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J. Biol. Chem., Vol. 282, Issue 30, 21889-21900, July 27, 2007

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Sequential Opening of Mitochondrial Ion Channels as a Function of Glutathione Redox Thiol Status^*

From the Institute of Molecular Cardiobiology, Department of Medicine, The Johns Hopkins University, Baltimore, Maryland 21205

Mitochondrial membrane potential (_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 _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 _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 _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, _m depolarized reversibly, and a matrix-localized fluorescent marker was retained; however, decreasing the GSH/GSSG to 50:1 irreversibly depolarized _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.

Received for publication, April 3, 2007 , and in revised form, April 30, 2007.

^* This work was supported by National Institutes of Health Grant R37-HL54598 (to B. O'R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S3.

¹ Present address: Klinik für Innere Medizin III, Universitätsklinikum des Saarlandes 66421 Homburg/Saar, Germany.

² To whom correspondence should be addressed: The Johns Hopkins University, Institute of Molecular Cardiobiology, 720 Rutland Ave., 1059 Ross Bldg., Baltimore, MD 21205. Tel.: 410-614-0034; E-mail: bor{at}jhmi.edu.

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