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

2007; Elsevier BV; Volume: 282; Issue: 30 Linguagem: Inglês

10.1074/jbc.m702841200

ISSN

1083-351X

Autores

Miguel A. Aon, Sonia Cortassa, Christoph Maack, Brian O’Rourke,

Tópico(s)

Mitochondrial Function and Pathology

Resumo

Mitochondrial membrane potential (ΔΨm) 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 ΔΨm 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 ΔΨm depolarization, although irreversible PTP activation is induced by extensive thiol oxidation. Mitochondrial dysfunction in response to diamide occurred in stages, progressing from oscillations in ΔΨm 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, ΔΨm depolarized reversibly, and a matrix-localized fluorescent marker was retained; however, decreasing the GSH/GSSG to 50:1 irreversibly depolarized ΔΨm 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. Mitochondrial membrane potential (ΔΨm) 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 ΔΨm 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 ΔΨm depolarization, although irreversible PTP activation is induced by extensive thiol oxidation. Mitochondrial dysfunction in response to diamide occurred in stages, progressing from oscillations in ΔΨm 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, ΔΨm depolarized reversibly, and a matrix-localized fluorescent marker was retained; however, decreasing the GSH/GSSG to 50:1 irreversibly depolarized ΔΨm 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. The dual nature of oxygen as a vital electron acceptor in oxidative phosphorylation and as a dangerously reactive molecule has created pressure for the cell to evolve powerful antioxidant defenses that convert reactive oxygen species (ROS) 3The abbreviations used are: ROS, reactive oxygen species; NBD chloride, 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole; CM-H2DCFDA, 5-(-6)-chloromethyl-2′,7′-dichlorohydrofluorescein diacetate; GR, glutathione reductase; PTP, permeability transition pore; IMAC, inner membrane anion channel; THD, transhydrogenase; 4Cl-DZP, 4′-chlorodiazepam; BCNU, carmustine; DIC, dicarboxylate; Trx, thioredoxin; DMEM, Dulbecco's modified Eagle's medium; TMRM, tetramethylrhodamine methyl ester; MCB, monochlorobimane; GSB, glutathione S-bimane; CsA, cyclosporin A; CM-DCF, chloromethyl dichlorofluorescein.3The abbreviations used are: ROS, reactive oxygen species; NBD chloride, 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole; CM-H2DCFDA, 5-(-6)-chloromethyl-2′,7′-dichlorohydrofluorescein diacetate; GR, glutathione reductase; PTP, permeability transition pore; IMAC, inner membrane anion channel; THD, transhydrogenase; 4Cl-DZP, 4′-chlorodiazepam; BCNU, carmustine; DIC, dicarboxylate; Trx, thioredoxin; DMEM, Dulbecco's modified Eagle's medium; TMRM, tetramethylrhodamine methyl ester; MCB, monochlorobimane; GSB, glutathione S-bimane; CsA, cyclosporin A; CM-DCF, chloromethyl dichlorofluorescein. 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. In addition to the redox couples involved in mitochondrial electron transport (the nicotinamide adenine dinucleotides NADH/NAD+, and the flavins FADH2/FAD), the three main cellular redox pairs participating in intracellular reactions include reduced/oxidized glutathione (GSH/GSSG), thioredoxin (Trx(SH)2/TrxSS), and NADPH/NADP+, with the latter providing the thermodynamic driving force behind the glutathione and thioredoxin systems (1Schafer F.Q. Buettner G.R. Free Radic. Biol. Med. 2001; 30: 1191-1212Crossref PubMed Scopus (3641) Google Scholar). The GSH/GSSG pool is the largest of the cell (2Chance B. Sies H. Boveris A. Physiol. Rev. 1979; 59: 527-605Crossref PubMed Scopus (4819) Google