ATP Synthase K+- and H+-Fluxes Drive ATP Synthesis and Enable Mitochondrial K+-"Uniporter" Function: I. Characterization of Ion Fluxes.

ATP synthase (F1Fo) synthesizes daily our body's weight in ATP, whose production-rate can be transiently increased several-fold to meet changes in energy utilization. Using purified mammalian F1Fo-reconstituted proteoliposomes and isolated mitochondria, we show F1Fo can utilize both ΔΨm-driven H+- and K+-transport to synthesize ATP under physiological pH = 7.2 and K+ = 140 mEq/L conditions. Purely K+-driven ATP synthesis from single F1Fo molecules measured by bioluminescence photon detection could be directly demonstrated along with simultaneous measurements of unitary K+ currents by voltage clamp, both blocked by specific Fo inhibitors. In the presence of K+, compared to osmotically-matched conditions in which this cation is absent, isolated mitochondria display 3.5-fold higher rates of ATP synthesis, at the expense of 2.6-fold higher rates of oxygen consumption, these fluxes being driven by a 2.7:1 K+: H+ stoichiometry. The excellent agreement between the functional data obtained from purified F1Fo single molecule experiments and ATP synthase studied in the intact mitochondrion under unaltered OxPhos coupling by K+ presence, is entirely consistent with K+ transport through the ATP synthase driving the observed increase in ATP synthesis. Thus, both K+ (harnessing ΔΨm) and H+ (harnessing its chemical potential energy, ΔµH) drive ATP generation during normal physiology.

ATP Synthase K+- and H+-fluxes Drive ATP Synthesis and Enable Mitochondrial K+-"Uniporter" Function: II. Ion and ATP Synthase Flux Regulation.

We demonstrated that ATP synthase serves the functions of a primary mitochondrial K+ "uniporter," i.e., the primary way for K+ to enter mitochondria. This K+ entry is proportional to ATP synthesis, regulating matrix volume and energy supply-vs-demand matching. We show that ATP synthase can be upregulated by endogenous survival-related proteins via IF1. We identified a conserved BH3-like domain of IF1 which overlaps its "minimal inhibitory domain" that binds to the ß-subunit of F1. Bcl-xL and Mcl-1 possess a BH3-binding-groove that can engage IF1 and exert effects, requiring this interaction, comparable to diazoxide to augment ATP synthase's H+ and K+ flux and ATP synthesis. Bcl-xL and Mcl-1, but not Bcl-2, serve as endogenous regulatory ligands of ATP synthase via interaction with IF1 at this BH3-like domain, to increase its chemo-mechanical efficiency, enabling its function as the recruitable mitochondrial KATP-channel that can limit ischemia-reperfusion injury. Using Bayesian phylogenetic analysis to examine potential bacterial IF1-progenitors, we found that IF1 is likely an ancient (∼2 Gya) Bcl-family member that evolved from primordial bacteria resident in eukaryotes, corresponding to their putative emergence as symbiotic mitochondria, and functioning to prevent their parasitic ATP consumption inside the host cell.

Computational modeling of mitochondrial K+- and H+-driven ATP synthesis.

ATP synthase (F1Fo) is a rotary molecular engine that harnesses energy from electrochemical-gradients across the inner mitochondrial membrane for ATP synthesis. Despite the accepted tenet that F1Fo transports exclusively H+, our laboratory has demonstrated that, in addition to H+, F1Fo ATP synthase transports a significant fraction of ΔΨm-driven charge as K+ to synthesize ATP. Herein, we utilize a computational modeling approach as a proof of principle of the feasibility of the core mechanism underlying the enhanced ATP synthesis, and to explore its bioenergetic consequences. A minimal model comprising the 'core' mechanism constituted by ATP synthase, driven by both proton (PMF) and potassium motive force (KMF), respiratory chain, adenine nucleotide translocator, Pi carrier, and K+/H+ exchanger (KHEmito) was able to simulate enhanced ATP synthesis and respiratory fluxes determined experimentally with isolated heart mitochondria. This capacity of F1Fo ATP synthase confers mitochondria with a significant energetic advantage compared to K+ transport through a channel not linked to oxidative phosphorylation (OxPhos). The K+-cycling mechanism requires a KHEmito that exchanges matrix K+ for intermembrane space H+, leaving PMF as the overall driving energy of OxPhos, in full agreement with the standard chemiosmotic mechanism. Experimental data of state 4➔3 energetic transitions, mimicking low to high energy demand, could be reproduced by an integrated computational model of mitochondrial function that incorporates the 'core' mechanism. Model simulations display similar behavior compared to the experimentally observed changes in ΔΨm, mitochondrial K+ uptake, matrix volume, respiration, and ATP synthesis during the energetic transitions at physiological pH and K+ concentration. The model also explores the role played by KHEmito in modulating the energetic performance of mitochondria. The results obtained support the available experimental evidence on ATP synthesis driven by K+ and H+ transport through the F1Fo ATP synthase.

