω6 and ω3 profiles (13 vs. 1), with the Std cells showing an intermediate value (~5). The (MUFA + PUFA)/ SFA ratio and the unsaturation index were approximately similar in AA cells and DHA cells (1.7 vs. 1.8 and 219 vs. 240, respectively). Compared with the Std cells, these two parameters were increased by 22% and ~35%.

3.2. AA cells showed lower cholesterol biosynthesis compared to DHA cells We next determined whether the incubation of cardiomyocytes with AA or DHA modified cellular cholesterol biosynthesis in 3 independent culture preparations. As shown in Fig. 2, the AA cells displayed a 3.0-fold lower cholesterol synthesis compared to Std cells, whereas DHA membrane incorporation caused a minor decrease (− 7%) that was not statistically significant. As a result, the AA cells displayed 2.7fold lower cholesterol biosynthesis than the DHA cells. These results suggest that the AA increase in membranes caused a significant reduction of cholesterol biosynthesis in cardiomyocytes, whereas the DHA had no significant impact.

3.3. AA cells showed lower free cholesterol masses and higher cholesteryl ester masses compared to DHA cells We then investigated the impact of AA vs. DHA membrane incorporation on cholesterol masses in 3 independent culture preparations. As illustrated in Fig. 3, the increased membrane AA incorporation caused a significant decrease of FC masses (− 43%), whereas DHA induced a weak but not significant increase of FC masses (+15%). Both PUFAs induced increases in CE masses, but the effect of AA was more prominent; CE represented 39% of the total cholesterol in the AA cells, whereas they represented 13% in DHA cells and 9% in standard cells. As a result, the AA cells showed lower total cholesterol masses (1.4-fold), lower FC masses (2-fold) and higher CE masses (2-fold) than DHA cells. We also observed qualitative differences in the ω6 and ω3 profiles of the CE subspecies. The AA cells contained more saturated (29.4% vs. 16.4%) and more ω6 FA (65.9% vs. 38.7%), whereas they contained much less ω3 (0.7% vs. 35.2%) as FA esterifying cholesterol compared to the DHA cells.

3.4. AA cells had a lower free cholesterol to phospholipid ratio and higher triglyceride levels compared to DHA cells We also investigated the impact of AA and DHA membrane incorporation on the FC:PL molar ratio, which is an important factor in determining the membrane fluidity and also in determining the availability of the FC for cholesterol efflux. As illustrated in Table 1, both FA treatments did not induce any significant changes in the levels of PL. As a result, the AA cells showed a significantly lower FC:PL ratio (− 38%), whereas the DHA cells showed a significantly higher FC:PL ratio (+33%). Thus, the AA cells had a 53% lower FC:PL ratio than the DHA cells. In addition, we determined the impact of AA and DHA membrane incorporation on cellular triglyceride levels. Both PUFA treatments induced increases in TG masses, but the effect of AA was more prominent; TG masses were 2.7-fold higher in the AA cells and 1.7-fold higher in the DHA cells. As a result, the AA cells had 60% higher cellular TG levels than DHA cells.

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40

Std cells

DHA cells

AA cells

Fatty acid (% of total FA)

30

20

10

0 C16:0

C16:1

C18:0

C18:1

C18:2 ω6

C20:4 ω6

C22:4 ω6

C20:5 ω3

C22:5 ω3

C22:6 ω3

Fig. 1. Fatty acid composition of membrane phospholipids from standard cells and AA- or DHA-enriched cardiomyocytes. Cells were incubated for 80 h with either 90 μM arachidonic acid (AA) or docosahexaenoic acid (DHA) in the experimental media; Std cells were maintained in the standard culture medium. After an overnight equilibration followed by a lipid extraction, phospholipids were separated and methylated for gas chromatography analysis. Each fatty acid (FA) was expressed as the percentage of total FA, and the data are expressed as the mean ± SEM of 9 determinations (3 dishes per culture in 3 different preparations). Std, standard cells.

