Abstract 38: Demonstration of Mitochondrial Membrane Depolarization with FRET Spectroscopy in the Intact Heart
Current methods for real time detection of mitochondrial membrane depolarization in the intact heart are very limited.
Objective: To utilize fluorescence resonance energy transfer (FRET) as a sensitive new method to distinguish mitochondrial membrane depolarization within the intact heart.
Methods: Isolated perfused male Sprague Dawley rat hearts were perfused at 85 mm Hg with temperature controlled (37.4° C) modified Krebs Henseleit buffer. A fiberoptic spectrometer measured fluorescence at the left ventricular wall. Hearts were loaded with mitotracker green (MTG), which localizes to the mitochondria, and then with tetramethyl rhodamine methyl ester (TMRM), a mitochondrial membrane specific probe that changes fluorescent intensity in response to mitochondrial membrane potential (ΔΨ). Excitation of MTG resulted in both the expected MTG emission and also a strong emission from TMRM, indicating that FRET occurred when the two probes were co-localized in the mitochondria. Hearts were infused with increasing concentrations of carbonyl cyanide p-(tri-fluromethoxy) phenyl-hydrazone (FCCP), an uncoupler of oxidative phosphorylation, at 30 nM, 100 nM and 300 nM. NADH, TMRM, MTG and FRET signals were simultaneously monitored to determine if changes in the mitochondrial membrane potential could be detected in the presence of uncoupling of the electron transport chain. Fluorophore signal changes at specific time points were expressed as Δ F/F0.
Results: After loading of both probes, excitation of MTG yielded emission in the TMRM emission wavelengths which constituted FRET. No changes in NADH were noted with 30 nM and 100 nM FCCP. However, at 300 nM there was a significant increase in NADH (p<0.001). A simultaneous increase in MTG and a significant decrease in FRET were seen with both 100 nM and 300 nM FCCP infusion (p<0.001 vs. baseline). Changes in FRET showed a dose response effect with increasing concentrations of FCCP (F/F0 = 0.989 ± 0.003 at 30 nM FCCP, F/F0 = 0.973 ± 0.013 at 100 nM FCCP and F/F0 = 0.926 ± 0.020 at 300 nM FCCP).
Conclusions: Utilizing the fluorescent probe pair of TMRM and Mitotracker green, FRET can be used as a sensitive measure of mitochondrial membrane depolarization in the intact heart.