Network dynamics: quantitative analysis of complex behavior in metabolism, organelles, and cells, from experiments to models and back.

Advancing from two core traits of biological systems: multilevel network organization and nonlinearity, we review a host of novel and readily available techniques to explore and analyze their complex dynamic behavior within the framework of experimental-computational synergy. In the context of concrete biological examples, analytical methods such as wavelet, power spectra, and metabolomics-fluxomics analyses, are presented, discussed, and their strengths and limitations highlighted. Further shown is how time series from stationary and nonstationary biological variables and signals, such as membrane potential, high-throughput metabolomics, O2 and CO2 levels, bird locomotion, at the molecular, (sub)cellular, tissue, and whole organ and animal levels, can reveal important information on the properties of the underlying biological networks. Systems biology-inspired computational methods start to pave the way for addressing the integrated functional dynamics of metabolic, organelle and organ networks. As our capacity to unravel the control and regulatory properties of these networks and their dynamics under normal or pathological conditions broadens, so is our ability to address endogenous rhythms and clocks to improve health-span in human aging, and to manage complex metabolic disorders, neurodegeneration, and cancer. WIREs Syst Biol Med 2017, 9:e1352. doi: 10.1002/wsbm.1352 For further resources related to this article, please visit the WIREs website.

Reversal of Mitochondrial Transhydrogenase Causes Oxidative Stress in Heart Failure.

Mitochondrial reactive oxygen species (ROS) play a central role in most aging-related diseases. ROS are produced at the respiratory chain that demands NADH for electron transport and are eliminated by enzymes that require NADPH. The nicotinamide nucleotide transhydrogenase (Nnt) is considered a key antioxidative enzyme based on its ability to regenerate NADPH from NADH. Here, we show that pathological metabolic demand reverses the direction of the Nnt, consuming NADPH to support NADH and ATP production, but at the cost of NADPH-linked antioxidative capacity. In heart, reverse-mode Nnt is the dominant source for ROS during pressure overload. Due to a mutation of the Nnt gene, the inbred mouse strain C57BL/6J is protected from oxidative stress, heart failure, and death, making its use in cardiovascular research problematic. Targeting Nnt-mediated ROS with the tetrapeptide SS-31 rescued mortality in pressure overload-induced heart failure and could therefore have therapeutic potential in patients with this syndrome.

From metabolomics to fluxomics: a computational procedure to translate metabolite profiles into metabolic fluxes.

We describe a believed-novel procedure for translating metabolite profiles (metabolome) into the set of metabolic fluxes (fluxome) from which they originated. Methodologically, computational modeling is integrated with an analytical platform comprising linear optimization, continuation and dynamic analyses, and metabolic control. The procedure was tested with metabolite profiles obtained from ex vivo mice Langendorff-heart preparations perfused with glucose. The metabolic profiles were analyzed using a detailed kinetic model of the glucose catabolic pathways including glycolysis, pentose phosphate (PP), glycogenolysis, and polyols to translate the glucose metabolome of the heart into the fluxome. After optimization, the ability of the model to simulate the initial metabolite profile was confirmed, and metabolic fluxes as well as the structure of control and regulation of the glucose catabolic network could be calculated. We show that the step catalyzed by phosphofructokinase together with ATP demand and glycogenolysis exert the highest control on the glycolytic flux. The negative flux control exerted by phosphofructokinase on the PP and polyol pathways revealed that the extent of glycolytic flux directly affects flux redirection through these pathways, i.e., the higher the glycolytic flux the lower the PP and polyols. This believed-novel methodological approach represents a step forward that may help in designing therapeutic strategies targeted to diagnose, prevent, and treat metabolic diseases.

Restoring redox balance enhances contractility in heart trabeculae from type 2 diabetic rats exposed to high glucose.