3.5. AA cells showed lower ABCA1 and ABCG1 cholesterol efflux capacity compared with DHA cells We evaluated the impact of AA and DHA membrane incorporation on cholesterol efflux from cardiomyocytes in a large number of independent experiments by measuring the isotopic cholesterol efflux mediated by several acceptors involving different pathways. As illustrated in Fig. 4A, the AA membrane increase induced a significant decrease in cholesterol efflux to apo AI (−33%), whereas DHA slightly but significantly increased the cholesterol efflux to apo AI (+ 15%). As a result, the AA cells showed 40% lower ABCA1-mediated cholesterol efflux values than the DHA cells. Additionally, the AA membrane increase caused a significant decrease in the cholesterol efflux to HDL (−20%), 75

Std cells

DHA cells

AA cells

whereas the DHA membrane increase had no significant impact (−0.7%) (Fig. 4B). As a result, the AA cells showed a 19% lower cholesterol efflux to HDL than the DHA cells. Similarly, the AA cells showed a 19% decrease in cholesterol efflux to 5% normolipidemic serum compared to DHA cells (data not shown). The variations in efflux to HDL were essentially attributed to a decrease in the ABCG1 pathway in AAenriched cardiomyocytes because none of the FA significantly altered the cholesterol efflux mediated by the aqueous diffusion process (Fig. 4C). Notably, there was no evidence that FA supplementations induced differences in cell viability or cell injury as determined by the cellular protein content and the activity of LDH released into the culture media (data not shown). Taken together, the incorporation of AA in the membrane induced a major decrease in the ABCA1 functionality associated with a moderate decrease in ABCG1, whereas DHA slightly increased the ABCA1 functionality without altering the ABCG1 pathway.

cpm cholesterol/g protein

3.6. AA cells and DHA cells showed similar ABCA1 and ABCG1 expressions **** **** 50

25

0 Fig. 2. Impact of AA or DHA membrane incorporation on endogenous cholesterol synthesis from cardiomyocytes. Cells were incubated for 80 h with either 90 μM arachidonic acid (AA) or docosahexaenoic acid (DHA) in the experimental media; Std cells were maintained untreated in the standard culture medium. All cells were then exposed for 18 h to tritiated acetate in serum-free media. The cholesterol biosynthesis level was expressed as cpm of free cholesterol/μg protein and the results are expressed as the mean ± SEM of 9 determinations (3 dishes per culture in 3 different preparations). Std, standard cells, ****P b 0.0001.

To better understand the mechanisms involved in the differences between the ΑΑ and DHA cells in cholesterol efflux to apo AI and HDL, we measured ABCA1 and ABCG1 expression in 4 independent culture preparations. As shown in Fig. 5A–B, the ABCA1 and ABCG1 mRNA levels were similar in the AA cells and DHA cells. We observed that the DHA cells showed lower ABCG1 mRNA levels than the Std cells. Moreover, no variation in ABCA1 or ABCG1 protein levels was observed between the ΑΑ and DHA cells (Fig. 5C–D). We observed that both the DHA and AA cells showed lower ABCG1 protein levels than the Std cells. These results suggest that the decreases in cholesterol efflux to apo AI or HDL induced by the AA membrane increase were not caused by a decrease in ABC transporter expression. 3.7. Treatment of cardiomyocytes by LXR/RXR agonists reduced the cholesterol efflux differences between AA cells and DHA cells We previously reported [14] that LXR/RXR agonists caused a major effect on ABCA1- and ABCG1-mediated cholesterol efflux, which was increased on average 3.6 and 1.7-fold, respectively. Interestingly, in cardiomyocytes overexpressing ABC transporters, the AA cells only

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Std cells

DHA cells

1417

AA cells

**

200 **

****

nmol/mg protein

150

**** ****

100

**** *** ****

50

0 TC

FC

CE

Fig. 3. Impact of AA or DHA membrane incorporation on cholesterol masses of cardiomyocytes. Cells were incubated for 80 h with either 90 μM arachidonic acid (AA) or docosahexaenoic acid (DHA) in the experimental media; Std cells were maintained untreated in the standard culture medium. After an overnight equilibration, the cell lipids were extracted and the masses of total cholesterol (TC), free cholesterol (FC) or cholesteryl ester (CE) quantified by HPLC were expressed as nmol/mg protein. Values are expressed as the mean ± SEM of 9 determinations (3 dishes per culture in 3 different preparations). Std, standard cells, **P b 0.01, ***P b 0.001 and ****P b 0.0001.

showed a 16% decrease in cholesterol efflux to apo AI compared to the DHA cells (9.25 ± 0.56% vs. 11.02 ± 0.57%; P = 0.02). Moreover, the efflux to HDL was not significantly different between ω6 and ω3 profiles (10.49 ± 0.29% vs. 11.49 ± 0.41% for the AA and DHA cells, respectively). Similarly, the AA and DHA cells showed closed cholesterol efflux values to 5% normolipidemic serum (data not shown). Taken together, the overexpression of ABCA1 and ABCG1 strongly reduced the differences between the AA and DHA cells for their capacity to export cholesterol. 4. Discussion This study was designed to evaluate the chronic effect of the membrane enrichment in ω3 and ω6 PUFAs on cholesterol homeostasis in cardiomyocytes. Although cholesterol-rich microdomains are important for several crucial functions of cardiomyocytes such as membrane domain rigidity, ion exchangers, β-adrenergic signaling pathways

Table 1 Cellular phospholipid and triglyceride contents and free cholesterol to phospholipid ratio from standard cells and AA- or DHA-enriched cardiomyocytes.

Phospholipid (μg/mg protein) FC:PL (molar ratio) Triglycerides (μg/mg protein)

Std cells

DHA cells

AA cells

202.3 ± 9.1

181.4 ± 33.2

188.7 ± 14.2

0.375 ± 0.019 18.6 ± 4.1

0.498 ± 0.091⁎ 31.6 ± 4.9⁎⁎

0.232 ± 0.017⁎⁎⁎⁎, c 50.7 ± 6.7⁎⁎⁎, b

Cells were incubated for 80 h with either 90 μM arachidonic acid (AA) or docosahexaenoic acid (DHA) in the experimental media; Std cells were maintained untreated in the standard culture medium. After an overnight equilibration, the cell lipids were extracted and the phospholipid and triglyceride contents were determined enzymatically; free cholesterol masses were quantified by HPLC. Lipids are expressed as μg/mg protein determined using the BCA method. Values are expressed as the mean ± SEM of 8 determinations (2 dishes per culture in 4 different culture preparations) for PL and mean ± SEM of 6 determinations (2 dishes per culture in 3 different culture preparations) for TG and FC:PL molar ratio. Std, standard cells; FC, free cholesterol; PL, phospholipids; TG, triglycerides. ⁎ P b 0.05 vs. standard. ⁎⁎ P b 0.01 vs. standard. ⁎⁎⁎ P b 0.001vs. standard. ⁎⁎⁎⁎ P b 0.0001 vs. standard. b P b 0.01 AA cells vs. DHA cells. c P b 0.001 AA cells vs. DHA cells.

[15–17], Ca2+ current regulation [18] or MP53 protein-mediated cardiac membrane repair [19], the regulation of cholesterol metabolism has been poorly investigated in this cell type. Several years ago, Shmeeda et al. reported that neonatal cardiomyocytes could synthesize significant levels of cholesterol but poorly internalized external cholesterol supplied by lipoproteins [28]. Our group recently described the regulation of cholesterol metabolism in cultured cardiomyocytes. These studies emphasized the involvement of the cholesterol efflux pathway in preserving cholesterol homeostasis under conditions in which cholesterol availability is decreased [14]. More recently, Campia et al. reported that the cardioactive glycosides digoxin and oubain increase the synthesis of cholesterol. In addition, these glycosides also increase the efflux of cholesterol through the ABCA1 pathway [29]. This suggests that glycosides finely control the level of cholesterol by providing sufficient cholesterol, crucial for the maintenance of membrane homeostasis and the proper activity of transporters, channels and receptors. This is accomplished by preventing the intracellular accumulation of cholesterol through accelerating its release to apo AI. The manipulation of the FA composition of the media led to a corresponding significant alteration of the PL fatty acid profile similar to those observed in vitro in our previous reports [8,12,13,30] and to those observed in vivo in the heart after an ω6 vs. an ω3 diet [24,31]. The primary result was the high difference in the ω6/ω3 ratio between the AA and DHA cells, demonstrating the membrane content increase of either AA or DHA according to the PUFA supplied. Compared to the Std cells, the AA and DHA cells both showed similar increases in the (MUFA + PUFA)/SFA ratio and the unsaturation index suggesting that both PUFA treatments create a more fluid membrane environment. However, the fluidizing effect of an increase in the levels of polyunsaturated FAs in the PL may also be modulated by a change in cholesterol content. Compared to the Std cells, AA but not DHA enrichment caused a significant decrease (2.7-fold) in de novo cholesterol synthesis from tritiated acetate when maintained in serum-free media. Incubation of J774 mouse macrophages with AA was reported to significantly decrease cellular cholesterol biosynthesis in a dose-dependent manner, whereas DHA had no effect [32]. Consistent with these findings, we propose that AA may specifically reduce endogenous cholesterol synthesis by reducing SREBP-1a (sterol regulatory element-binding protein 1a) levels because its expression was strongly reduced in various cell types after treatment with AA [33–36]. In contrast, inconsistent results have been reported for the effect of DHA on SREBP-1 expression [36,37].

