Monitoring mitochondrial electron fluxes using NAD(P)H-flavoprotein fluorometry reveals complex action of isoflurane on cardiomyocytes.
Mitochondrial bioenergetic studies mostly rely on isolated mitochondria thus excluding the regulatory role of other cellular compartments important for the overall mitochondrial function. In intact cardiomyocytes, we followed the dynamics of electron fluxes along specific sites of the electron transport chain (ETC) by simultaneous detection of NAD(P)H and flavoprotein (FP) fluorescence intensities using a laser-scanning confocal microscope. This method was used to delineate the effects of isoflurane, a volatile anesthetic and cardioprotective agent, on the ETC. Comparison to the effects of well-characterized ETC inhibitors and uncoupling agent revealed two distinct effects of isoflurane: uncoupling-induced mitochondrial depolarization and inhibition of ETC at the level of complex I. In correlation, oxygen consumption measurements in cardiomyocytes confirmed a dose-dependent, dual effect of isoflurane, and in isolated mitochondria an obstruction of the ETC primarily at the level of complex I. These effects are likely responsible for the reported mild stimulation of mitochondrial reactive oxygen species (ROS) production required for the cardioprotective effects of isoflurane. In conclusion, isoflurane exhibits complex effects on the ETC in intact cardiomyocytes, altering its electron fluxes, and thereby enhancing ROS production. The NAD(P)H-FP fluorometry is a useful method for exploring the effect of drugs on mitochondria and identifying their specific sites of action within the ETC of intact cardiomyocytes.
Sedlic
Filip
F
Department of Anesthesiology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA.
Pravdic
Danijel
D
Hirata
Naoyuki
N
Mio
Yasushi
Y
Sepac
Ana
A
Camara
Amadou K
AK
Wakatsuki
Tetsuro
T
Bosnjak
Zeljko J
ZJ
Bienengraeber
Martin
M
D018685:Anesthetics, Inhalation / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004579:Electron Transport / Q000187:drug effects; D042967:Electron Transport Complex I / Q000378:metabolism; D005420:Flavoproteins / Q000378:metabolism*; D005470:Fluorometry / Q000379:methods; D007530:Isoflurane / Q000494:pharmacology*; D008297:Male; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D018613:Microscopy, Confocal; D008929:Mitochondria, Heart / Q000378:metabolism*; D008954:Models, Biological; D032383:Myocytes, Cardiac / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D009249:NADP / Q000378:metabolism*; D010084:Oxidation-Reduction / Q000187:drug effects; D010101:Oxygen Consumption / Q000187:drug effects; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000378:metabolism
D018685:Anesthetics, Inhalation; D005420:Flavoproteins; D017382:Reactive Oxygen Species; D009249:NADP; D007530:Isoflurane; D042967:Electron Transport Complex I