Sequential opening of mitochondrial ion channels as a function of glutathione redox thiol status

Abstract

Mitochondrial membrane potential (DeltaPsi(m)) depolarization contributes to cell death and electrical and contractile dysfunction in the post-ischemic heart. An imbalance between mitochondrial reactive oxygen species production and scavenging was previously implicated in the activation of an inner membrane anion channel (IMAC), distinct from the permeability transition pore (PTP), as the first response to metabolic stress in cardiomyocytes. The glutathione redox couple, GSH/GSSG, oscillated in parallel with DeltaPsi(m) and the NADH/NAD(+) redox state. Here we show that depletion of reduced glutathione is an alternative trigger of synchronized mitochondrial oscillation in cardiomyocytes and that intermediate GSH/GSSG ratios cause reversible DeltaPsi(m) depolarization, although irreversible PTP activation is induced by extensive thiol oxidation. Mitochondrial dysfunction in response to diamide occurred in stages, progressing from oscillations in DeltaPsi(m) to sustained depolarization, in association with depletion of GSH. Mitochondrial oscillations were abrogated by 4'-chlorodiazepam, an IMAC inhibitor, whereas cyclosporin A was ineffective. In saponin-permeabilized cardiomyocytes, the thiol redox status was systematically clamped at GSH/GSSG ratios ranging from 300:1 to 20:1. At ratios of 150:1-100:1, DeltaPsi(m) depolarized reversibly, and a matrix-localized fluorescent marker was retained; however, decreasing the GSH/GSSG to 50:1 irreversibly depolarized DeltaPsi(m) and induced maximal rates of reactive oxygen species production, NAD(P)H oxidation, and loss of matrix constituents. Mitochondrial GSH sensitivity was altered by inhibiting either GSH uptake, the NADPH-dependent glutathione reductase, or the NADH/NADPH transhydrogenase, indicating that matrix GSH regeneration or replenishment was crucial. The results indicate that GSH/GSSG redox status governs the sequential opening of mitochondrial ion channels (IMAC before PTP) triggered by thiol oxidation in cardiomyocytes.

FIGURE 1. Diamide-triggered mitochondrial oscillations in intact cardiomyocytes

Freshly isolated guinea pig cardiomyocytes were loaded with TMRM (ΔΨ_m probe) and CM-H₂DCFDA (ROS probe) at 37 °C and imaged by two-photon laser scanning fluorescence microscopy in a perfusion chamber. A, after exposure to the thiol-oxidizing agent diamide (0.1 mm), the oxidation of the ROS probe steadily increased until mitochondrial oscillations were spontaneously triggered. Cycles of ΔΨ_m depolarization-repolarization and NADH oxidation-reduction continued in-phase, usually for more than 10 min before the irreversible collapse of ΔΨ_m and maximal NADH oxidation occurred followed by rapid cell contracture. The 1st and 2nd arrows mark the onsets of ΔΨ_m oscillation and sustained depolarization associated with cell contracture, respectively. Images were collected every 3.5 s. B, mitochondrial benzodiazepine receptor antagonist 4Cl-DZP (64 μm) stopped the diamide-elicited oscillations and stabilized ΔΨ_m. ΔΨ_m oscillation and cell death resumed after washout in the continued presence of diamide. C, cardiomyocyte loaded with TMRM (100 nm) and MCB (50 μm) was treated with 0.1 mm diamide under similar conditions as in A and B. Mitochondrial oscillations were triggered after an ∼20% decrease in the GSB fluorescence. Images were acquired every 30 s. D, montage of images taken from the time course depicted in C during an oscillatory cycle and during the irreversible collapse of ΔΨ_m.

FIGURE 2. Inhibition of diamide-induced ΔΨ_m oscillations by 4Cl-DZP but not CsA

A, freshly isolated cardiomyocytes were loaded with 100 nm TMRM and exposed to 0.1 mm diamide until oscillations were induced. Treatment with 4-Cl-DZP (64 μm) stopped the oscillations and stabilized ΔΨ_m in the polarized state. After washout of 4Cl-DZP, ΔΨ_m oscillations returned before the mitochondria irreversibly depolarized and the cell went into contracture. B, at moderate levels of GSH depletion by diamide, ΔΨ_m oscillations were not reversed by the PTP inhibitor CsA (1 μm). In fact, CsA had a nonspecific depolarizing effect that was the opposite what would be expected for PTP inhibition. More severe GSH depletion activated PTP later in the experiment, as indicated by the arrow. Images were collected every 3.5 s.