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Cholesterol efflux to apo A-I (%/4h)

6

Std cells

DHA cells

12

AA cells

Cholesterol efflux to HDL (%/4h)

1418

**** *** 4

****

2

0

Std cells

DHA cells

AA cells

**** 9

****

6

3

0

(%/30mi)

Cholesterol efflux to cyclodextrin

2

Std cells

DHA cells

AA cells

1

0 Fig. 4. Impact of AA or DHA membrane incorporation on cholesterol efflux from cardiomyocytes. Cells were incubated for 80 h with either 90 μM arachidonic acid (AA) or docosahexaenoic acid (DHA) in the experimental media; Std cells were maintained untreated in the standard culture medium. After an overnight equilibration, the cells were incubated with 10 μg/ml apo AI for 4 h (A), 25 μg/ml HDL-PL for 4 h (B) or with 1 mg/ml methyl-β-cyclodextrin for 30 min (C) as cholesterol acceptors. Fractional cholesterol efflux values are expressed as the mean ± SEM of 30 determinations (3 dishes per culture in 10 different preparations) for efflux to apo AI, the mean ± SEM of 27 determinations (3 dishes per culture in 9 different preparations) for efflux to HDL and the mean ± SEM of 15 determinations (3 dishes per culture in 5 different preparations) for efflux to methyl-β-cyclodextrin. Std, standard cells; apo AI, apolipoprotein AI; HDL-PL, HDL-phospholipids, ***P b 0.001 and ****P b 0.0001.

We next investigated the impact of AA and DHA incorporation on cholesterol masses measured after an overnight equilibration without any lipoprotein source. The results show that the AA cells displayed a 2-fold decrease in FC masses compared to the DHA cells. During the first 80 h of incubation with experimental media (containing calf and human sera), it is unlikely that the cardiomyocytes synthesize cholesterol or take up cholesterol from lipoproteins, this latter pathway being poorly expressed and regulated in these cells [14,28]. The lower FC masses observed in AA cells compared with DHA cells can be attributed to a decrease in cholesterol biosynthesis that occurs during the equilibration step because this decrease shows the same magnitude (2.7-fold). Conversely, the AA cells showed an increase in CE compared with the DHA cells (39% vs. 13% of total cholesterol was esterified in AA cells and DHA cells, respectively). It is unlikely that in cardiomyocytes, DHA decreased the intracellular esterification of cholesterol as reported in macrophages [38] because the CE fraction in the DHA cells was slightly increased compared to that of Std cells (approximately 9%), consistent with our previous work [14]. These results suggest that the higher CE content in the AA cells may result from the stimulation of the ACAT1 isoform found in cardiomyocytes [39,40]. We finally investigated the impact of membrane AA vs. DHA on various cholesterol efflux pathways. The AA cells showed lower ABCA1- and, to a lesser extent, lower ABCG1-mediated cholesterol efflux capacity than the DHA cells. ABCA1 exports cellular FC to lipidpoor apo AI-containing particles (pre-β HDL), whereas ABCG1 exports FC to PL-rich mature α HDL.

We excluded a potential effect of AA or DHA on the expression of ABCs (mRNA or protein) in our experimental conditions. Several groups elegantly showed that MUFA or ω6 or ω3 PUFA decreases cholesterol efflux from macrophages, either by increasing the degradation of ABCA1 or by suppressing the stimulatory effects of oxysterols and retinoids on ABCA1 and ABCG1 expression [41,42]. However, these studies investigated the acute FA impact on cholesterol efflux from macrophages, and the results cannot be compared with our approach, which is a chronic PUFA treatment in cardiomyocytes. Because of the extensive modifications in the cell phospholipid PUFA composition induced by AA or DHA incorporation, the observed variations in cholesterol efflux may be attributed to changes in membrane fluidity. The full ABCA1 transporter is highly sensitive to variations in membrane physicochemical properties [43,44]. We have previously reported in macrophages that decreasing the membrane fluidity by incorporating trans elaidic or palmitic acids may diminish ABCA1 function [45]. However, in addition to FA composition, the fluidity of a membrane is determined by its cholesterol content, and the cholesterol to phospholipid molar ratio is a good reflection of membrane fluidity. Bastiaanse et al. demonstrated in neonatal rat cardiomyocytes that an increase in the FC:PL ratio was associated with a diminished fluidity, whereas a decrease in the FC:PL ratio was associated with an augmented fluidity [46]. In our study, AA induced a strong decrease in the FC:PL ratio (−38%) and an increase in the unsaturation index, suggesting that the membrane is more fluid. This excess membrane fluidity may be related to the alteration of cholesterol efflux. The impact of DHA is