Scholar, 3Rebrin I. Kamzalov S. Sohal R.S. Free Radic. Biol. Med. 2003; 35: 626-635Crossref PubMed Scopus (214) Google Scholar) and is considered to be a major indicator of the cellular redox status. GSH is a tripeptide (l-γ-glutamyl-l-cysteinyl-glycine) that is converted from its disulfide form, GSSG, by an NADPH-dependent reductase (glutathione reductase (GR)) whose activity increases in response to an increase in GSSG concentration. GSH provides reducing power for a variety of thiol modifications of disulfide bridges, thioethers, and thioesters and is a substrate for protein glutathionylation (4Shelton M.D. Chock P.B. Mieyal J.J. Antioxid. Redox. Signal. 2005; 7: 348-366Crossref PubMed Scopus (326) Google Scholar, 5Sies H. Free Radic. Biol. Med. 1999; 27: 916-921Crossref PubMed Scopus (1361) Google Scholar). Overall, the balance of GSH and GSSG provides a dynamic indicator of oxidative stress (6Jones D.P. Methods Enzymol. 2002; 348: 93-112Crossref PubMed Scopus (636) Google Scholar). Mitochondria, as the central site of oxygen consumption in the cell during the process of oxidative metabolism, are also a main source of ROS production. At the level of the respiratory chain, the highly positive reduction potential of the oxygen/superoxide anion (O2/O2-˙) redox pair combined with the proximity of other redox pairs such as the ubiquinone (CoQ)/ubisemiquinone radical (CoQ-˙) can result in O2-˙ generation (1Schafer F.Q. Buettner G.R. Free Radic. Biol. Med. 2001; 30: 1191-1212Crossref PubMed Scopus (3641) Google Scholar). Under normal conditions, there is a balance between ROS formation and antioxidant activity; however, under pathological conditions oxidative stress can occur as a consequence of either increased ROS production or by depletion of the antioxidant pool (6Jones D.P. Methods Enzymol. 2002; 348: 93-112Crossref PubMed Scopus (636) Google Scholar, 7Droge W. Physiol. Rev. 2002; 82: 47-95Crossref PubMed Scopus (7493) Google Scholar, 8Halliwell B. Thomas C.E. Kalyanaraman B. Oxygen Radicals and the Disease Process. Harwood Academic, Amsterdam, Netherlands1997: 1-14Google Scholar). Higher organisms have adapted to the presence of potentially toxic ROS within cells not only by building up antioxidant defenses but also by harnessing ROS as signaling molecules for regulating the activity of the cell. Changes in the cellular redox environment, both oxidative and reductive, can trigger redox cascades altering signal transduction, DNA, RNA and protein synthesis, enzyme regulation, gene expression, and cell cycle progression (9Abate C. Patel L. Rauscher III, F.J. Curran T. Science. 1990; 249: 1157-1161Crossref PubMed Scopus (1374) Google Scholar, 10Arrigo A.P. Free Radic. Biol. Med. 1999; 27: 936-944Crossref PubMed Scopus (427) Google Scholar, 11Powis G. Gasdaska J.R. Baker A. Adv. 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Such is the case for the ryanodine receptor of the sarcoplasmic reticulum of muscle cells, which possesses sensitive sulfhydryl groups that influence the rate of Ca2+ release depending upon the thiol redox status (16Xia R. Stangler T. Abramson J.J. J. Biol. Chem. 2000; 275: 36556-36561Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Considerable evidence also shows that agents altering GSH concentration affect transcription of detoxification enzymes, cell proliferation, and apoptosis (17Hall A.G. Eur. J. Clin. Investig. 1999; 29: 238-245Crossref PubMed Scopus (301) Google Scholar, 18Hwang C. Sinskey A.J. Lodish H.F. Science. 