Hearts from type 2 diabetic (T2DM) subjects are chronically subjected to hyperglycemia and hyperlipidemia, both thought to contribute to oxidizing conditions and contractile dysfunction. How redox alterations and contractility interrelate, ultimately diminishing T2DM heart function, remains poorly understood. Herein we tested whether the fatty acid palmitate (Palm), in addition to its energetic contribution, rescues function by improving redox [glutathione (GSH), NAD(P)H, less oxidative stress] in T2DM rat heart trabeculae subjected to high glucose. Using cardiac trabeculae from Zucker Diabetic Fatty (ZDF) rats, we assessed the impact of low glucose (EG) and high glucose (HG), in absence or presence of Palm or insulin, on force development, energetics, and redox responses. We found that in EG ZDF and lean trabeculae displayed similar contractile work, yield of contractile work (Ycw), representing the ratio of force time integral over rate of O2 consumption. Conversely, HG had a negative impact on Ycw, whereas Palm, but not insulin, completely prevented contractile loss. This effect was associated with higher GSH, less oxidative stress, and augmented matrix GSH/thioredoxin (Trx) in ZDF mitochondria. Restoration of myocardial redox with GSH ethyl ester also rescued ZDF contractile function in HG, independently from Palm. These results support the idea that maintained redox balance, via increased GSH and Trx antioxidant activities to resist oxidative stress, is an essential protective response of the diabetic heart to keep contractile function.

Integrating mitochondrial energetics, redox and ROS metabolic networks: a two-compartment model.

To understand the mechanisms involved in the control and regulation of mitochondrial reactive oxygen species (ROS) levels, a two-compartment computational mitochondrial energetic-redox (ME-R) model accounting for energetic, redox, and ROS metabolisms is presented. The ME-R model incorporates four main redox couples (NADH/NAD(+), NADPH/NADP(+), GSH/GSSG, Trx(SH)(2)/TrxSS). Scavenging systems-glutathione, thioredoxin, superoxide dismutase, catalase-are distributed in mitochondrial matrix and extra-matrix compartments, and transport between compartments of ROS species (superoxide: O(2)(⋅-), hydrogen peroxide: H(2)O(2)), and GSH is also taken into account. Model simulations are compared with experimental data obtained from isolated heart mitochondria. The ME-R model is able to simulate: i), the shape and order of magnitude of H(2)O(2) emission and dose-response kinetics observed after treatment with inhibitors of the GSH or Trx scavenging systems and ii), steady and transient behavior of ΔΨ(m) and NADH after single or repetitive pulses of substrate- or uncoupler-elicited energetic-redox transitions. The dynamics of the redox environment in both compartments is analyzed with the model following substrate addition. The ME-R model represents a useful computational tool for exploring ROS dynamics, the role of compartmentation in the modulation of the redox environment, and how redox regulation participates in the control of mitochondrial function.

GSH or palmitate preserves mitochondrial energetic/redox balance, preventing mechanical dysfunction in metabolically challenged myocytes/hearts from type 2 diabetic mice.

In type 2 diabetes, hyperglycemia and increased sympathetic drive may alter mitochondria energetic/redox properties, decreasing the organelle's functionality. These perturbations may prompt or sustain basal low-cardiac performance and limited exercise capacity. Yet the precise steps involved in this mitochondrial failure remain elusive. Here, we have identified dysfunctional mitochondrial respiration with substrates of complex I, II, and IV and lowered thioredoxin-2/glutathione (GSH) pools as the main processes accounting for impaired state 4→3 energetic transition shown by mitochondria from hearts of type 2 diabetic db/db mice upon challenge with high glucose (HG) and the ß-agonist isoproterenol (ISO). By mimicking clinically relevant conditions in type 2 diabetic patients, this regimen triggers a major overflow of reactive oxygen species (ROS) from mitochondria that directly perturbs cardiac electro-contraction coupling, ultimately leading to heart dysfunction. Exogenous GSH or, even more so, the fatty acid palmitate rescues basal and ß-stimulated function in db/db myocyte/heart preparations exposed to HG/ISO. This occurs because both interventions provide the reducing equivalents necessary to counter mitochondrial ROS outburst and energetic failure. Thus, in the presence of poor glycemic control, the diabetic patient's inability to cope with increased cardiac work demand largely stems from mitochondrial redox/energetic disarrangements that mutually influence each other, leading to myocyte or whole-heart mechanical dysfunction.

