Antioxidant Effects of Exogenous Mitochondria: The Role of Outer Membrane Integrity

The administration of isolated mitochondria is a promising strategy for protecting cells from oxidative damage. This study aimed to identify mitochondrial characteristics that contribute to stronger protective effects. We compared two types of mitochondria isolated from C6 cells with similar ATP-producing capacity but differing in outer membrane integrity. To evaluate their stability in extracellular conditions, we examined their behavior in serum. Both types underwent mitochondrial permeability transition to a similar extent; however, under intracellular-like conditions after serum incubation, mitochondria with intact membranes retained more polarized mitochondria. Notably, mitochondria with intact outer membranes were internalized more efficiently than those with damaged membranes. In H9c2 cells, both types of mitochondria similarly increased intracellular ATP levels 1 h after administration under all tested conditions. When co-administered with H₂O₂, both suppressed oxidative damage to a comparable degree, as indicated by similar H₂O₂-scavenging activity in solution, comparable intracellular ROS levels, and equivalent preservation of electron transport chain activity. However, at higher H₂O₂ concentrations, cells treated with mitochondria possessing intact outer membranes exhibited greater survival 24 h after co-administration. Furthermore, when mitochondria were added after H₂O₂-induced damage and their removal, intact mitochondria conferred superior cell survival compared to damaged ones. These findings suggest that while both mitochondrial types exert comparable antioxidant effects, outer membrane integrity prior to administration plays a critical role in enhancing cell survival under conditions of oxidative stress.