1992; 257: 1496-1502Crossref PubMed Scopus (1589) Google Scholar) or necrosis, depending on the severity of the oxidative challenge. GSH is synthesized in the cytoplasm and is exchanged with other intracellular compartments, with overall cellular GSH/GSSG ratios ranging between 30:1 and 300:1, but with the exception that the endoplasmic reticulum maintains a relatively oxidative environment with ratios of 1:1 to 3:1 (14Gilbert H.F. Adv. Enzymol. Relat. Areas Mol. Biol. 1990; 63: 69-172PubMed Google Scholar, 18Hwang C. Sinskey A.J. Lodish H.F. Science. 1992; 257: 1496-1502Crossref PubMed Scopus (1589) Google Scholar). Mitochondria do not synthesize GSH but are able to transport and accumulate up to ∼15% of the total cellular GSH (1Schafer F.Q. Buettner G.R. Free Radic. Biol. Med. 2001; 30: 1191-1212Crossref PubMed Scopus (3641) Google Scholar, 19Meister A. Anderson M.E. Annu. Rev. Biochem. 1983; 52: 711-760Crossref PubMed Scopus (5966) Google Scholar). As both a source and target of ROS production, the importance of the antioxidant capacity of mitochondria has been emphasized in recent years. An established target of oxidative stress in mitochondria is the permeability transition pore (PTP), a major player in initiating both apoptotic and necrotic cell death. Oxidative stress through oxidation of intracellular GSH and other critical sulfhydryl groups favors the PTP opening (20Chernyak B.V. Biosci. Rep. 1997; 17: 293-302Crossref PubMed Scopus (58) Google Scholar, 21Costantini P. Chernyak B.V. Petronilli V. Bernardi P. J. Biol. Chem. 1996; 271: 6746-6751Abstract Full Text Full Text PDF PubMed Scopus (472) Google Scholar, 22Petronilli V. Costantini P. Scorrano L. Colonna R. Passamonti S. Bernardi P. J. Biol. Chem. 1994; 269: 16638-16642Abstract Full Text PDF PubMed Google Scholar). Previously, we demonstrated that metabolic stress in the form of substrate deprivation (23O'Rourke B. Ramza B.M. Marban E. Science. 1994; 265: 962-966Crossref PubMed Scopus (226) Google Scholar, 24Romashko D.N. Marban E. O'Rourke B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1618-1623Crossref PubMed Scopus (179) Google Scholar) or localized ROS generation (25Aon M.A. Cortassa S. Marban E. O'Rourke B. J. Biol. Chem. 2003; 278: 44735-44744Abstract Full Text Full Text PDF PubMed Scopus (435) Google Scholar) can trigger cell-wide oscillations or collapse of ΔΨm in isolated cardiomyocytes and that cyclical GSH oxidation occurred in parallel. The magnitude and extent of oxidative stress on the mitochondrial network was a key predictor of whether synchronized cell-wide mitochondrial oscillations occurred (25Aon M.A. Cortassa S. Marban E. O'Rourke B. J. Biol. Chem. 2003; 278: 44735-44744Abstract Full Text Full Text PDF PubMed Scopus (435) Google Scholar, 26Cortassa S. Aon M.A. Winslow R.L. O'Rourke B. Biophys. J. 2004; 87: 2060-2073Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). A reproducible threshold of oxidation of an ROS-sensitive reporter preceded the first global ΔΨm transition of the mitochondrial network, and we referred to this sensitive state as "mitochondrial criticality" (27Aon M.A. Cortassa S. O'Rourke B. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 4447-4452Crossref PubMed Scopus (173) Google Scholar). This complex self-organizing phenomenon was scaled to alter the electrical and Ca2+ handling properties of the whole cell and contributed to the arrhythmias induced by ischemia-reperfusion in the whole heart (28Akar F.G. Aon M.A. Tomaselli G.F. O'Rourke B. J. Clin. Investig. 2005; 115: 3527-3535Crossref PubMed Scopus (261) Google Scholar, 29Aon M.A. Cortassa S. Akar F.G. O'Rourke B. Biochim. Biophys. Acta. 2006; 1762: 232-240Crossref PubMed Scopus (126) Google Scholar). Further investigation into the mechanism of this phenomenon revealed that several inhibitors of the mitochondrial inner membrane anion channel (IMAC) could reversibly suppress or prevent the mitochondrial ROS-induced ROS release response, thereby stabilizing ΔΨm (28Akar F.G. Aon M.A. Tomaselli G.F. O'Rourke B. J. Clin. Investig. 