MITOCHONDRIAL-MEMBRANE POTENTIAL IN SINGLE LIVING ADULT-RAT CARDIAC MYOCYTES EXPOSED TO ANOXIA OR METABOLIC INHIBITION

The relation between mitochondrial membrane potential (Delta Psi(m)) a nd cell function was investigated in single adult rat cardiac myocytes during anoxia and reoxygenation. Delta Psi(m) was studied by loading myocytes with JC-1 ro-1,1',3,3'-tetra-ethylbenzimidazolylcarbocyanine iodide), a fluorescent probe characterized by two emission peaks (539 and 597 nm with excitation at 490 nm) corresponding to monomer and agg regate forms of the dye. 2. De-energizing conditions applied to mitoch ondria, cell suspensions or single cells decreased the aggregate emiss ion and increased the monomer emission. This latter result cannot be e xplained by changes of JC-1. concentration in the aqueous mitochondria l matrix phase indicating that hydrophobic interaction of the probe wi th membranes has to be taken into account to explain JC-1. fluorescenc e properties in isolated mitochondria or intact cells. 3. A different sensitivity of the two JC-1 forms to Delta Psi(m) changes was shown in isolated mitochondria by the effects of ADP and FCCP and the calibrat ion with K+ diffusion potentials. The monomer emission was responsive to values of Delta Psi(m) below 140 mV, which hardly modified the aggr egate emission. Thus JC-1 represents a unique double sensor which can provide semi-quantitative information in both low and high potential r anges. 4. At the onset of glucose-free anoxia the epifluorescence of i ndividual myocytes studied in the single excitation (490 nm)-double em ission (530 and 590 nm) mode showed a gradual decline of the aggregate emission, which reached a plateau while electrically stimulated (0.2 Hz) contraction was still retained. The subsequent failure of contract ion was followed by the rise of the emission at 530 nm, corresponding to the monomer form of the dye, concomitantly with the development of rigor contracture. 5. The onset of the rigor was preceded by the incre ase in intracellular Mg2+ concentration ([Mg2+](1)) monitored by mag-i ndo-1 epifluorescence. Since under these experimental conditions intra cellular [Ca2+] and pH are fairly stable, the increase in [Mg2+](i) wa s likely to be produced by a decrease in ATP content. 6. The inhibitio n of mitochondrial ATPase induced by oligomycin during anoxia was asso ciated with a rapid and simultaneous change of both the components of JC-l fluorescence, suggesting that Delta Psi(m) instead of producing A TP, is generated by glycolytic ATP during anoxia. 7. The readmission o f oxygen induced a rapid decrease of the monomer emission and a slower increase of the aggregate emission. These fluorescence changes were n ot necessarily associated with the recovery of mechanical function. Ev en in those cells which hypercontracted during reoxygenation a further addition of FCCP induced a decrease in Delta Psi(m) and an increase i n [Mg2+](1), indicating that residual mitochondrial function and ATP p roduction can exist even in irreversibly damaged cells.