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

We developed a computational model of mitochondrial energetics that includes Ca²⁺, 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.     

A Computational Model of Reactive Oxygen Species and Redox Balance in Cardiac Mitochondria

Elevated levels of reactive oxygen species (ROS) play a critical role in cardiac myocyte signaling in both healthy and diseased cells. Mitochondria represent the predominant cellular source of ROS, specifically the activity of complexes I and III. The model presented here explores the modulation of electron transport chain ROS production for state 3 and state 4 respiration and the role of substrates and respiratory inhibitors. Model simulations show that ROS production from complex III increases exponentially with membrane potential (ΔΨ_m) when in state 4. Complex I ROS release in the model can occur in the presence of NADH and succinate (reverse electron flow), leading to a highly reduced ubiquinone pool, displaying the highest ROS production flux in state 4. In the presence of ample ROS scavenging, total ROS production is moderate in state 3 and increases substantially under state 4 conditions. The ROS production model was extended by combining it with a minimal model of ROS scavenging. When the mitochondrial redox status was oxidized by increasing the proton permeability of the inner mitochondrial membrane, simulations with the combined model show that ROS levels initially decline as production drops off with decreasing ΔΨ_m and then increase as scavenging capacity is exhausted. Hence, this mechanistic model of ROS production demonstrates how ROS levels are controlled by mitochondrial redox balance.     

Distinct Alterations in Mitochondrial Mass and Function Characterize Different Models of Apoptosis

Recent studies have shown that reduction in mitochondrial membrane potential (ΔΨ_m) and generation of reactive oxygen species are early events in apoptosis. In this study, we present two different models of apoptotic cell death, Chinese hamster ovary (CHO) cells treated with aphidicolin and dexamethasone-treated 2B4 T-cell hybridoma cells, which display opposing mitochondrial changes. CHO cells arrested at G1/S with aphidicolin have a progressive increase in mitochondria mass and number, assessed by flow cytometry and fluorescent microscopy with mitochondria-specific probes. The increase in mitochondrial mass was not accompanied by a gain in net cellular mitochondrial membrane potential, consistent with an accumulation of relatively depolarized mitochondria. Fluorescent microscopy demonstrated an increased content of low ΔΨ_mmitochondria in aphidicolin-treated CHO cells, but high ΔΨ_mmitochondria were also present and remained stable in number. Mitochondrial mass correlated with decreased clonogenicity of aphidicolin-treated CHO cells. Cycloheximide prevented both the proliferation of mitochondria and subsequent cell death. In contrast, dexamethasone treatment of 2B4 T-cell hybridoma cells caused a decrease in ΔΨ_mwithout mitochondrial proliferation. Cycloheximide and Bcl-2 overexpression inhibited the loss of ΔΨ_m, as well as apoptosis. In both models, cell death was associated with a decrease in mitochondrial potential relative to mitochondrial mass, suggesting that an accumulation of damaged or dysfunctional mitochondria had occurred.     

Time and charge/pH-dependent activation of K+ channel-mediated K+ influx and K+/H+ exchange in guinea pig heart isolated mitochondria; role in bioenergetic stability