2005; 115: 3527-3535Crossref PubMed Scopus (261) Google Scholar). A computational model also supported a mechanistic scheme involving the activation of IMAC by ROS (26Cortassa S. Aon M.A. Winslow R.L. O'Rourke B. Biophys. J. 2004; 87: 2060-2073Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). In this study, we investigate the role played by the glutathione redox potential in the approach to mitochondrial criticality and the collapse of ΔΨm in the mitochondrial network. 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. Cardiomyocyte Isolation and Handling—Cardiomyocytes were prepared from adult guinea pig hearts using a Langendorff perfusion system as described before (23O'Rourke B. Ramza B.M. Marban E. Science. 1994; 265: 962-966Crossref PubMed Scopus (226) Google Scholar). The freshly isolated cells were handled as described before (25Aon M.A. Cortassa S. Marban E. O'Rourke B. J. Biol. Chem. 2003; 278: 44735-44744Abstract Full Text Full Text PDF PubMed Scopus (435) Google Scholar, 26Cortassa S. Aon M.A. Winslow R.L. O'Rourke B. Biophys. J. 2004; 87: 2060-2073Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). Briefly, after isolation, cells were either immediately used for imaging or stored in Dulbecco's modified Eagle's medium (10-013 DMEM, Mediatech, Inc., Herndon, VA) containing 5% fetal bovine serum and 1% penicillin/streptomycin in a 5% CO2 incubator at 37 °C for at least 2 h before assaying them. Experimental recordings of the freshly isolated cells (with or without storage in DMEM) started after suspending them in Tyrode solution containing (in mm) the following: 140 NaCl, 5 KCl, 1 MgCl2, 10 HEPES, 1 CaCl2, pH 7.5 (adjusted with NaOH), supplemented with 10 mm glucose or the addition of diamide when indicated. The perfusion chamber containing the cardiomyocytes was thermostatically controlled at 37 °C with unrestricted access to atmospheric oxygen on the stage of a Nikon E600FN upright microscope. Fluorescent Probes and Loading Conditions—Tetramethylrhodamine ethyl (or methyl) ester (TMRM) accumulates across polarized membranes with a Nernstian distribution. ROS production was monitored with the ROS-sensitive fluorescent probe 5-(-6)-chloromethyl-2′,7′-dichlorohydrofluorescein diacetate (CM-H2DCFDA, Invitrogen) (25Aon M.A. Cortassa S. Marban E. O'Rourke B. J. Biol. Chem. 2003; 278: 44735-44744Abstract Full Text Full Text PDF PubMed Scopus (435) Google Scholar). Reduced glutathione (GSH) was measured in myocytes loaded with the membrane permeant indicator monochlorobimane (MCB) (26Cortassa S. Aon M.A. Winslow R.L. O'Rourke B. Biophys. J. 2004; 87: 2060-2073Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 30Kosower N.S. Kosower E.M. Methods Enzymol. 1987; 143: 76-84Crossref PubMed Scopus (158) Google Scholar). This probe reports the level of GSH as the fluorescent product glutathione S-bimane (GSB) according to the reversible reaction, MCB + GSH ⇔ GSB. We have determined that concentrations up to 50 μm MCB do not deplete the intracellular pool of GSH to a level that compromises cell viability. The concentrations of the probes were 100 nm TMRM, 2 μm CM-H2DCFDA, and 50 μm MCB, and they were loaded into the cells for at least 20 min at 37 °C. The response of the MCB fluorescence to changes in GSH was quantified by the addition of exogenous GSH (see also below and Supplemental Material). Two-photon Microscopy—The equipment and methods utilized for imaging isolated cardiomyocytes were as described previously (25Aon M.A. Cortassa S. Marban E. O'Rourke B. J. Biol. Chem. 2003; 278: 