Glutathione/thioredoxin systems modulate mitochondrial H2O2 emission: an experimental-computational study.

The net emission of hydrogen peroxide (H(2)O(2)) from mitochondria results from the balance between reactive oxygen species (ROS) continuously generated in the respiratory chain and ROS scavenging. The relative contribution of the two major antioxidant systems in the mitochondrial matrix, glutathione (GSH) and thioredoxin (Trx), has not been assessed. In this paper, we examine this key question via combined experimental and theoretical approaches, using isolated heart mitochondria from mouse, rat, and guinea pig. As compared with untreated control mitochondria, selective inhibition of Trx reductase with auranofin along with depletion of GSH with 2,4-dinitrochlorobenzene led to a species-dependent increase in H(2)O(2) emission flux of 17, 11, and 6 fold in state 4 and 15, 7, and 8 fold in state 3 for mouse, rat, and guinea pig mitochondria, respectively. The maximal H(2)O(2) emission as a percentage of the total O(2) consumption flux was 11%/2.3% for mouse in states 4 and 3 followed by 2%/0.25% and 0.74%/0.29% in the rat and guinea pig, respectively. A minimal computational model accounting for the kinetics of GSH/Trx systems was developed and was able to simulate increase in H(2)O(2) emission fluxes when both scavenging systems were inhibited separately or together. Model simulations suggest that GSH/Trx systems act in concert. When the scavenging capacity of either one of them saturates during H(2)O(2) overload, they relieve each other until complete saturation, when maximal ROS emission occurs. Quantitatively, these results converge on the idea that GSH/Trx scavenging systems in mitochondria are both essential for keeping minimal levels of H(2)O(2) emission, especially during state 3 respiration, when the energetic output is maximal. This suggests that the very low levels of H(2)O(2) emission observed during forward electron transport in the respiratory chain are a result of the well-orchestrated actions of the two antioxidant systems working continuously to offset ROS production.

Mitochondrial energetics, pH regulation, and ion dynamics: a computational-experimental approach.

We developed a computational model of mitochondrial energetics that includes Ca(2+), proton, Na(+), and phosphate dynamics. The model accounts for distinct respiratory fluxes from substrates of complex I and complex II, pH effects on equilibrium constants and enzyme kinetics, and the acid-base equilibrium distributions of energy intermediaries. We experimentally determined NADH and ΔΨ(m) in guinea pig mitochondria during transitions from de-energized to energized, or during state 2/4 to state 3 respiration, or into hypoxia and uncoupling, and compared the results with those obtained in model simulations. The model quantitatively reproduces the experimentally observed magnitude of ΔΨ(m), the range of NADH levels, respiratory fluxes, and respiratory control ratio upon transitions elicited by sequential additions of substrate and ADP. Simulation results are also able to mimic the change in ΔΨ(m) upon addition of phosphate to state 4 mitochondria, leading to matrix acidification and ΔΨ(m) polarization. The steady-state behavior of the integrated mitochondrial model qualitatively simulates the dependence of respiration on the proton motive force, and the expected flux-force relationships existing between respiratory and ATP synthesis fluxes versus redox and phosphorylation potentials. This upgraded mitochondrial model provides what we believe are new opportunities for simulating mitochondrial physiological behavior during dysfunctional states involving changes in pH and ion dynamics.

Metabolic control analysis applied to mitochondrial networks.

To understand the control and regulation of mitochondrial energy metabolism a generalized matrix method of Metabolic Control Analysis has been applied to a computational model of mitochondrial energetics. The computational model of Cortassa et al. (2003) encompasses oxidative phosphorylation, the tricarboxylic acid (TCA) cycle, and ion dynamics across the inner mitochondrial membrane. Control of respiration and ATP synthesis fluxes were found to be distributed among various mitochondrial processes. Control is shared by processes associated with ATP synthesis and ATP/ADP transport, as well as by Ca(2+) dynamics. The analysis of flux control coefficients and response coefficients has led to the notion of control by diffuse loops, that points to the regulatory interactions exerted by processes that are mechanistically only indirectly related with each other. The approach we have utilized demonstrates how properties of integrated systems may be understood through applications of computational modeling and control analysis.

Calcium sensitivity, force frequency relationship and cardiac troponin I: critical role of PKA and PKC phosphorylation sites.

