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, 285 (34), 26494-505

Mitochondrial Complex II Prevents Hypoxic but Not Calcium- And Proapoptotic Bcl-2 Protein-Induced Mitochondrial Membrane Potential Loss

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Mitochondrial Complex II Prevents Hypoxic but Not Calcium- And Proapoptotic Bcl-2 Protein-Induced Mitochondrial Membrane Potential Loss

Brian J Hawkins et al. J Biol Chem.

Abstract

Mitochondrial membrane potential loss has severe bioenergetic consequences and contributes to many human diseases including myocardial infarction, stroke, cancer, and neurodegeneration. However, despite its prominence and importance in cellular energy production, the basic mechanism whereby the mitochondrial membrane potential is established remains unclear. Our studies elucidate that complex II-driven electron flow is the primary means by which the mitochondrial membrane is polarized under hypoxic conditions and that lack of the complex II substrate succinate resulted in reversible membrane potential loss that could be restored rapidly by succinate supplementation. Inhibition of mitochondrial complex I and F(0)F(1)-ATP synthase induced mitochondrial depolarization that was independent of the mitochondrial permeability transition pore, Bcl-2 (B-cell lymphoma 2) family proteins, or high amplitude swelling and could not be reversed by succinate. Importantly, succinate metabolism under hypoxic conditions restores membrane potential and ATP levels. Furthermore, a reliance on complex II-mediated electron flow allows cells from mitochondrial disease patients devoid of a functional complex I to maintain a mitochondrial membrane potential that conveys both a mitochondrial structure and the ability to sequester agonist-induced calcium similar to that of normal cells. This finding is important as it sets the stage for complex II functional preservation as an attractive therapy to maintain mitochondrial function during hypoxia.

