Arachidonic acid supplementation enhances hydrogen peroxide induced oxidative injury of neonatal rat cardiac myocytes.

OBJECTIVE

Selective fatty acid supplementation of non-myocardial cells has been reported to enhance oxidative injury. The purpose of this study was to extend this research by determining if supplementation of isolated cardiac myocytes with arachidonic acid increases myocyte sensitivity to H2O2 induced lipid peroxidation and cytotoxicity.

METHODS

Myocytes were supplemented with arachidonic acid by complexing it with calf serum and adding it to myocytes at a final concentration of 0, 50, or 100 microM for 36 h. Myocytes were then exposed to 50 or 100 microM H2O2 for 1 h and lipid peroxidation and cytotoxicity were assessed by measuring thiobarbituric acid reactive substances and lactate dehydrogenase release into culture medium. To determine if unesterified arachidonic acid contributed to oxidative injury, 100 microM arachidonic acid was added directly to cultured myocytes in serum-free buffer. Vitamin E and desferrioxamine were assessed for their ability to protect against oxidative injury induced by unesterified arachidonic acid or H2O2.

RESULTS

Supplementation with 100 microM arachidonic acid for 36 h increased the arachidonic acid content of phospholipids from 0.58(SD 0.06) to 0.90(0.19) nmol.nmol-1 lipid phosphorus. A 1 h exposure to H2O2 induced lipid peroxidation and cytotoxicity in a concentration dependent manner. Pretreatment of myocytes with 10 microM vitamin E or 1 mM desferrioxamine prevented H2O2 induced effects. Arachidonic acid supplementation at 50 or 100 microM significantly enhanced lipid peroxidation induced by 100 microM H2O2, whereas cytotoxicity was increased only in myocytes supplemented with 100 microM arachidonic acid. Addition of unesterified arachidonic acid directly to cultured myocytes caused marked cytotoxicity and lipid peroxidation which were prevented by vitamin E or desferrioxamine.

CONCLUSIONS

Arachidonic acid supplementation of cardiac myocytes modifies fatty acid content of phospholipids and enhances the susceptibility of myocytes to H2O2 induced oxidative injury.