A new method of quantifying glutathione levels in freshly isolated single superfused rat cardiomyocytes

Abstract

Introduction: Glutathione (GSH) is an important antioxidant in the heart whose content changes during cardiac insults. However, there are currently no methods for continuously monitoring free cytoplasmic GSH levels in single isolated and superfused cardiomyocytes exposed to normal and pathological conditions. Methods: GSH was measured using CellTracker™ Blue CMAC (Molecular Probes), a member of a new family of thiol-sensitive dyes. The fluorescence of 5 μM CellTracker™ Blue CMAC was measured in various solutions containing glutathione- S-transferase and in freshly isolated single superfused cardiomyocytes using an inverted fluorescence microscope. The cardiomyocytes were isolated by standard procedures and loaded with either CellTracker™ BlueCMAC or monochlorobimane by 15 min of shaking incubation in the dark at room temperature followed by centrifugation with resuspension of the cells in dye-free media. Cell volume was calculated from the ³H₂O and [¹⁴C]sucrose space. Results: CellTrackerk™ Blue CMAC fluorescence was linearly proportional to 0–100 μM GSH, as described by the equation: Y= 182.2 (X) + 681.6 (r²=.99, P < .001). Fluorescence was not affected by changing the glutathione-S-transferase level, the calcium concentration, or the pH, neither was the fluorescence quenched by H₂O₂ or cyanide. Exposure of freshly isolated single superfused cardiomyocytes to oxidative stress in the presence of 0–1mM H₂O₂ caused a progressive decrease in cellular GSH. In contrast, brief exposure to metabolic inhibition in the presence of 2.5mMNaCN evoked a significant increase in cardiomyocyte GSH followed by a return to control levels during washoff. In comparison to monochlorobimane, cells loaded with CellTracker™ Blue CMAC gave a stronger signal with better cellular retention of the probe. Discussion: These results suggest that CellTracker™ BlueCMAC fluorescence will be a good tool for measuring GSH in freshly isolated single superfused cardiomyocytes because it shows the expected changes to oxidative stress and metabolic inhibition, and is reversible.