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Objective:To study the mitochondrial redox state in experimental animals to sensitively detect early signs of mitochondrial function in pathophysiological conditions, such as ischemia. Methods:Fluorescence of nicotinamide adenine dinucleotide (phosphate), or NAD(P)H, the principal electron donor in mitochondrial respiration responsible for vital ATP supply of cardiomyocytes, is studied for non-invasive fluorescent probing of the mitochondrial function. Examination of NAD(P)H fluorescence in living cardiomyocytes following excitation by UV-pulsed laser diode and detection by spectrally-resolved time-correlated single photon counting (TCSPC),is based on the simultaneous measurement of the fluorescence spectra and lifetime. Results:The dynamic characteristics of NAD(P)H fluorescence decay in living rat cardiomyocytes show that at least a 3-exponential decay model, with 0.4-0.7 ns, 1.2-1.9 ns and 8.0-13.0 ns lifetimes, is necessary to describe cardiomyocyte autofluorescence (AF). Decay-associated spectra (DSA) revealed the presence of 4 spectrally-distinct populations of NADH molecules in cardiomyocytes with spectral maximum at 470 nm for short-lifetime pool for the first time,and emission peaks at 450 nm, 470 nm and 490 nm for intermediate and long-lifetime pools. Increased mitochondrial NADH content ratio by ketone bodies enhanced the AF intensity, without the significant change in fluorescent lifetimes. Rotenone, the inhibitor of Complex I of the mitochondrial respiratory chain, increased AF and shortened the average fluorescence lifetime. Dinitrophenol (DNP),an uncoupling agent of the mitochondrial oxidative phosphorylation, lowered AF,broadened the spectral shoulder at 520 nm and increased the average lifetime. These effects,comparable to the changes in the concentration and in the rate of dehydrogenation of NADH in vitro,were also examined under ischemia-mimetic conditions.Conclusion:Our findings anticipate a contribution of both conformational NADH changes and energy transfer from NADH to lipoamide dehydrogenase (LipDH)-bound flavins,to explain observed fluorescence kinetics.Presented spectrally resolved fluorescence lifetime approach provides promising new tool for analysis of mitochondrial NAD(P)H in living cardiomyocytes,and hence for investigation of energy metabolism and mitochondrial dysfunction at a cellular level.