Mitochondria play an important role not only in producing energy for the cell but also for regulating mitochondrial and cell function depending on the cell's needs and environment. Uptake of cations, anions, and substrates requires a stable, polarized transmembrane charge potential (ΔΨ_m). Chemiosmosis requires ion exchangers to remove Na⁺, K⁺, Ca²⁺, PO₄^3?, and other charged species that enter mitochondria. Knowledge of the kinetics of mitochondrial (m) cation channels and exchangers is important in understanding their roles in regulating mitochondrial chemiosmosis and bioenergetics. The influx/efflux of K⁺, the most abundant mitochondrial cation, alters mitochondrial volume and shape by bringing in anions and H₂O by osmosis. The effects of K⁺ uptake through ligand-specific mK⁺ channels stimulated/inhibited by agonists/antagonists on mitochondrial volume (swelling/contraction) are well known. However, a more important role for K⁺ influx is likely its effects on H⁺ cycling and bioenergetics facilitated by mitochondrial (m) K⁺/H⁺ exchange (mKHE), though the kinetics and consequences of K⁺ efflux by KHE are not well described. We hypothesized that a major role of K⁺ influx/efflux is stimulation of respiration via the influx of H⁺ by KHE. We proposed to modulate KHE activity by energizing guinea pig heart isolated mitochondria and by altering the mK⁺ cycle to capture changes in mitochondrial volume, pH_m, ΔΨ_m, and respiration that would reflect a role for H⁺ influx via KHE to regulate bioenergetics. To test this, mitochondria were suspended in a 150 mM K⁺ buffer at pH 6.9, or in a 140 mM Cs⁺ buffer at pH 7.6 or 6.9 with added 10 mM K⁺, minimal Ca²⁺ and free of Na⁺. O₂ content was measured by a Clark electrode, and pH_m, ΔΨ_m, and volume, were measured by fluorescence spectrophotometry and light-scattering. Adding pyruvic acid (PA) alone caused increases in volume and respiration and a rapid decrease in the transmembrane pH gradient (ΔpH_m = pH_in–pH_ext) at pH_ext 6.9> > 7.6, so that ΔΨ_m was charged and maintained. BK_Ca agonist NS1619 and antagonist paxilline modified these effects, and KHE inhibitor quinine and K⁺ ionophore valinomycin depolarized ΔΨ_m. We postulate that K⁺ efflux-induced H⁺ influx via KHE causes an inward H⁺ leak that stimulates respiration, but at buffer pH 6.9 also utilizes the energy of ΔpH_m, the smaller component of the overall proton motive force, ΔμH⁺. Thus ΔpH_m establishes and maintains the ΔΨ_m required for utilization of substrates, entry of all cations, and for oxidative phosphorylation. Thus, K⁺ influx/efflux appears to play a pivotal role in regulating energetics while maintaining mitochondrial ionic balance and volume homeostasis.     

Enhanced charge-independent mitochondrial free Ca2 + and attenuated ADP-induced NADH oxidation by isoflurane: Implications for cardioprotection

Modulation of mitochondrial free Ca^2 + ([Ca^2 +]_m) is implicated as one of the possible upstream factors that initiates anesthetic-mediated cardioprotection against ischemia–reperfusion (IR) injury. To unravel possible mechanisms by which volatile anesthetics modulate [Ca^2 +]_m and mitochondrial bioenergetics, with implications for cardioprotection, experiments were conducted to spectrofluorometrically measure concentration-dependent effects of isoflurane (0.5, 1, 1.5, 2 mM) on the magnitudes and time-courses of [Ca^2 +]_m and mitochondrial redox state (NADH), membrane potential (ΔΨ_m), respiration, and matrix volume. Isolated mitochondria from rat hearts were energized with 10 mM Na⁺- or K⁺-pyruvate/malate (NaPM or KPM) or Na⁺-succinate (NaSuc) followed by additions of isoflurane, 0.5 mM CaCl₂ (≈ 200 nM free Ca^2 + with 1 mM EGTA buffer), and 250 μM ADP. Isoflurane stepwise: (a) increased [Ca^2 +]_m in state 2 with NaPM, but not with KPM substrate, despite an isoflurane-induced slight fall in ΔΨ_m and a mild matrix expansion, and (b) decreased NADH oxidation, respiration, ΔΨ_m, and matrix volume in state 3, while prolonging the duration of state 3 NADH oxidation, respiration, ΔΨ_m, and matrix contraction with PM substrates. These findings suggest that isoflurane's effects are mediated in part at the mitochondrial level: (1) to enhance the net rate of state 2 Ca^2 + uptake by inhibiting the Na⁺/Ca^2 + exchanger (NCE), independent of changes in ΔΨ_m and matrix volume, and (2) to decrease the rates of state 3 electron transfer and ADP phosphorylation by inhibiting complex I. These direct effects of isoflurane to increase [Ca^2 +]_m, while depressing NCE activity and oxidative phosphorylation, could underlie the mechanisms by which isoflurane provides cardioprotection against IR injury at the mitochondrial level.     