44735-44744Abstract Full Text Full Text PDF PubMed Scopus (435) Google Scholar, 26Cortassa S. Aon M.A. Winslow R.L. O'Rourke B. Biophys. J. 2004; 87: 2060-2073Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). Images were recorded using a two-photon laser scanning microscope (Bio-Rad MRC-1024MP) with excitation at 740 nm (or 780 nm for GSB imaging). The red emission of TMRM was collected at 605 ± 25 nm; the green emission of CM-DCF was recorded at 525 ± 25 nm, and the blue GSB emission was recorded at 480 ± 20 nm. The NADH emission was collected as the total fluorescence <490 nm. Because of the overlap of NADH and GSB emissions (at steady state, NADH emission was approximately one-third that of GSB (26Cortassa S. Aon M.A. Winslow R.L. O'Rourke B. Biophys. J. 2004; 87: 2060-2073Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 31Lemar K.M. Passa O. Aon M.A. Cortassa S. Muller C.T. Plummer S. O'Rourke B. Lloyd D. Microbiology. 2005; 151: 3257-3265Crossref PubMed Scopus (79) Google Scholar)), separate experiments were conducted in which we monitored either ΔΨm, ROS, and NADH or ΔΨm and GSB, with cells loaded with TMRM and CM-DCF or TMRM and MCB, respectively. Images were collected simultaneously at the indicated time intervals (3.5, 5, or 30 s) and stored as described before (25Aon M.A. Cortassa S. Marban E. O'Rourke B. J. Biol. Chem. 2003; 278: 44735-44744Abstract Full Text Full Text PDF PubMed Scopus (435) Google Scholar). Permeabilization Procedure of Cardiomyocytes—Myocytes, which had been stored in DMEM for at least 2 h (which facilitates repletion of the cellular glutathione pool), were resuspended in the experimental solution, and the fluorescent probes were loaded directly in the perfusion chamber on the stage of the microscope for at least 20 min at 37 °C before starting the experiment. The permeabilizing solution (PS) contained 25 μg/ml saponin (to selectively permeabilize the sarcolemma but not the mitochondrial membranes) and (in mm) 130 potassium methanesulfonate, 20 KCl, 0.5 MgCl2, 10 HEPES-Na, 0.1 EGTA, 3 ATP, 5 pyruvate; the pH was adjusted to 7.2 with KOH and was perfused into the chamber for 2.5 min. Controls were performed to determine the efficiency of permeabilization by comparing the effects of solutions with or without saponin but containing the same GSH/GSSG ratios as shown in the results (with GSH constant at 3 mm). In the absence of saponin, there was no response of the mitochondria to lowering the GSH/GSSG ratio to <50:1. In the first set of experiments GSH/GSSG was varied in the presence of a fixed GSH level (at either 1, 3, or 4 mm) by increasing the GSSG concentration. In other experiments, as indicated under "Results," the GSSG concentration was fixed at 10 μm, and the GSH was varied. The initial permeabilization of the cells was always carried out in the presence of a 300:1 GSH/GSSG ratio. TMRM, CM-H2DCFDA, or MCB was included in the perfusion solution in most experiments to avoid any loss of the mitochondria-localized dyes in the course of the experiment in permeabilized cells subjected to changes in the GSH/GSSG ratio. A main aim of the permeabilization was to preserve mitochondrial integrity along with facilitating the access to cells of the perfusion solution. Several controls were run in order to verify mitochondrial function and the extent of permeabilization. These are described in the Supplemental Material. Kinetic Experiments with Intact and Permeabilized Cardiomyocytes—Kinetic experiments with intact or permeabilized cardiomyocytes were carried out at 37 °C in a thermostatically controlled flow chamber mounted on the stage of the upright microscope (Nikon E600FN) attached to the multiphoton laser scanning