Transgenic models with pseudo phosphorylation mutants of troponin I, PKA sites at Ser 22 and 23 (cTnIDD(22,23) mice) or PKC sites at Ser 42 and 44 (cTnIAD(22,23)DD(42,44)) displayed differential force-frequency relationships and afterload relaxation delay in vivo. We hypothesized that cTnI PKA and PKC phosphomimics impact cardiac muscle rate-related developed twitch force and relaxation kinetics in opposite directions. cTnIDD(22,23) transgenic mice produce a force frequency relationship (FFR) equivalent to control NTG albeit at lower peak [Ca(2+)](i), while cTnIAD(22,23)DD(42,44) TG mice had a flat FFR with normal peak systolic [Ca(2+)](i), thus suggestive of diminished responsiveness to [Ca(2+)](i) at higher frequencies. Force-[Ca(2+)](i) hysteresis analysis revealed that cTnIDD(22,23) mice have a combined enhanced myofilament calcium peak response with an enhanced slope of force development and decline per unit of [Ca(2+)](i), whereas cTnIAD(22,23)DD(42,44) transgenic mice showed the opposite. The computational ECME model predicts that the TG lines may be distinct from each other due to different rate constants for association/dissociation of Ca(2+) at the regulatory site of cTnC. Our data indicate that cTnI phosphorylation at PKA sites plays a critical role in the FFR by increasing relative myofilament responsiveness, and results in a distinctive transition between activation and relaxation, as displayed by force-[Ca(2+)](i) hysteresis loops. These findings may have important implications for understanding the specific contribution of cTnI to beta-adrenergic inotropy and lusitropy and to adverse contractile effects of PKC activation, which is relevant during heart failure development.

Modeling cardiac action potential shortening driven by oxidative stress-induced mitochondrial oscillations in guinea pig cardiomyocytes.

Ischemia-induced shortening of the cardiac action potential and its heterogeneous recovery upon reperfusion are thought to set the stage for reentrant arrhythmias and sudden cardiac death. We have recently reported that the collapse of mitochondrial membrane potential (DeltaPsi(m)) through a mechanism triggered by reactive oxygen species (ROS), coupled to the opening of sarcolemmal ATP-sensitive potassium (K(ATP)) channels, contributes to electrical dysfunction during ischemia-reperfusion. Here we present a computational model of excitation-contraction coupling linked to mitochondrial bioenergetics that incorporates mitochondrial ROS-induced ROS release with coupling between the mitochondrial energy state and electrical excitability mediated by the sarcolemmal K(ATP) current (I(K,ATP)). Whole-cell model simulations demonstrate that increasing the fraction of oxygen diverted from the respiratory chain to ROS production triggers limit-cycle oscillations of DeltaPsi(m), redox potential, and mitochondrial respiration through the activation of a ROS-sensitive inner membrane anion channel. The periods of transient mitochondrial uncoupling decrease the cytosolic ATP/ADP ratio and activate I(K,ATP), consequently shortening the cellular action potential duration and ultimately suppressing electrical excitability. The model simulates emergent behavior observed in cardiomyocytes subjected to metabolic stress and provides a new tool for examining how alterations in mitochondrial oxidative phosphorylation will impact the electrophysiological, contractile, and Ca(2+) handling properties of the cardiac cell. Moreover, the model is an important step toward building multiscale models that will permit investigation of the role of spatiotemporal heterogeneity of mitochondrial metabolism in the mechanisms of arrhythmogenesis and contractile dysfunction in cardiac muscle.

Control and regulation of integrated mitochondrial function in metabolic and transport networks.

The pattern of flux and concentration control coefficients in an integrated mitochondrial energetics model is examined by applying a generalized matrix method of control analysis to calculate control coefficients, as well as response coefficients The computational model of Cortassa et al. encompasses oxidative phosphorylation, the TCA cycle, and Ca(2+) dynamics. Control of ATP synthesis, TCA cycle, and ANT fluxes were found to be distributed among various mitochondrial processes. Control is shared by processes associated with ATP/ADP production and transport, as well as by Ca(2+) dynamics. The calculation also analyzed the control of the concentrations of key regulatory ions and metabolites (Ca(2+), NADH, ADP). The approach we have used demonstrates how properties of integrated systems may be understood through applications of computational modeling and control analysis.

Control and regulation of mitochondrial energetics in an integrated model of cardiomyocyte function.