FIGURE 3. ROS production, ΔΨ_m, and NADH in saponin-permeabilized cardiomyocytes

Myocytes stored in DMEM were resuspended in the experimental solution in the perfusion chamber and loaded with TMRM (100 nm) and CM-H₂DCFDA (2 μm) for at least 20 min. After loading, the excess dye was washed out, and the cells were briefly superfused with a permeabilizing solution (see “Experimental Procedures”) and then continuously perfused with an intracellular solution containing different GSH/GSSG ratios as indicated. ΔΨ_m, oxidation of the ROS probe, and NADH redox state were simultaneously monitored using two-photon fluorescence excitation. TMRM was included in the medium to avoid depletion of the probe during depolarization-repolarization cycles. A, representative raw trace of CM-DCF signal at different ratios, from which the rates of ROS production are calculated in the steady portion of the signal. The bar within the range 150 - 100:1 indicates where IMAC-dependent ΔΨ_m oscillations happen. B, rate of oxidation of the ROS probe (F/F₀/unit time), NADH (in % of initial fluorescence before permeabilization), and TMRM (ΔΨ_m, in % of initial fluorescence before permeabilization) obtained from seven different cells in independent experiments (mean ± S.E.) at different GSH/GSSG ratios (3 mm GSH concentration). The arrows in A and B denote the point at which ΔΨ_m irreversibly collapsed. C and D, representative montage of images of a permeabilized cardiomyocyte loaded with the ΔΨ_m and ROS sensors at different GSH/GSSG ratios: 300:1 (C, top left), 200:1 (C, top right), 150:1 (C, bottom left), 100:1 (C, bottom right), and 50:1 (D). Notice the ΔΨ_m oscillation (in the range 150:1 to 100:1) and the irreversible depolarization with exit of the ROS probe from mitochondria (50:1).

FIGURE 4. ROS production, ΔΨ_m, and NADH in permeabilized cardiomyocytes in the presence of IMAC or PTP inhibitors

Myocytes were handled, loaded with the ΔΨ_m and ROS sensors, and permeabilized as described in the legend of Fig. 3. Rate of oxidation of the ROS probe (F/F₀/unit time), NADH (in % of initial fluorescence before permeabilization) (A), and ΔΨ_m (in % of initial TMRM fluorescence before permeabilization) (B) obtained from four cells in the absence or the presence of 60 μm 4CL-DZP or 1 μm CsA (mean ± S.E.; 2 experiments) at different GSH/GSSG ratios (3 mm GSH concentration). The arrows in A and B denote the point at which ΔΨ_m irreversibly collapsed. Key to symbols: con, control; csa, cyclosporin A; 4dzp, 4Cl-DZP.

FIGURE 5. Relationship between glutathione redox potential, GSH/GSSG ratio, and GSH pool size

The glutathione redox potential, E_hc, was calculated according to Equation 1 in the text. The main panel shows how the total concentration of GSH influences the E_hc midpoint potential and how the pool size influences the % oxidation of the GSH pool. For example, if 10% of the GSH is oxidized to GSSG (horizontal dashed line), then for an initial GSH concentration of 10 mm, E_hc = -230 mV; for 4 mm, E_hc = -218 mV; for 3 mm, E_hc = -214 mV; and for 1 mm, E_hc = -200 mV. Thus, a cell with 10 mm GSH will have a higher reduction potential and a higher reducing capacity than one containing 1 mm GSH. The inset shows the reduction potential of NADP⁺/NADPH, whose negative redox potential (-400 mV (6)) makes it a key electron donor for the GSH system, for other redox systems (1), and for biosynthetic reactions. In general, NADPH is a cofactor in reductive (biosynthetic) reactions and serves as a source of electrons, whereas NAD⁺-dependent reactions are oxidative (catabolic) reactions where NAD⁺ serves as a sink for electrons. As opposed to the NADH/NAD⁺ ratio (= 100:1), the NADPH/NADP⁺ ratio is much lower (1:100) in cells and tissues (for review see Refs. 1, 6). The numbers at the top left of the inset correspond to GSH concentrations whose redox potential is represented in the plot (three continuous lines), and NADPH redox potential is shown in dashed line.

