Does n-3 PUFA supplementation favour free-radical damage? Different effects of EPA And DHA in cultured cardiomyocytes.

Introduction. Numerous epidemiological studies pointed out the key role played by dietary fats in the development of cardiovascular diseases; in particular, the lower incidence of coronary dysfunction was correlated with n-3 polyunsaturated fatty acids (PUFAs) [1], which beneficial influence had been mainly explained through their vascular hypolipemiant, antiagregant and hypotensive actions. Recently, a direct effect of n-3 PUFAs on the expression of genes related to cardiovascular diseases has been demonstrated in the heart [2]. The putative role of free radicals (FRs) in the pathogenesis of heart disorders had been stressed in many studies [3]. These FRs exert their noxious effects through peroxidative damages of membrane and of intracellular components, resulting in significant changes to the cellular metabolism and functions leading ultimately to cell death. At the cellular scale, the lipid peroxidation seems dependent on the cell fatty acid composition [4]. In this context, a diet rich in PUFA may favour free-radical mediated lipid peroxidation. However, the few studies dealing with this issue led to discordant results, and the specific influence of each class of PUFA remains ignored. The purpose of this study was to evaluate the influence of n - 3 PUFA supplementation on oxidative damage in cultured cardiomyocytes, in both basal conditions and after an H2O2-induced stress. Materials and Methods. Primary rat cardiomyocyte cultures were prepared from newborn Wistar rats as described [2]. The standard culture medium was composed of Ham's F10 medium supplemented with 10% fetal calf serum, 10% horse serum and antibiotics. From this standard culture medium, two fatty acid-enriched media were prepared, one supplemented with eicosapentaenoic acid (EPA, C20:5n - 3; 60 M), the other with docosahexaenoic acid (DHA, C22:6n - 3; 60 M). At confluence, some cells of each group received 0.1mM H2O2. After 1 h, cells were scraped off, and the following parameters were determined: phospholipid (PL) fatty acid composition, LDH release in culture media, cell vitality (MTT test), conjugated diene level, cytosolic total antioxidant activity (TAA), catalase activity, glutathione peroxidase activity. Results. PL fatty acid composition was deeply modified by n-3 PUFA supplementation, with an enrichment in EPA or DHA relative molar content. Differences observed in LDH release in basal condition could be accounted to these modifications. H2O2 exposure increased LDH release and decreased cell vitality in all groups, and to a higher extent in PUFA supplemented cells. Surprisingly, H2O2 exposure increased conjugated diene level only in control cells, and not in PUFA supplemented ones. In the former group, after the oxidative stress TAA decreased to a higher extent than in the latter ones. This partial maintenance of antioxidant defences could be accounted, at least in part, to the significant increase in catalase activity observed in PUFA supplemented cardiomyocytes in both basal and oxidative conditions. Although there is an evident relation between the chemical nature of PUFAs and their sensitivity to peroxidation, the demonstration of this theoretical link has received little experimental support and the cross relationship between the PUFA content of cells and the entity of oxidative stress remains largely elusive. Lamers et al. [5] showed that ischemia-reperfused hearts from PUFA-fed pigs release less MDA than hearts from saturated fats-fed animals, and our data suggest that PUFA enrichment may positively influence cell protection against FRs. 1. Ruxton CHS et al (2004) J Hum Nutr Dietet 17, 449; 2. Bordoni A et al (2007) FEBS Lett 581, 923; 3. Griendling KK & FitzGerald GA (2003) Circulation 108, 1912; 4. North JA et al (1994) Am J Physiol 267, C177; 5. Lamers JMJ et al (1988) J Mol Cell Cardiol 20, 605.