Optical detection of mitochondrial NADH content in intact human myotubes.
Time-gated fluorescence spectroscopy in combination with non-radiative energy transfer was used on intact human skeletal myotubes for the determination of the mitochondrial NADH content which is considered to be a sensitive indicator of mitochondrial function. To mimic dysfunction of the mitochondrial energy metabolism, complexes I or III of the respiratory chain were inhibited by drugs. In the absence of the fluorescent mitochondrial marker rhodamine (R123), the NADH autofluorescence (i.e. a signal monitoring cytoplasmic plus mitochondrial NADH) remained unchanged upon inhibition of complex I by rotenone, and was increased by a factor of 2 upon inhibition of complex III by antimycin. In the presence of R123, the autofluorescence of NADH was reduced indicating non-radiative energy transfer from NADH to R123. The ratio of the R123 fluorescence signals obtained with the two excitation wavelengths of 355 nm and 488 nm was taken as a measure of mitochondrial NADH. Relative NADH changes were estimated in the presence of the above-mentioned inhibitors. Upon complex I inhibition, mitochondrial NADH was increased by a factor of 1.5. Upon inhibition of complex III, mitochondrial NADH was increased by a factor of 2. We conclude that time-gated spectroscopy combined with non-radiative energy transfer is an appropriate tool for probing mitochondrial enzyme complex deficiencies.
Gschwend
M H
MH
Abteilung für Allgemeine Physiologie, Universität Ulm, Germany. michael.gschwend@medizin.uni-ulm.de
Rüdel
R
R
Strauss
W S
WS
Sailer
R
R
Brinkmeier
H
H
Schneckenburger
H
H
D000968:Antimycin A / Q000031:analogs & derivatives* / Q000494:pharmacology; D046508:Culture Techniques; D004579:Electron Transport; D042967:Electron Transport Complex I; D014450:Electron Transport Complex III / Q000037:antagonists & inhibitors; D004735:Energy Transfer; D005456:Fluorescent Dyes; D006801:Humans; D008931:Mitochondria, Muscle / Q000187:drug effects / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000187:drug effects / Q000378:metabolism*; D009243:NAD / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000037:antagonists & inhibitors; D020112:Rhodamine 123; D012402:Rotenone / Q000494:pharmacology; D013050:Spectrometry, Fluorescence / Q000379:methods
D005456:Fluorescent Dyes; D012402:Rotenone; D009243:NAD; C032456:antimycin; D020112:Rhodamine 123; D000968:Antimycin A; D009247:NADH, NADPH Oxidoreductases; D042967:Electron Transport Complex I; D014450:Electron Transport Complex III