Nicotinamide adenine dinucleotide fluorescence spectroscopy and imaging of isolated cardiac myocytes.
Nicotinamide adenine dinucleotide (NADH) plays a critical role in oxidative phosphorylation as the primary source of reducing equivalents to the respiratory chain. Using a modified fluorescence microscope, we have obtained spectra and images of the blue autofluorescence from single rat cardiac myocytes. The optical setup permitted rapid acquisition of fluorescence emission spectra (390-595 nm) or intensified digital video images of individual myocytes. The spectra showed a broad fluorescence centered at 447 +/- 0.2 nm, consistent with mitochondrial NADH. Addition of cyanide resulted in a 100 +/- 10% increase in fluorescence, while the uncoupler FCCP resulted in a 82 +/- 4% decrease. These two transitions were consistent with mitochondrial NADH and implied that the myocytes were 44 +/- 6% reduced under the resting control conditions. Intracellular fluorescent structures were observed that correlated with the distribution of a mitochondrial selective fluorescent probe (DASPMI), the mitochondrial distribution seen in published electron micrographs, and a metabolic digital subtraction image of the cyanide fluorescence transition. These data are consistent with the notion that the blue autofluorescence of rat cardiac myocytes originates from mitochondrial NADH.
Eng
J
J
Laboratory of Cardiac Energetics, National Heart, Lung and Blood Institute, Bethesda, Maryland 20892.
Lynch
R M
RM
Balaban
R S
RS
D000818:Animals; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D002470:Cell Survival; D002478:Cells, Cultured; D003486:Cyanides / Q000494:pharmacology; D006321:Heart / Q000187:drug effects; D006352:Heart Ventricles / Q000378:metabolism; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D009206:Myocardium / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure; D009243:NAD / Q000378:metabolism*; D010101:Oxygen Consumption; D051381:Rats; D011919:Rats, Inbred Strains
D003486:Cyanides; D009243:NAD; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone