40 research outputs found
Integrated high-content quantification of intracellular ROS levels and mitochondrial morphofunction
Oxidative stress arises from an imbalance between the production of reactive oxygen species (ROS) and their removal by cellular antioxidant systems. Especially under pathological conditions, mitochondria constitute a relevant source of cellular ROS. These organelles harbor the electron transport chain, bringing electrons in close vicinity to molecular oxygen. Although a full understanding is still lacking, intracellular ROS generation and mitochondrial function are also linked to changes in mitochondrial morphology. To study the intricate relationships between the different factors that govern cellular redox balance in living cells, we have developed a high-contentmicroscopy-based strategy for simultaneous quantification of intracellular ROS levels and mitochondrial morphofunction. Here, we summarize the principles of intracellular ROS generation and removal, and we explain the major considerations for performing quantitative microscopy analyses of ROS and mitochondrial morphofunction in living cells. Next, we describe our workflow, and finally, we illustrate that a multiparametric readout enables the unambiguous classification of chemically perturbed cells as well as laminopathy patient cells
Membrane potential and delta pH dependency of reverse electron transport-associated hydrogen peroxide production in brain and heart mitochondria
Succinate-driven reverse electron transport (RET) is one of the main sources of mitochondrial reactive oxygen species (mtROS) in ischemia-reperfusion injury. RET is dependent on mitochondrial membrane potential (ÎÏm) and transmembrane pH difference (ÎpH), components of the proton motive force (pmf); a decrease in ÎÏm and/or ÎpH inhibits RET. In this study we aimed to determine which component of the pmf displays the more dominant effect on RET-provoked ROS generation in isolated guinea pig brain and heart mitochondria respiring on succinate or α-glycerophosphate (α-GP). ÎÏm was detected via safranin fluorescence and a TPP+ electrode, the rate of H2O2 formation was measured by Amplex UltraRed, the intramitochondrial pH (pHin) was assessed via BCECF fluorescence. Ionophores were used to dissect the effects of the two components of pmf. The K+/H+ exchanger, nigericin lowered pHin and ÎpH, followed by a compensatory increase in ÎÏm that led to an augmented H2O2 production. Valinomycin, a K+ ionophore, at low [K+] increased ÎpH and pHin, decreased ÎÏm, which resulted in a decline in H2O2 formation. It was concluded that ÎÏm is dominant over âpH in modulating the succinate- and α-GP-evoked RET. The elevation of extramitochondrial pH was accompanied by an enhanced H2O2 release and a decreased âpH. This phenomenon reveals that from the pH component not âpH, but rather absolute value of pH has higher impact on the rate of mtROS formation. Minor decrease of ÎÏm might be applied as a therapeutic strategy to attenuate RET-driven ROS generation in ischemia-reperfusion injury