O2 affects mitochondrial functionality ex vivo

Mitochondria have originated in eukaryotic cells by endosymbiosis of a specialized prokaryote approximately 2 billion years ago. They are essential for normal cell function by providing energy through their role in oxidizing carbon substrates. Glutathione (GSH) is a major thiol-disulfide redox buffer of the cell including the mitochondrial matrix and intermembrane space. We have generated cardiomyocyte-specific Grx1-roGFP2 GSH redox potential (E-GSH) biosensor mice in the past, in which the sensor is targeted to the mitochondrial matrix. Using this mouse model a distinct E-GSH of the mitochondrial matrix ( - 278.9 +/- 0.4 mV) in isolated cardio myocytes is observed. When analyzing the E GSH in isolated mitochondria from the transgenic hearts, however, the E(GSH )in the mitochondrial matrix is significantly oxidized ( - 247.7 +/- 8.7 mV). This is prevented by adding N-Ethylmaleimide during the mitochondria isolation procedure, which precludes disulfide bond formation. A similar reducing effect is observed by isolating mitochondria in hypoxic (0.1-3% 0 2 ) conditions that mimics mitochondrial pO(2) levels in cellulo. The reduced E-GSH is accompanied by lower ROS production, reduced complex III activity but increased ATP levels produced at baseline and after stimulation with succinate/ADP. Altogether, we demonstrate that oxygenation is an essential factor that needs to be considered when analyzing mitochondrial function ex vivo.