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.
Highlights
JULY 27, 2007 VOLUME 282 NUMBER 30 cule has created pressure for the cell to evolve powerful antioxidant defenses that convert reactive oxygen species (ROS)3 into harmless products to maintain a predominantly reduced redox environment
In addition to the redox couples involved in mitochondrial electron transport, the three main cellular redox pairs participating in intracellular reactions include reduced/oxidized glutathione (GSH/GSSG), thioredoxin (Trx(SH)2/TrxSS), and NADPH/ NADPϩ, with the latter providing the thermodynamic driving force behind the glutathione and thioredoxin systems [1]
We have previously shown that cell-wide oscillations in mitochondrial energetics in adult ventricular myocytes are preceded by a threshold level of oxidation of the ROS probe CM-H2DCF [25,26,27]
Summary
JULY 27, 2007 VOLUME 282 NUMBER 30 cule has created pressure for the cell to evolve powerful antioxidant defenses that convert reactive oxygen species (ROS) into harmless products to maintain a predominantly reduced redox environment. This depends on the following two factors: the reduction potential of the electron carriers, and the reducing capacity (i.e. the total concentration of the reduced species) of linked redox couples present in the cytoplasm or in the intraorganellar compartments (e.g. the mitochondrial matrix) of the cell. We show that the glutathione redox status determines the rate of mitochondrial ROS production and that the changes in the absolute concentrations of GSH and GSSG as well as the GSH/GSSG ratio, trigger, at moderate ratios (150:1 to 100:1), the reversible opening of IMAC and, at a lower ratio (50:1), irreversible PTP activation
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