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2

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Fig. 5. Impact of AA or DHA membrane incorporation on ABCA1 and ABCG1 expression in cardiomyocytes. Cells were incubated for 80 h with either 90 μM arachidonic acid (AA) or docosahexaenoic acid (DHA) in the experimental media; Std cells were maintained untreated in the standard culture medium. After an overnight equilibration, ABCA1 or ABCG1 mRNA was quantified by RT-PCR, normalized to 18S mRNA and expressed as fold variations over the standard condition (A and B). Values are expressed as the mean ± SEM of 12 determinations (3 dishes per culture in 4 different preparations). ABCA1 or ABCG1 proteins were determined by Western blotting using polyclonal antibodies; 30 μg of total cell lysate was loaded in each lane and β-actin was used as loading control (C and D). Data are expressed as the ratio of ABCA1 to β-actin or as the ratio of ABCG1 to β-actin and given as the mean ± SEM of 6 determinations (2 dishes per culture in 3 different preparations). Std, standard cells; NS, not significant, *P b 0.05 and **P b 0.01.

more difficult to predict because it induced an increase in the FC:PL ratio (+33%), which may counteract the increase in the level of unsaturation. More importantly, the FC:PL ratio may reflect the availability of FC in the membrane for cholesterol efflux. Consequently, the lower FC:PL ratio observed in the AA cells may be related to the decreased cholesterol efflux. However, in addition to these hypotheses involving PL, we cannot rule out the possibility that PUFAs may modulate cholesterol efflux through quantitative and/or qualitative modifications of the nonphosphorous lipids. In addition to a much higher CE content compared to the DHA cells, the AA cells showed more saturated and ω6 (but less ω3) FA esterifying cholesterol. Although the cardiomyocytes cannot properly be considered as cholesterol loaded, this profile may be associated with anisotropic lipid droplets, and thus with a decreased rate of CE hydrolysis and efflux of cholesterol from cells by creating less fluid cellular CE droplets [47,48]. We also observed that AA incorporation induced a strong increase in the cellular TG content, whereas DHA had a more moderate effect. Consistent with a previous report for cholesterol-loaded human macrophages, the marked increase in TG induced by AA may inhibit ABCA1 function without altering its expression level [49]. Conversely, it was recently reported that EPA and DHA are highly incorporated into triacylglycerols in neonatal rat cardiomyocytes [50], which may facilitate the cholesterol efflux by enhancing CE mobilization from mixed droplets [51].

Finally, we also cannot exclude the possibility that PUFAs modulate ABCA1-mediated cholesterol efflux by the production of eicosanoids and/or cAMP. Several groups have shown that the functionality of ABCA1 depends on the phosphorylation state of the protein, which is modulated by different pathways including PKA [52,53]. We and others demonstrated that DHA supplementation led to a decrease in stimulated cAMP production in cardiomyocytes, most likely by influencing intracellular steps of the signal transduction pathway such as phosphodiesterases, phosphatatases or PKA [10,54]. It was also reported that ω3 PUFA incorporation into cardiomyocyte membranes induced a decrease in eicosanoid production [1]. Interestingly, the effects of AA and DHA membrane incorporation on cholesterol efflux were almost abolished when ABC transporters were overexpressed by LXR/RXR agonists, suggesting that the mechanism responsible for these variations was counteracted by the large number of transporter molecules. Although a complete understanding of the mechanisms will require more detailed investigation, a high level of AA in the membrane reduces the atheroprotective pathway of cholesterol efflux through the ABCA1 transporter, whereas DHA incorporation increases this crucial step. In conclusion, this study demonstrates that the incorporation of AA into the membranes of cardiomyocytes decreased the FC turnover, by dramatically decreasing the endogenous cholesterol synthesis and the ABCA1- and ABCG1-cholesterol efflux pathways. In contrast, DHA incorporation had more moderate effects on these processes and only slightly

