Oxidized Ferric and Ferryl Forms of Hemoglobin Trigger Mitochondrial Dysfunction and Injury in Alveolar Type I Cells

Am J Respir Cell Mol Biol. 2016 Aug;55(2):288-98. doi: 10.1165/rcmb.2015-0197OC.


Lung alveoli are lined by alveolar type (AT) 1 cells and cuboidal AT2 cells. The AT1 cells are likely to be exposed to cell-free hemoglobin (Hb) in multiple lung diseases; however, the role of Hb redox (reduction-oxidation) reactions and their precise contributions to AT1 cell injury are not well understood. Using mouse lung epithelial cells (E10) as an AT1 cell model, we demonstrate here that higher Hb oxidation states, ferric Hb (HbFe(3+)) and ferryl Hb (HbFe(4+)) and subsequent heme loss play a central role in the genesis of injury. Exposures to HbFe(2+) and HbFe(3+) for 24 hours induced expression of heme oxygenase (HO)-1 protein in E10 cells and HO-1 translocation in the purified mitochondrial fractions. Both of these effects were intensified with increasing oxidation states of Hb. Next, we examined the effects of Hb oxidation and free heme on mitochondrial bioenergetic function by measuring changes in the mitochondrial transmembrane potential and oxygen consumption rate. In contrast to HbFe(2+), HbFe(3+) reduced basal oxygen consumption rate, indicating compromised mitochondrial activity. However, HbFe(4+) exposure not only induced early expression of HO-1 but also caused mitochondrial dysfunction within 12 hours when compared with HbFe(2+) and HbFe(3+). Exposure to HbFe(4+) for 24 hours also caused mitochondrial depolarization in E10 cells. The deleterious effects of HbFe(3+) and HbFe(4+) were reversed by the addition of scavenger proteins, haptoglobin and hemopexin. Collectively, these data establish, for the first time, a central role for cell-free Hb in lung epithelial injury, and that these effects are mediated through the redox transition of Hb to higher oxidation states.

Keywords: heme oxygenase; hemoglobin; hemolysis; lung epithelium; mitochondria.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adult
  • Alveolar Epithelial Cells / drug effects
  • Alveolar Epithelial Cells / metabolism*
  • Alveolar Epithelial Cells / pathology*
  • Animals
  • Cell Line
  • Heme Oxygenase-1 / metabolism
  • Hemoglobins / isolation & purification
  • Hemoglobins / pharmacology*
  • Humans
  • Iron / metabolism*
  • Membrane Potential, Mitochondrial / drug effects
  • Mice
  • Mitochondria / drug effects
  • Mitochondria / metabolism*
  • Models, Biological
  • Oxidation-Reduction / drug effects
  • Protein Transport / drug effects
  • Up-Regulation / drug effects


  • Hemoglobins
  • Iron
  • Heme Oxygenase-1