Short-Term Differentiation of Glioblastoma Stem Cells Induces Hypoxia Tolerance

Glioblastoma is the most common and malignant brain cancer. In spite of surgical removal, radiation and chemotherapy, this cancer recurs within short time and median survival after diagnosis is less than a year. Glioblastoma stem cells (GSCs) left in the brain after surgery is thought to explain the inevitable recurrence of the tumor. Although hypoxia is a prime factor contributing to treatment resistance in many cancers, its effect on GSC has been little studied. Especially how differentiation influences the tolerance to acute hypoxia in GSCs is not well explored. We cultured GSCs from three patient biopsies and exposed these and their differentiated (1- and 4-weeks) progeny to acute hypoxia while monitoring intracellular calcium and mitochondrial membrane potential (ΔΨ_m). Undifferentiated GSCs were not hypoxia tolerant, showing both calcium overload and mitochondrial depolarization. One week differentiated cells were the most tolerant to hypoxia, preserving intracellular calcium stability and ΔΨ_m during 15 min of acute hypoxia. After 4 weeks of differentiation, mitochondrial mass was significantly reduced. In these cells calcium homeostasis was maintained during hypoxia, although the mitochondria were depolarized, suggesting a reduced mitochondrial dependency. Basal metabolic rate increased by differentiation, however, low oxygen consumption and high ΔΨ_m in undifferentiated GSCs did not provide hypoxia tolerance. The results suggest that undifferentiated GSCs are oxygen dependent, and that limited differentiation induces relative hypoxia tolerance. Hypoxia tolerance may be a factor involved in high-grade malignancy. This warrants a careful approach to differentiation as a glioblastoma treatment strategy.     

Redox regulation of ER and mitochondrial Ca2+ signaling in cell survival and death

Physiological signaling by reactive oxygen species (ROS) and their pathophysiological role in cell death are well recognized. This review focuses on two ROS targets that are key to local Ca²⁺ signaling at the ER/mitochondrial interface – notably, inositol trisphosphate receptors (IP₃Rs) and the mitochondrial calcium uniporter (MCU). Both transport systems are central to molecular mechanisms in cell survival and death. Methods for the measurement of the redox state of these proteins and for the detection of ROS nanodomains are described. Recent results on the redox regulation of these proteins are reviewed.     

Decrease in mitochondrial energy coupling by thyroid hormones: a physiological effect rather than a pathological hyperthyroidism consequence

The effect of the in vivo thyroid status on mitochondrial membrane potential (ΔΨ_m) in isolated rat hepatocytes was studies by means of a cytofluorimetric technique and the ΔΨ_m-specific probe JC-1. It is shown that the ΔΨ_m level decreases in the order hypothyroid>euthyroid>hyperthyroid. Polarographic measurement of the hepatocyte respiratory rates revealed an opposite trend of values: the highest respiratory rate in hepatocytes from hyperthyroid animals, the lowest in those from hypothyroid ones. This means that mitochondrial energy coupling is highest in hypothyroid hepatocytes and lowest in hyperthyroid hepatocytes. 6-Ketocholestanol added to hepatocytes failed to counterbalance the uncoupling effect of thyroid hormones on ΔΨ_m and respiration rate. Under the same conditions, 6-ketocholestanol appeared to be effective in recoupling of respiration uncoupled by low concentrations of the artificial protonophore FCCP. The mechanism and possible physiological functions of the thyroid hormone-induced decrease in mitochondrial energy coupling are discussed.     

MitoWave: Spatiotemporal analysis of mitochondrial membrane potential fluctuations during I/R

Mitochondria exhibit unstable inner membrane potentials (ΔΨ_m) when subjected to stress, such as during ischemia/reperfusion (I/R). Understanding the mechanism of ΔΨ_m instability involves characterizing and quantifying this phenomenon in an unbiased and reproducible manner. Here, we describe a simple analytical workflow called “MitoWave” that combines wavelet transform methods and image segmentation to unravel dynamic ΔΨ_m changes in the cardiac mitochondrial network during I/R. In vitro ischemia was affected by placing a glass coverslip on a monolayer of neonatal mouse ventricular myocytes for 1 h and removing the coverslip to allow for reperfusion, revealing complex oscillatory ΔΨ_m. MitoWave analysis was then used to identify individual mitochondrial clusters within the cells and track their intrinsic oscillation frequencies over the course of reperfusion. Responses segregated into five typical behaviors were quantified by MitoWave that were corroborated by visual inspection of the time series. Statistical analysis of the distribution of oscillating mitochondrial clusters during reperfusion showed significant differences between the five different outcomes. Features such as the time point of ΔΨ_m depolarization during I/R, area of mitochondrial clusters, and time-resolved frequency components during reperfusion were determined per cell and per mitochondrial cluster. Mitochondria from neonatal mouse ventricular myocytes subjected to I/R oscillate in the frequency range of 8.6–45 mHz, with a mean of 8.73 ± 4.35 mHz. Oscillating clusters had smaller areas ranging from 49.8 ± 1.2 μm², whereas nonoscillating clusters had larger areas 66 ± 1.5 μm². A negative correlation between frequency and mitochondrial cluster area was observed. We also observed that late ΔΨ_m loss during ischemia correlated with early ΔΨ_m stabilization after oscillation on reperfusion. Thus, MitoWave analysis provides a semiautomated method to quantify complex time-resolved mitochondrial behavior in an easy-to-follow workflow, enabling unbiased, reproducible quantitation of complex nonstationary cellular phenomena.     