system. A constant flow was controlled with a peristaltic pump. With the fluorescent dye carboxyfluorescein (not taken up by the cells), we determined that the delay time for a compound to increase from 10 to 90% of its final concentration was 60 s for the chamber utilized. TMRM- and CM-DCF-loaded or TMRM- and MCB-loaded cardiomyocytes were perfused with normal modified Tyrode's solution (as described above for cardiomyocyte isolation) containing 1 mm Ca2+ in the absence or presence of different reagents (e.g. diamide). Quantification of Intracellular GSH—GSH levels in intact cells were monitored after incubating the cells with MCB. To determine the intracellular concentration of GSH (in mm), we performed in vitro calibrations by serially diluting a 10 mm GSB stock solution that was loaded into glass microcapillaries and placed in the same field as MCB-loaded myocytes. Purified glutathione S-transferase (GST, from rabbit liver; Sigma) was used to catalyze (37 °C) the reaction of GSH and MCB in an assay mixture consisting of the following (in mm): 10 PO4KH2/10 HEPES, pH 7.1, 0.2 MCB, 10 GSH, and 3 units of GST. The time course of the reaction was monitored with fluorometry at maximal emission of the fluorescent adduct GSB (λem = 390 nm; λex = 480 nm). We used a fluorometer (Photon Technology International) equipped with an adjustable photomultiplier and a thermostated cuvette with stirring. To calibrate the two-photon microscope for the measurement of intracellular GSH, we filled the microcapillaries (50 μm diameter) with known amounts of GSB obtained as described above. The capillaries were imaged by two-photon microscopy in the range 1-10 mm with a linear response obtained in the range 1-5 mm (supplemental Fig. S3A). This calibration standard was used as a reference for measuring intracellular GSH in myocytes loaded with MCB. The two-photon imaging of the myocytes loaded with 50 μm MCB was performed in the presence of a 3 mm GSB-filled microcapillary as an internal standard (supplemental Fig. S3B). The steady state cell GSB fluorescence signal was measured and referred to the internal standard of 3 mm GSB. Using this method we found an ∼2.7 mm concentration of GSH in isolated cardiomyocytes from guinea pig in high K+ or DMEM (see supplemental Fig. S3C). 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 (25Aon M.A. Cortassa S. Marban E. O'Rourke B. J. Biol. Chem. 2003; 278: 44735-44744Abstract Full Text Full Text PDF PubMed Scopus (435) Google Scholar, 26Cortassa S. Aon M.A. Winslow R.L. O'Rourke B. Biophys. J. 2004; 87: 2060-2073Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 27Aon M.A. Cortassa S. O'Rourke B. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 4447-4452Crossref PubMed Scopus (173) Google Scholar). As an alternative method to induce oxidative stress in the myocyte, we tested whether a pro-oxidative shift in the thiol status of glutathione, the largest cellular redox pool, influenced mitochondrial ROS accumulation and triggered ΔΨm oscillations. Several intracellular variables related to the mitochondrial functional status (ΔΨm, NADH, ROS, and GSH) were monitored in intact cardiomyocytes in the absence or presence of the thiol oxidant diamide (0.1 mm), which effectively depletes the reduced glutathione pool (5Sies H. Free Radic. Biol. Med. 1999; 27: 916-921Crossref PubMed Scopus (1361) Google Scholar, 32Kosower N.S. Kosower E.M. Methods Enzymol. 