P-258 - Monitoring intracellular hydrogen peroxide changes in skeletal muscle fibres by HyPer, a genetically encoded hydrogen peroxide biosensor

Abstract

Numerous pathophysiological processes are driven by cellular signalling pathways where hydrogen peroxide (H2O2) seems to play an important role as a molecular signal. We have used a genetically encoded hydrogen peroxide biosensor, HyPer, in order to monitor and quantify intracellular H2O2 changes. We combined different approaches, based in direct microinjection of HyPer DNA construct in the flexor digitorum brevis muscle following by application of electroporation in this muscle, and achieved the expression of biosensor HyPer in isolated single skeletal muscle fibres. Using live cell fluorescence microscopy image analysis, we monitored intracellular flow of H2O2 in fibres that expressed HyPer. This is due to HyPer fluorescence properties, mainly the increase of HyPer fluorescence when this reacts with H2O2. Different experimental conditions were examined: i) H2O2 extracellular exposure, ii) exposure to an enzymatic system that generates H2O2, glucose oxidase (GOX), and iii) exposure to a reducing agent, dithiothreitol. Changes of extracellular H2O2 concentration were reflected in changes of fluorescence emitted by HyPer intracellularly. This reveal that there is a H2O2 flux across cellular membrane and HyPer biosensor is a suitable tool to detect and register this H2O2 flux.