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induced an increase in the FC pool size. The reduced FC masses observed in AA-rich cardiomyocytes may have deleterious consequences for several crucial cellular functions. In addition, the cellular accumulation of CE and TG observed in AA-rich cells may also exert detrimental effects because accumulation of neutral lipids in the heart has been associated with cardiac dysfunction and heart failure [55]. We propose that our observations may partially contribute to the beneficial effects on the heart of a diet containing a high ω3/ω6 PUFA ratio. There are certain limitations to the present study. Although we excluded some hypotheses, we did not have the opportunity to identify the mechanisms by which incorporation of AA and DHA into the membrane modulated the cholesterol efflux pathways. Further experiments will be required to elucidate the mechanisms addressed here. For example, it would be of interest to use fluorescent probes to measure the changes in membrane fluidity and to study the metabolites formed from AA and DHA by cyclooxygenases and lipooxygenases. However, a better understanding of efflux modifications will require a thorough investigation and was out of the scope of this study. Moreover, it is difficult to predict the extent to which our data collected from cell culture experiments can be extrapolated to in vivo conditions. An elegant approach would be to isolate cardiomyocytes from rats consuming diets enriched or not (standard diet) with AA or DHA, and studying the different aspects of cholesterol homeostasis in these ex vivo conditions may provide new relevant and complementary information to the present study. Disclosures None declared. Acknowledgements The authors thank Athina Kalopissis (INSERM U1138, Paris, France) and George Rothblat (Lipid Research Group, Children's Hospital of Philadelphia, PA) for their help and advice during the revision of this manuscript. The authors also thank Sylviane Tardivel (EA 4529, UFR de Pharmacie, Châtenay-Malabry) for her technical assistance. References [1] F. Oudot, A. Grynberg, J.P. Sergiel, Eicosanoid synthesis in cardiomyocytes: influence of hypoxia, reoxygenation, and polyunsaturated fatty acids, Am. J. Physiol. 268 (1995) H308–H315. [2] A. Denys, A. Hichami, N.A. Khan, n-3 PUFAs modulate T-cell activation via protein kinase C-alpha and -epsilon and the NF-kappaB signaling pathway, J. Lipid Res. 46 (2005) 752–758. [3] M. Salehipour, E. Javadi, J.Z. Reza, M. Doosti, S. Rezaei, M. Paknejad, N. Nejadi, M. Heidari, Polyunsaturated fatty acids and modulation of cholesterol homeostasis in THP-1 macrophage-derived foam cells, Int. J. Mol. Sci. 11 (2010) 4660–4672. [4] A.P. Simopoulos, The importance of the omega-6/omega-3 fatty acid ratio in cardiovascular disease and other chronic diseases, Exp. Biol. Med. 233 (2008) 674–688. [5] P.C. Weber, Are we what we eat? Fatty acids in nutrition and in cell membranes: cell functions and disorders induced by dietary conditions, Svanoy Foundation, Svanoybukt, Norway, 1989. 9–18 (Report n°4). [6] A.P. Simopoulos, L.G. Cleland, Omega-6/omega-3 essential fatty acid ratio: the scientific evidence, World Rev. Nutr. Diet., 92, Karger, Basel, 2003. [7] M. de Lorgeril, S. Renaud, N. Mamelle, P. Salen, J.-L. Martin, I. Monjaud, J. Guidollet, P. Touboul, J. Delaye, Mediterranean alpha-linolenic acid-rich diet in secondary prevention of coronary heart disease, Lancet 343 (1994) 1454–1459. [8] A. Grynberg, G. Nalbone, J. Leonardi, H. Lafont, P. Athias, Eicosapentaenoic and docosahexaenoic acids in cultured rat ventricular myocytes and hypoxia-induced alterations of phospholipase-A activity, Mol. Cell. Biochem. 116 (1992) 75–78. [9] H.W. de Jonge, D.H. Dekkers, E.M. Bastiaanse, K. Bezstarosti, A. van der Laarse, J.M. Lamers, Eicosapentaenoic acid incorporation in membrane phospholipids modulates receptor-mediated phospholipase C and membrane fluidity in rat ventricular myocytes in culture, J. Mol. Cell. Cardiol. 28 (1996) 1097–1108. [10] A. Grynberg, A. Fournier, J.P. Sergiel, P. Athias, Effect of docosahexaenoic acid and eicosapentaenoic acid in the phospholipids of rat heart muscle cells on adrenoceptor responsiveness and mechanism, J. Mol. Cell. Cardiol. 27 (1995) 2507–2520. [11] A. Grynberg, A. Fournier, J.P. Sergiel, P. Athias, Membrane docosahexaenoic acid vs. eicosapentaenoic acid and the beating function of the cardiomyocyte and its regulation through the adrenergic receptors, Lipids 31 (1996) S205–S210 (Suppl.).

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