PLA2dependence of diaphragm mitochondrial formation of reactive oxygen species

Abstract

Contraction-induced respiratory muscle fatigue and sepsis-related reductions in respiratory muscle force-generating capacity are mediated, at least in part, by reactive oxygen species (ROS). The subcellular sources and mechanisms of generation of ROS in these conditions are incompletely understood. We postulated that the physiological changes associated with muscle contraction (i.e., increases in calcium and ADP concentration) stimulate mitochondrial generation of ROS by a phospholipase A2(PLA2)-modulated process and that sepsis enhances muscle generation of ROS by upregulating PLA2activity. To test these hypotheses, we examined H2O2generation by diaphragm mitochondria isolated from saline-treated control and endotoxin-treated septic animals in the presence and absence of calcium and ADP; we also assessed the effect of PLA2inhibitors on H2O2formation. We found that 1) calcium and ADP stimulated H2O2formation by diaphragm mitochondria from both control and septic animals; 2) mitochondria from septic animals demonstrated substantially higher H2O2formation than mitochondria from control animals under basal, calcium-stimulated, and ADP-stimulated conditions; and 3) inhibitors of 14-kDa PLA2blocked the enhanced H2O2generation in all conditions. We also found that administration of arachidonic acid (the principal metabolic product of PLA2activation) increased mitochondrial H2O2formation by interacting with complex I of the electron transport chain. These data suggest that diaphragm mitochondrial ROS formation during contraction and sepsis may be critically dependent on PLA2activation.