The role of mitochondrial membrane potential in ischemic heart failure

The molecular events occurring during myocardial infarction and cardioprotection are described with an emphasis on the changes of the mitochondrial membrane potential (ΔΨ_m). The low ΔΨ_m values of the normal beating heart (100–140 mV) are explained by the allosteric ATP-inhibition of cytochrome c oxidase (CcO) through feedback inhibition by ATP at high [ATP]/[ADP] ratios. During ischemia the mechanism is reversibly switched off by signaling through reactive oxygen species (ROS). At reperfusion high ΔΨ_m values cause a burst of ROS production leading to apoptosis and/or necrosis. Ischemic preconditioning is suggested to cause additional phosphorylation of CcO, protecting the enzyme from immediate dephosphorylation via ROS signaling.     

PPAR‐β facilitating maturation of hepatic‐like tissue derived from mouse embryonic stem cells accompanied by mitochondriogenesis and membrane potential retention

Relatively little is known about mitochondria metabolism in differentiating embryonic stem (ES) cells. Present research focused on several elements of cellular energy metabolism in hepatic‐like tissue derived from mouse ES cells. We demonstrated that mitochondrial location patterns and mitochondrial membrane potential (ΔΨ_m) existed in subsequent differentiation of the tissue. Mitochondriogenesis appeared at the early stage and kept a normal ΔΨ_m in differentiated mature hepatocytes. Peroxisome proliferator‐activated receptor‐α (PPAR‐α) expression was transitorily increased at the beginning, and kept a relatively low level later, which accompanied by expression of PPAR‐γ coactivator (PGC)‐1α, a master regulator of mitochondrial biogenesis. PPAR‐β expression showed robust up‐regulation in the late differentiation course. Enhanced co‐expressions of PPAR‐β and albumin with catalysis of UDP‐glucuronosyltransferases (UGTs) were observed at mature stage. While PPAR‐γ expression changed little before and after differentiation. Mitochondriogenesis could be accelerated by PPAR‐α specific agonist WY14643 and abolished by its antagonist GW6471 at the early stage. Neither of them affected mitochondrial ΔΨ_m and albumin generation in the differentiated hepatocytes. Furthermore, maturation of hepatic‐like tissue and mitochondriogenesis in hepatocyte could be efficiently stimulated by PPAR‐β specific agonist L165041 and abolished by PPAR‐β specific antagonist GSK0660, but not affected by PPAR‐γ specific agonist GW1929. In conclusion, the derived hepatic tissue morphologically possessed cellular energy metabolism features. PPAR‐α seemed only necessary for early mitochondriogenesis, while less important for ΔΨ_m retention in the mature tissue derived. The stimulation of PPAR‐β but not ‐γ enhanced hepatogenesis, hepatocytes maturation, and mitochondriogenesis. PPAR‐β took an important role in cellular energy metabolism of hepatogenesis. J. Cell. Biochem. 109: 498–508, 2010. © 2009 Wiley‐Liss, Inc.     

Altered Spatiotemporal Dynamics of the Mitochondrial Membrane Potential in the Hypertrophied Heart

Chronically elevated levels of oxidative stress resulting from increased production and/or impaired scavenging of reactive oxygen species are a hallmark of mitochondrial dysfunction in left ventricular hypertrophy. Recently, oscillations of the mitochondrial membrane potential (ΔΨ_m) were mechanistically linked to changes in cellular excitability under conditions of acute oxidative stress produced by laser-induced photooxidation of cardiac myocytes in vitro. Here, we investigate the spatiotemporal dynamics of ΔΨ_m within the intact heart during ischemia-reperfusion injury. We hypothesize that altered metabolic properties in left ventricular hypertrophy modulate ΔΨ_m spatiotemporal properties and arrhythmia propensity.     

Modelling biochemical features of mitochondrial neuropathology

Background

The neuropathology of mitochondrial disease is well characterised. However, pathophysiological mechanisms at the level of biochemistry and cell biology are less clear. Progress in this area has been hampered by the limited accessibility of neurologically relevant material for analysis.