Figures

FIGURE 1.
FIGURE 1.
Complex II cannot restore mitochondrial function following irreversible ΔΨm loss. A, complex I/F0F1-ATPase inhibition (rotenone/oligomycin) triggered ΔΨm that was reversed by succinate (Succ; 10 mm) but not the PTP blocker CsA (5 μm). B, in contrast, Ca2+-induced mitochondrial depolarization (200 μm) is blocked by CsA but not succinate. C, succinate-dependent ΔΨm maintenance is abolished by mitochondrial swelling triggered by the microbial antibiotic alamethacin (20 μg/ml). Note: B and C were performed in the presence of 2 mm succinate. D, succinate did not restore ΔΨm in response to Bcl-2 family protein tBid-induced (10 μg/ml) depolarization. succinate restored ΔΨm in both (E) wild type and (F) bax−/−bak−/− double-deficient murine embryonic fibroblasts ollowing ΔΨm loss. G, mitochondrial (Mito) cytochrome c release from the mitochondria in response to Ca2+, tBid, and the inhibitor combination of rotenone/oligomycin; Rot, rotenone; WT, wild-type.
FIGURE 2.
FIGURE 2.
Mitochondrial complex II establishes the membrane potential in permeabilized cells. Succinate (Succ; 2 mm) but not malate/glutamate (Mal/Glut; 1 mm/1 mm) maintained ΔΨm in digitonin-permeabilized RPMVECs under control cells (A) and in the presence of the F0F1-ATPase inhibitor oligomycin (Oligo; 20 μg/ml) (B). ATP (100 μm) was effective only in the absence of oligomycin. Complex I inhibition by rotenone (Rot; 20 μm) (C) does not affect succinate-mediated ΔΨm maintenance but the complex II inhibitor malonate (Mao; 2 μm) (D) abolished succinate-dependent ΔΨm. Succinate afforded no protection from complex III inhibition by myxothiazol (5 μm) (E) or antimycin A (20 μm) (F). Arrows indicate the addition of different molecules at various intervals. The mitochondrial uncoupler CCCP was added at the indicated time to dissipate the membrane potential.
FIGURE 3.
FIGURE 3.
Complex II substrate restores ΔΨm during complex I and F0F1-ATPase inhibition. A, differential effects of complex II (succinate (Succ); 10 mm) and complex I (malate/glutamate (Mal/Glut); 5 mm/5 mm, malate/pyruvate (Mal/Pyr); 5 mm/5 mm) substrates on reestablishment of ΔΨm in murine myoblast (C2C12) cell line. B, ΔΨm depolarization caused by complex I and F0F1-ATPase inhibition (rotenone/oligomycin (Rot/Oligo); 20 μm/20 μg/ml) could be reversed by succinate (10 mm) in MPMVECs. C, the citric acid entry substrate acetyl CoA (AcetylCoA; 5 mm), and the succinate precursor α-ketoglutarate did not restore ΔΨm in human PMVECs. D, malonate (Mao)-induced ΔΨm depolarization was not reversed by combination of the complex I substrates malate/pyruvate (5 mm/5 mm) and the cofactor NAD+ (100 μm). E, an increase in NADH production in response to malate/glutamate (5 mm/5 mm) did not restore ΔΨm in response to complex II inhibition by malonate (2 mm). F, the complex IV substrates TMPD/ascorbate do not effectively reestablish ΔΨm following Ros/oligomycin-induced ΔΨm loss. Oligomycin was added at 80 s in B, C, D, and F. Arrows indicate the addition of different molecules at various intervals.
FIGURE 4.
FIGURE 4.
Establishment of ΔΨm in human fibroblasts from patients with mitochondrial disorders. A, control human fibroblasts (CF9) exhibit ΔΨm loss in response to rotenone/oligomycin that is reversed by succinate (Succ; 10 mm). Similar responses were observed in human clinical samples from LHON (B) and KSS (C) patients. CF9 control (D) and LHON (E) and KSS (F) patient samples display a similar mitochondrial morphology as determined by the cationic dye TMRE. Mitochondrial depolarization, as detected by the presence of TMRE in the nucleus, occurs in response to malonate (Mao; 5 mm) but not rotenone (20 μm) in CF9 (G), LHON (H), and KSS (I) cells. CCCP was used as a positive control. J, quantitation of nuclear TMRE fluorescence increase in response to rotenone, Mao, and CCCP in CF9, LHON, and KSS cells. Values indicate mean ± S.E. (n = 3). K, mitochondrial Ca2+ uptake is similar in all cell types in response to the protease-activated receptor agonist peptide TRAP (25 μm). L, whole cell mitochondrial oxygen consumption of CF9, LHON, and KSS cells (8 × 106). Stopper indicates when the oxygen consumption chamber was sealed. Sodium azide (NaN3) was used to inhibit cellular oxygen consumption at complex IV as a positive control. Values indicate oxygen consumption as nmol oxygen/min/8 × 106 cells (mean ± S.E.; n = 3). f.a.u., fluorescence arbitrary units.
FIGURE 5.
FIGURE 5.
Differential effects of complex-specific metabolism on ΔΨm and oxygen consumption. A, successive assessment of the relative changes in ΔΨm following addition of the complex I substrates malate/pyruvate (Mal/Pyr; inhibited by rotenone), the complex II substrate succinate (inhibited by Myx), and the complex IV substrates TMPD/ascorbate (Asc). Values represent the mean value (± S.E.) of 10 data points immediately prior to addition of the respective inhibitor (n = 4). B, oxygen measured via a Clark-type electrode following successive additions as indicated in permeabilized cells. Rate of oxygen consumption for complex II was less than both complexes I and IV. Rate of oxygen consumption was measured and is displayed as nmol oxygen/min/6 × 106 cells (mean ± S.E.; n = 12). The trace is a single experiment that is representative of mean oxygen consumption. Rot, rotenone.
FIGURE 6.
FIGURE 6.
Preservation of mitochondrial function and energy metabolism by succinate-mediated metabolism during hypoxia. Both chemical- (A) and hypoxic (B)-induced ΔΨm loss is reversed by succinate (Succ; 10 mm) but not malate/pyruvate (5 mm/5 mm) in freshly isolated permeabilized, mature murine cardiomyocytes and C2C12 cells, respectively. C, hypoxic status was confirmed by HIF-1α stabilization in C2C12 cells following 5 h of hypoxia (NS; nonspecific). D, primary adult cardiomyocyte mitochondrial complex I and complex II activity were unaltered during hypoxia. E, intact C2C12 or freshly isolated (F) adult cardiomyocytes were subjected to hypoxic conditions for 5 h, and cells were loaded with the ΔΨm indicator TMRE. Normoxic and succinate-supplemented hypoxic cells retained ΔΨm. In contrast, hypoxia alone or supplemented with malate/pyruvate (Mal/Pyr) caused mitochondrial depolarization. F and G, average fluorescence intensity of mitochondrial TMRE retention as described in F and G following normoxic and hypoxic conditions. H, ATP level maintenance by succinate supplementation in response to hypoxia. All values in D, G, H, and I indicate mean ± S.E. (n = 3). Rot, rotenone. f.a.u., fluorescence arbitrary units.

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