Abstract 2619: A FRET Spectroscopy Approach to Localization of Mitochondrial ROS Formation during Ischemia and Reperfusion in the Intact Heart
Current methods for real time detection of reactive oxygen species (ROS) in the intact heart have provided limited opportunity to distinguish between compartmental sources. We have developed a fluorescence resonance energy transfer (FRET) method designed to distinguish mitochondrial ROS formation and used this method to test the hypothesis that ROS formed during both myocardial ischemia and reperfusion are primarily of mitochondrial origin.
Methods: Isolated adult rat hearts (n = 4) were studied using Langendorff perfusion. A fiberoptic spectrometer measured fluorescence on the left ventricular wall. Hearts were preloaded with Mitotracker Orange (MTO) for localization of mitochondria and then with Dihydrofluorescein, a non-specific ROS probe that oxidizes to fluorescein (Fluor). Excitation of Fluor resulted in both the expected Fluor emission and a strong emission from MTO, indicating the occurrence of FRET when the two probes were co-localized in the mitochondria. Hearts were subjected to 20 minutes of global ischemia and 20 minutes of reperfusion (85 mm Hg). Fluorophore signal changes at specific time points were expressed as ΔF/F0.
Results: During the ischemic phase there was a rapid rise in the Fluor signal, reaching a peak (ΔF/F0 = +0.69 ± 0.07 at 5.9 ± 0.9 min) which decayed slowly during ischemia to +0.42 ± 0.09 at 20 minutes ischemia. In contrast, the FRET signal rose slowly and continuously, attaining +0.49 ± 0.04 at 5.9 minutes of ischemia and rising to +0.65 ± 0.06 at 20 minutes ischemia. During reperfusion, there was a sharp secondary rise in the Fluor signal (0.68 ± 0.13) which peaked at 1.4 ± 0.2 minutes and was completely absent in the FRET signal. The FRET signal decayed to +0.32 ± 0.07 by 1.4 minutes into reperfusion and returned to baseline by 10 minutes.
Conclusions: The data are consistent with FRET occurring between these two fluorophores during ischemia, suggesting localized ROS production in the mitochondria. The lack of FRET signal during reperfusion suggests other possible origins of dihydrofluorescein oxidation in the reperfused heart.