Understanding the regulation and control of complex networks of reactions requires analytical tools that take into account the interactions between individual network components controlling global network function. Here, we apply a generalized matrix method of control analysis to calculate flux and concentration control coefficients, as well as response coefficients, in an integrated model of excitation-contraction (EC) coupling and mitochondrial energetics (ECME model) in the cardiac ventricular myocyte. Control and regulation of oxygen consumption (V(O2)) was first assessed in a mitochondrion model, and then in the integrated cardiac myocyte model under resting and working conditions. The results demonstrate that in the ECME model, control of respiration is distributed among cytoplasmic ATPases and mitochondrial processes. The magnitude of control by cytoplasmic ATPases increases under working conditions. The model prediction that the respiratory chain exerts strong positive control on V(O2) (control coefficient 0.89) was corroborated experimentally in cardiac trabeculae utilizing the inhibitor titration method. In the model, mitochondrial respiration displayed the highest response coefficients with respect to the concentration of cytoplasmic ATP. This was due to the high elasticity of ANT flux toward ATP in the cytoplasm. The analysis reveals the complex interdependence of sarcolemmal, cytoplasmic, and mitochondrial processes that contribute to the control of energy supply and demand in the heart. Moreover, by visualizing the structure of control of the metabolic network of the myocyte, we provide support for the emerging concept of control by diffuse loops, in which action on the network (e.g., by a pharmacological agent) may bring about changes in processes without obvious direct mechanistic links between them.

The scale-free dynamics of eukaryotic cells.

Temporal organization of biological processes requires massively parallel processing on a synchronized time-base. We analyzed time-series data obtained from the bioenergetic oscillatory outputs of Saccharomyces cerevisiae and isolated cardiomyocytes utilizing Relative Dispersional (RDA) and Power Spectral (PSA) analyses. These analyses revealed broad frequency distributions and evidence for long-term memory in the observed dynamics. Moreover RDA and PSA showed that the bioenergetic dynamics in both systems show fractal scaling over at least 3 orders of magnitude, and that this scaling obeys an inverse power law. Therefore we conclude that in S. cerevisiae and cardiomyocytes the dynamics are scale-free in vivo. Applying RDA and PSA to data generated from an in silico model of mitochondrial function indicated that in yeast and cardiomyocytes the underlying mechanisms regulating the scale-free behavior are similar. We validated this finding in vivo using single cells, and attenuating the activity of the mitochondrial inner membrane anion channel with 4-chlorodiazepam to show that the oscillation of NAD(P)H and reactive oxygen species (ROS) can be abated in these two evolutionarily distant species. Taken together these data strongly support our hypothesis that the generation of ROS, coupled to redox cycling, driven by cytoplasmic and mitochondrial processes, are at the core of the observed rhythmicity and scale-free dynamics. We argue that the operation of scale-free bioenergetic dynamics plays a fundamental role to integrate cellular function, while providing a framework for robust, yet flexible, responses to the environment.

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

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.

Diallyl disulphide depletes glutathione in Candida albicans: oxidative stress-mediated cell death studied by two-photon microscopy.

Using two-photon scanning laser microscopy, we investigated the effect of an Allium sativum (garlic) constituent, diallyl disulphide (DADS), on key physiological functions of the opportunistic pathogen Candida albicans. A short 30 min exposure to 0.5 mM DADS followed by removal induced 70% cell death (50% necrotic, 20% apoptotic) within 2 h, increasing to 75% after 4 h. The early intracellular events associated with DADS-induced cell death were monitored with two-photon fluorescence microscopy to track mitochondrial membrane potential (Deltapsi(m)), reactive oxygen species (ROS) and NADH or reduced glutathione (GSH) under aerobic conditions. DADS treatment decreased intracellular GSH and elevated intracellular ROS levels. Additionally, DADS induced a marked decrease of Deltapsi(m) and lowered respiration in cell suspensions and isolated mitochondria. In vitro kinetic experiments in cell-free extracts suggest that glutathione-S-transferase (GST) is one of the intracellular targets of DADS. Additional targets were also identified, including inhibition of a site or sites between complexes II-IV in the electron transport chain, as well as the mitochondrial ATP-synthase. The results indicate that DADS is an effective antifungal agent able to trigger cell death in Candida, most probably by eliciting oxidative stress as a consequence of thiol depletion and impaired mitochondrial function.