H2O2 biosensors HyPer2, HyPer3 and GFP2-Orp1 detect rapid pH changes due to environmental CO2 fluctuations, in addition to intracellular H2O2, in isolated skeletal muscle fibres

Abstract

[EN] Hydrogen peroxide (H2O2) is one of the Reactive Oxygen Species (ROS) that seems to play an essential role in pathophysiological processes. H2O2 might act as a signaling molecule and modulate different crucial cellular signaling pathways, such as the glucose uptake in skeletal muscle, where H2O2 has been proposed to play an important role. HyPer2, HyPer3 and GFP2-Orp1 are hydrogen peroxide biosensors. We use these biosensors to monitor intracellular H2O2 in single skeletal muscle fibres isolated from the flexor digitorum brevis (FDB) mouse muscle. Previously, the coding sequences of these biosensors were microinjected and electroporated in FDB. Isolated fibres in culture that expressed one of the biosensors were settled incubation chamber coupled to the fluorescence microscope. The chamber maintains temperature (37ºC), environmental CO2 (5%) and humidity. Different time course experimental conditions were performed where fibres were exposed to different agents (insulin, interleukin 1β, H2O2, DTT) and intracellular H2O2 flux was registered in real time using fluorescence microscopy imaging analysis. We observed that when there were environmental CO2 (5%) fluctuations, due to initial medium stabilization or occasional interruption of CO2 supply, the biosensors showed changes in the fluorescence emission, which were registered. The main consequence of CO2 fluctuations is the change in the pH of medium. The main part of the biosensor structure is a fluorescent protein, YFP in de case of HyPer2 and HyPer3, and GFP2 in GFP2-Orp1. It has been reported that these fluorescent proteins are sensitive to pH and this might be a disadvantage for the biosensors. However, we believe that this pH sensitivity should be considered as an additional property of this biosensors, since they provide information in real time about the rapid changes of pH due to environmental fluctuation of CO2 and likely other gases such as O2 or N2.