Following ischaemia and reperfusion of isolated rat hearts, the level of tissue damage and functional recovery is aggravated by glutathione depletion (Blaustein et al. 1989) and improved by glutathione addition (Seiler et al. 1996). This suggests a strong correlation between glutathione level and outcome and also highlights the importance of having an accurate method of monitoring the glutathione content of heart cells during various insults and interventions. In this study we have assessed the ability of a new fluorescent dye, CellTracker Blue, for measuring the concentration of reduced glutathione (GSH) in single isolated and perfused cardiomyocytes.
Male Wistar rats were humanely killed by cervical dislocation and the hearts dissected. Ventricular cardiomyocytes were isolated as described previously (Williams et al. 2001). Loading of 2.4 µM CellTracker Blue was achieved by incubating the cells for 10 min with the dye in the dark at room temperature, followed by centrifugation and suspension of the pellet in fresh dye-free solution. Changes in CellTracker Blue fluorescence during oxidative stress were measured in Tyrode solution plus 1 mM H2O2 on a fluorescence microscope with excitation at 350 nm and emission at 460 nm. All cells were maintained at 37°C and stimulated at 0.2 Hz. A GSH calibration curve was obtained and the effect of different calcium, pH and glutathione-S-transferase concentration was measured during perfusion with dye-containing solutions.
CellTracker Blue was sensitive to GSH concentration and showed good cellular retention. Assuming a normalised fluorescence of 100 ± 5 % at pH 7.0, the fluorescence was not significantly altered by changes in pH, being 100.4 ± 5.5 % at pH 7.2 and 112.5 ± 10.9 % at pH 7.5 (n = 3, means ± S.E.M., ANOVA). There was also no significant difference in fluorescence when 0.2, 0.4 or 0.8 U ml-1 of glutathione-S-transferase or when 0, 100 nM or 10 mM calcium was used. Under normal conditions, assuming a cell volume of 1.9 µl (Boyett et al. 1991), the mean GSH concentration of single isolated and perfused rat cardiomyocytes was 1.7 ± 0.1 mM. During oxidative stress, the GSH concentration decreased significantly to 1.1 ± 0.2 mM (P < 0.02, Student’s paired t test, n = 7, mean ± S.E.M.).
This work suggests CellTracker Blue may make a good tool for monitoring the GSH concentration in isolated cells.
This work was supported by the British Heart Foundation.