FIGURE 6. Effects of glutathione redox status and pool size on mitochondrial ROS production, NADH, and ΔΨ_m in permeabilized myocytes

The rates of oxidation of the matrix localized CM-H₂DCF (ROS probe) and the NADH autofluorescence at different GSH/GSSG ratios are compared for total GSH concentrations of 1 mm (A), 3 mm (B), and 4 mm (C). Data are plotted as a function of the glutathione reduction potential for the GSH/GSSG solutions, calculated as shown in Fig. 5. Note that the midpoint of the curve describing the acceleration of ROS production lies in a region where there is a relatively small change in E_hc. Moreover, the redox potential at which ΔΨ_m collapses differs significantly depending on the GSH pool size, indicating that the mechanism of mitochondrial dysfunction is not strictly dependent on the reduction potential of the cytoplasmic solution. The arrows in A-C denote the ratio at which ΔΨ_m irreversibly collapsed.

FIGURE 7. Effects of GSH or GSSG concentration on mitochondrial ROS production in permeabilized myocytes

GSH/GSSG ratio was varied between 300:1 and 20:1 with either a GSH fixed at 3 mm or with GSSG fixed at 10 μm. Increasing GSSG at a fixed GSH induced corresponding increases in the rate of oxidation of the ROS probe while clamping GSSG to 10 μm inhibited ROS production and shifted mitochondrial ΔΨ_m depolarization (arrows) to a lower ratio (20:1).

FIGURE 8. Mitochondrial redox status and ROS production in response to inhibition of enzymes responsible for mitochondrial GSH regeneration and GSH transport

NADH (as % of initial level) and ROS production in saponin-permeabilized cardiomyocytes were exposed to different GSH/GSSG ratios. A, concentration-dependent effects of the glutathione reductase inhibitor BCNU. B, concentration-dependent effects of the NADH/NADPH transhydrogenase inhibitor NBD chloride. C, rate of oxidation of the ROS probe (F/F₀/unit time) and NADH (in % of initial fluorescence before permeabilization) in the absence or the presence of 200 μm butyl malonate obtained from four cells (mean ± S.E.; two experiments). The rates of ROS production and NADH oxidation were similar to the control in the presence of 100 μm butyl malonate (not shown). The arrows denote the GSH/GSSG ratio at which ΔΨ_m collapsed irreversibly. ΔΨ_m collapses at increasingly higher (more reduced redox potentials) GSH/GSSG ratios in the presence of increasing concentrations of the inhibitors. D, timing of the depletion of reduced glutathione levels and ΔΨ_m induced by decreasing the GSH/GSSG ratio in saponin-permeabilized cardiomyocytes. Cells were loaded with 100 nm TMRM and 50 μm MCB and then permeabilized to remove the cytoplasmic component of GSB. Inset, note that the rapid oxidation of the mitochondrial GSB signal occurs ∼30 s before the irreversible collapse of ΔΨ_m. Key to symbols: con, control; bmal, butyl malonate.

FIGURE 9. Coupled glutathione, NADH, and NADPH redox cycles in the mitochondrial matrix

This scheme shows the carrier-mediated mitochondrial GSH uptake and the main enzymatic steps involved in the regeneration of GSH and NADPH in the mitochondrial matrix. DIC, dicarboxylate carrier (its inhibition by butyl malonate, Bmal, is indicated); OGC, 2-oxoglutarate carrier; GPX, glutathione peroxidase; GR, NADPH-dependent glutathione reductase (its inhibition by carmustine, BCNU, is indicated); THD, transhydrogenase (its inhibition by NBD chloride, NBD chloride, is indicated); ΔμH, proton electrochemical gradient; CAT, catalase; SOD, Mn-superoxide dismutase; PSSG, protein-SSG mixed disulfides; PSH, protein-SH. ROS is highlighted in light gray: ${\mathrm{O}}_2^{\stackrel{‒}{\cdot }}$, superoxide anion; H₂O₂, hydrogen peroxide.