Glutamate loading of cardiomyocytes has been shown to decrease production of reactive oxygen species (ROS) and improve recovery during simulated ischaemia (Williams et al. 2001; King et al. 2003). However in these studies glutamate loading was achieved during isolation in conditions involving reduced calcium and enzyme perfusion. The aim of this study was twofold, firstly to investigate glutamate incubation as a new method of loading isolated cardiomyocytes, and secondly to characterize the response of these cells to a novel model of oxidative stress involving exogenous H2O2.
Male Wistar rats were humanely killed by cervical dislocation and the hearts dissected. Ventricular myocytes were isolated as described previously (Williams et al. 2001) and incubated with/without 5.12 mM potassium glutamate for 1, 2, 3 and 4 h at 20 °C. The cells were placed in suspension on the stage of a fluorescence microscope at 37 °C and stimulated at 0.2 Hz. Cells were superfused with a Tyrode solution containing 100 µM H2O2 with/without 5.12 mM potassium glutamate. Time taken for a field of myocytes to undergo arrhythmia was observed visually and confirmed using an edge-tracking device. Identical superfusion conditions were used to measure ROS. For this myocytes were loaded with 10 µM 5-(and-6)-chloromethyl-2â,7â-dichlorodihydrofluorescein diacetate (King et al. 2003) prior to glutamate incubation. The intracellular glutamate concentration of myocytes was measured using HPLC. Results are expressed as means ± S.E.M.
Incubating cardiomyocytes with glutamate for 4 h significantly increased their intracellular glutamate concentration compared with controls (920.9 ± 182.0 vs. 238.5 ± 37.7 nmol (mg protein)-1, n = 5, P < 0.05), whilst 1, 2 or 3 h incubations showed no significant increase (ANOVA). A 4 h glutamate incubation also significantly increased the time to arrhythmia in response to H2O2 (25.7 ± 3.4 vs. 14.6 ± 1.1 min, P < 0.05, ANOVA, n = 9-12). Fluorescence traces for a representative isolation with linear regression lines superimposed indicated a decreased rate of ROS production as the duration of incubation increased.
This work suggests that it is possible to load cardiomyocytes with glutamate by incubating them in glutamate-containing solutions for relatively long periods and that this appears to protect against oxidative stress.
This work was supported by the BHF.