Mitochondria-targeted paraquat and metformin mediate ROS production to induce multiple pathways of retrograde signaling: A dose-dependent phenomenon

The mitochondrial electron transport chain is a major source of reactive oxygen species (ROS) and is also a target of ROS, with an implied role in the stabilization of hypoxia-inducible factor (HIF) and induction of the AMPK pathway. Here we used varying doses of two agents, Mito-Paraquat and Mito-Metformin, that have been conjugated to cationic triphenylphosphonium (TPP(+)) moiety to selectively target them to the mitochondrial matrix compartment, thereby resulting in the site-specific generation of ROS within mitochondria. These agents primarily induce superoxide (O(2)(•–)) production by acting on complex I. In Raw264.7 macrophages, C2C12 skeletal myocytes, and HCT116 adenocarcinoma cells, we show that mitochondria-targeted oxidants can induce ROS (O(2)(•–) and H(2)O(2)). In all three cell lines tested, the mitochondria-targeted agents disrupted membrane potential and activated calcineurin and the Cn-dependent retrograde signaling pathway. Hypoxic culture conditions also induced Cn activation and HIF1α activation in a temporally regulated manner, with the former appearing at shorter exposure times. Together, our results indicate that mitochondrial oxidant-induced retrograde signaling is driven by disruption of membrane potential and activation of Ca(2+)/Cn pathway and is independent of ROS-induced HIF1α or AMPK pathways.