The effects of idebenone on mitochondrial bioenergetics

Abstract

We have studied the effects of idebenone on mitochondrial function in cybrids derived from one normal donor (HQB17) and one patient harboring the G3460A/MT-ND1 mutation of Leber's Hereditary Optic Neuropathy (RJ206); and in XTC.UC1 cells bearing a premature stop codon at amino acid 101 of MT-ND1 that hampers complex I assembly. Addition of idebenone to HQB17 cells caused mitochondrial depolarization and NADH depletion, which were inhibited by cyclosporin (Cs) A and decylubiquinone, suggesting an involvement of the permeability transition pore (PTP). On the other hand, addition of dithiothreitol together with idebenone did not cause PTP opening and allowed maintenance of the mitochondrial membrane potential even in the presence of rotenone. Addition of dithiothreitol plus idebenone, or of idebenol, to HQB17, RJ206 and XTC.UC1 cells sustained membrane potential in intact cells and ATP synthesis in permeabilized cells even in the presence of rotenone and malonate, and restored a good level of coupled respiration in complex I-deficient XTC.UC1 cells. These findings demonstrate that idebenol can feed electrons at complex III. If the quinone is maintained in the reduced state, a task that in some cell types appears to be performed by dicoumarol-sensitive NAD(P)H:quinone oxidoreductase 1 [Haefeli et al. (2011) PLoS One 6, e17963], electron transfer to complex III may allow reoxidation of NADH in complex I deficiencies.

Fig. 1

Effects of idebenone on mitochondrial membrane potential, Ca^2 + retention capacity and volume. A, the incubation medium contained 130 mM KCl, 10 mM MOPS-Tris, 1 mM Pi-Tris, 10 μM EGTA, 0.15 μM Rhodamine 123, pH 7.4 and 0.8 μM CsA (trace b only); where indicated, 5 mM succinate (Succ), 35 μM Ca^2 +, 50 μM idebenone (IdB) and 0.5 μM FCCP were added. B, the mitochondrial CRC was determined following the addition of 10 μM Ca^2 + pulses, and values were normalized to the CRC obtained in the absence of idebenone (CRC₀). Mitochondria were treated with the indicated concentrations of idebenone in the absence (closed symbols) or presence (open symbols) of 1 mM DTT. C, the incubation medium contained 0.25 M sucrose, 1 mM Pi-Tris, 10 mM MOPS-Tris, 20 μM EGTA-Tris, 5 mM glutamate-Tris, 2.5 mM malate-Tris. Where indicated 50 μM Ca^2 +, 20 μM N-ethylmaleimide (NEM) and 50 μM idebenone (IdB) were added.

Fig. 2

Effects of idebenone, CsA and decylubiquinone on mitochondrial TMRM accumulation and NAD(P)H levels. A, TMRM fluorescence and B, NAD(P)H fluorescence of HQB17 cells in the absence (open symbols, traces a) or presence of 1.6 μM CsA (closed squares, traces b) or 50 μM decylubiquinone (closed triangles, traces c). Where indicated 50 μM idebenone (IdB), 4 μM FCCP, 80 μM alamethicin (Ala) and 4 μM rotenone (R) were added. Data in both panels report one representative experiment of five (for idebenone and idebenone plus CsA) or three (idebenone plus decylubiquinone). The maximal S.E., which is omitted for clarity, was ± 10%.

Fig. 3

Effects of idebenone on respiration of HQB17 cells. HQB17 cells were incubated in Seahorse 24-well plates (20,000 cells/well) and respiration measured in the absence (open circles) or presence of 50 μM idebenone alone (gray circles) or with 1.6 μM CsA (closed symbols). Where indicated 1 μg/mL oligomycin (O), 0.2 μM FCCP (F), 1 μM rotenone (R), and 1 μM antimycin A (AA) were added. Data report one representative experiment of three, and the maximal S.E. was ± 11.3 pmol oxygen/min. OCR, oxygen consumption rate.

Fig. 4

Effects of DTT and idebenone on mitochondrial TMRM fluorescence in HQB17 cells. HQB17 cells were loaded with 10 nM TMRM. A, additions were 2 mM DTT, 5 μM oligomycin (O), 4 μM rotenone (R), 1 μM antimycin A (AA) and 4 μM FCCP (F); B, additions were 2 mM DTT plus 50 μM idebenone (DTT + IdB), 5 μM oligomycin (O), 4 μM rotenone (R), 1 μM antimycin A (AA) and 4 μM FCCP. Data are from three or six independent experiments for panels A and B, respectively.

Fig. 5

Effects of idebenol on mitochondrial TMRM fluorescence in HQB17, RJ206 and XTC.UC1 cells. HQB17 (A), RJ206 (B) and XTC.UC1 cells (C) were loaded with 10 nM TMRM in the absence (open symbols) or presence (closed symbols) of 1.6 μM CsA, and changes in fluorescence were monitored by fluorescence microscopy. Where indicated 50 μM idebenol (IdBH₂), 5 μM oligomycin plus 4 μM rotenone (O + R), 1 μM antimycin A (AA) and 4 μM FCCP were added. Data are from three, eleven and ten independent experiments for panels A, B, and C, respectively.

Fig. 6

Idebenol and DTT-reduced idebenone promote basal respiration in XTC.UC1, but not in HQB17 and RJ206 cells. Cellular OCR of HQB17 (A, A′), RJ206 (B,B′) and XTC.UC1 cells (C,C′) was measured in 24-well Seahorse plates (20,000 cells/well). Cells were incubated in the absence of added quinone (open squares; the dashed trace in panel A is taken from Fig. 3). A–C, cells were supplemented with 50 μM idebenol (closed squares); A′-C′, cells were supplemented with 1 mM DTT (open circles) or 1 mM DTT plus 50 μM idebenone (closed circles). Note the different scale in panels A and A′. Data are representative of at least seven independent experiments, and the maximal variation (in pmol oxygen/min) was 12.5 (panel A), 3.1 (panel B), 7.5 (panel C), 9.9 (panel A′), 4.5 (panel B′), 4.3 (panel C′).

Fig. 7

ATP synthesis in permeabilized HQB17, RJ206 and XTC.UC1 cells. HQB17 (A), RJ206 (B) and XTC.UC1 (C) cells were permeabilized with digitonin and rate of ATP synthesis was evaluated in the presence of 5 mM pyruvate and 5 mM malate (P/M), 50 μM idebenol (IdBH₂), 50 μM idebenone (IdB), 5 μM rotenone and 5 mM malonate (Rot/Malon) as indicated. Asterisks denote values significantly different from P/M alone (P < 0.05).