1995; 251: 123-133Crossref PubMed Scopus (285) Google Scholar). Diamide induced an increased rate of mitochondrial ROS production that, upon reaching a threshold level of oxidative stress, triggered oscillations in ΔΨm and NADH (Fig. 1A, 1st arrow). Further depletion of GSH in the presence of diamide resulted in the eventual irreversible depolarization of ΔΨm, followed ∼800 s later by cell contracture (Fig. 1A, 2nd arrow). A higher concentration of diamide (1 mm) provoked rapid ΔΨm loss and cell contracture without traversing the oscillatory domain (data not shown). Similar results were also obtained with another thiol-oxidizing agent, diallyldisulfide (31Lemar K.M. Passa O. Aon M.A. Cortassa S. Muller C.T. Plummer S. O'Rourke B. Lloyd D. Microbiology. 2005; 151: 3257-3265Crossref PubMed Scopus (79) Google Scholar), a component of garlic extract (data not shown). Exposure of the cells to 4′-chlorodiazepam (4Cl-DZP) during the diamide-induced ΔΨm oscillations stabilized ΔΨm, reversed the oxidation of the mitochondrial NADH pool, and slowed the rate of CM-H2DCF oxidation (Fig. 1B). This effect was reversible; after washout of 4Cl-DZP in the presence of diamide, the rate ROS production increased, the NADH pool oxidized, and ΔΨm depolarized, subsequently ending in cell contracture. ΔΨm oscillation and sustained loss of ΔΨm were correlated with the gradual depletion of the intracellular GSH pool, as monitored by a continuous decrease in the fluorescence levels of the GSB adduct (Fig. 1C). The effect of 4Cl-DZP to stabilize ΔΨm in the presence of diamide (Fig. 2A) was not reproduced with the PTP inhibitor cyclosporin A (CsA; Fig. 2B), indicating that 4Cl-DZP was inhibiting a distinct target on the inner membrane (25Aon M.A. Cortassa S. Marban E. O'Rourke B. J. Biol. Chem. 2003; 278: 44735-44744Abstract Full Text Full Text PDF PubMed Scopus (435) Google Scholar, 33Beavis A.D. J. Bioenerg. Biomembr. 1992; 24: 77-90Crossref PubMed Scopus (111) Google Scholar). In fact, although 4Cl-DZP had a repolarizing influence of ΔΨm that could be washed out to re-initiate oscillations, CsA exacerbated mitochondrial depolarization (probably a nonspecific toxic effect that is opposite to what would be expected for inhibition of PTP), with partial restoration of oscillation upon washout. Thus, moderate depletion of GSH with diamide triggers reversible 4Cl-DZP-sensitive oscillations in ΔΨm. Continued exposure to diamide for a long time severely depletes the GSH antioxidant pool and activates the PTP irreversibly (at the arrows in Figs. 1 and 2). Taken together, the results suggest that the mitochondrial oscillations elicited by diamide are consistent with the mechanism we have described previously for other triggers of metabolic stress, i.e. that mitochondrial oxidative stress triggers the activation of IMAC as part of a self-sustaining limit cycle oscillator (25Aon M.A. Cortassa S. Marban E. O'Rourke B. J. Biol. Chem. 2003; 278: 44735-44744Abstract Full Text Full Text PDF PubMed Scopus (435) Google Scholar, 26Cortassa S. Aon M.A. Winslow R.L. O'Rourke B. Biophys. J. 2004; 87: 2060-2073Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). Importantly, these results strongly suggest that GSH, and likely the glutathione redox potential, are main cellular variables that determine the approach of the mitochondrial network to criticality through an increase in oxidative stress. In the next section, we test this possibility in a permeabilized cell system. Varying GSH/GSSG—In the first series of experiments in saponin-permeabilized cardiomyocytes, cells were exposed progressively to GSH/GSSG ratios ranging from 300:1 to 50:1 effected by increasing the concentration of GSSG while keeping the GSH constant at 3 mm. This concentration of GSH was used because it was close to the intracellular [GSH] determined in freshly isolated cardiomyocytes using MCB as a probe (see under "Experimental Procedures" and Suppleme

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