Hemin causes mitochondrial dysfunction in endothelial cells through promoting lipid peroxidation: the protective role of autophagy

Higdon AN, Benavides GA, Chacko BK, Ouyang X, Johnson MS, Landar A, Zhang J, Darley-Usmar VM. Hemin causes mitochondrial dysfunction in endothelial cells through promoting lipid peroxidation: the protective role of autophagy. Am J Physiol Heart Circ Physiol 302: H1394-H1409, 2012. First published January 13, 2012; doi: 10.1152/ajpheart.00584.2011.-The hemolysis of red blood cells and muscle damage results in the release of the heme proteins myoglobin, hemoglobin, and free heme into the vasculature. The mechanisms of heme toxicity are not clear but may involve lipid peroxidation, which we hypothesized would result in mitochondrial damage in endothelial cells. To test this, we used bovine aortic endothelial cells (BAEC) in culture and exposed them to hemin. Hemin led to mitochondrial dysfunction, activation of autophagy, mitophagy, and, at high concentrations, apoptosis. To detect whether hemin induced lipid peroxidation and damaged proteins, we used derivatives of arachidonic acid tagged with biotin or Bodipy (Bt-AA, BD-AA). We found that in cells treated with hemin, Bt-AA was oxidized and formed adducts with proteins, which were inhibited by alpha-tocopherol. Hemin-dependent mitochondrial dysfunction was also attenuated by alpha-tocopherol. Protein thiol modification and carbonyl formation occurred on exposure and was not inhibited by alpha-tocopherol. Supporting a protective role of autophagy, the inhibitor 3-methyladenine potentiated cell death. These data demonstrate that hemin mediates cytotoxicity through a mechanism which involves protein modification by oxidized lipids and other oxidants, decreased respiratory capacity, and a protective role for the autophagic process. Attenuation of lipid peroxidation may be able to preserve mitochondrial function in the endothelium and protect cells from heme-dependent toxicity.