Scope of review

Here we discuss the recent development of a variety of model systems that have greatly extended our capacity to understand the biochemical features associated with mitochondrial neuropathology. These include animal and cell based models, with mutations in both nuclear and mitochondrial DNA encoded genes, which aim to recapitulate the neuropathology and cellular biochemistry of mitochondrial diseases.

Major conclusions

Analysis of neurological tissue and cells from these models suggests that although there is no unifying mode of pathogenesis, dysfunction of the oxidative phosphorylation (OXPHOS) system is often central. This can be associated with altered reactive oxygen species (ROS) generation, disruption of the mitochondrial membrane potential (ΔΨ_m) and inadequate ATP synthesis. Thus, other cellular processes such as calcium (Ca^2 +) homeostasis, cellular signaling and mitochondrial morphology could be altered, ultimately compromising viability of neuronal cells.

General significance

Mechanisms of neuronal dysfunction in mitochondrial disease are only just beginning to be characterised, are system dependent and complex, and not merely driven by energy deficiency. The diversity of pathogenic mechanisms emphasises the need for characterisation in a wide range of models, as different therapeutic strategies are likely to be needed for different diseases.This article is part of a Special Issue entitled Frontiers of Mitochondrial Research.     

Calcium and ATP handling in human NADH:Ubiquinone oxidoreductase deficiency

Proper cell functioning requires precise coordination between mitochondrial ATP production and local energy demand. Ionic calcium (Ca²⁺) plays a central role in this coupling because it activates mitochondrial oxidative phosphorylation (OXPHOS) during hormonal and electrical cell stimulation. To determine how mitochondrial dysfunction affects cytosolic and mitochondrial Ca²⁺/ATP handling, we performed life-cell quantification of these parameters in fibroblast cell lines derived from healthy subjects and patients with isolated deficiency of the first OXPHOS complex (CI). In resting patient cells, CI deficiency was associated with a normal mitochondrial ([ATP]_m) and cytosolic ([ATP]_c) ATP concentration, a normal cytosolic Ca²⁺ concentration ([Ca²⁺]_c), but a reduced Ca²⁺ content of the endoplasmic reticulum (ER). Furthermore, cellular NAD(P)H levels were increased, mitochondrial membrane potential was slightly depolarized, reactive oxygen species (ROS) levels were elevated and mitochondrial shape was altered. Upon stimulation with bradykinin (Bk), the peak increases in [Ca²⁺]_c, mitochondrial Ca²⁺ concentration ([Ca²⁺]_m), [ATP]_c and [ATP]_m were reduced in patient cells. In agreement with these results, ATP-dependent Ca²⁺ removal from the cytosol was slower. Here, we review the interconnection between cytosolic, endoplasmic reticular and mitochondrial Ca²⁺ and ATP handling, and summarize our findings in patient fibroblasts in an integrative model.     

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 (ΔΨ_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 ΔΨ_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 Ca²⁺ 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.     

Effects of Tl<Superscript>+</Superscript> on ion permeability,membrane potential and respiration of isolated rat liver mitochondria

It is known that permeability of the inner mitochondrial membrane is low to most univalent cations (K⁺, Na⁺, H⁺) but high to Tl⁺. Swelling, state 4, state 3, and 2,4-dinitrophenol (DNP)-stimulated respiration as well as the membrane potential (ΔΨ_mito) of rat liver mitochondria were studied in media containing 0–75 mM TlNO₃ either with 250 mM sucrose or with 125 mM nitrate salts of other monovalent cations (KNO₃, or NaNO₃, or NH₄NO₃). Tl⁺ increased permeability of the inner mitochondrial membrane to K⁺, Na⁺, and H⁺, that was manifested as stimulation of the swelling of nonenergized and energized mitochondria as well as via an increase of state 4 and dissipation of ΔΨ_mito. These effects of Tl⁺ increased in the order of sucrose <K⁺ <Na⁺ ≤ NH₄⁺. They were stimulated by inorganic phosphate and decreased by ADP, Mg²⁺, and cyclosporine A. Contraction of energized mitochondria, swollen in the nitrate media, was markedly inhibited by quinine. It suggests participation of the mitochondrial K⁺/H⁺ exchanger in extruding of Tl⁺-induced excess of univalent cations from the mitochondrial matrix. It is discussed that Tl⁺ (like Cd²⁺ and other heavy metals) increases the ion permeability of the inner membrane of mitochondria regardless of their energization and stimulates the mitochondrial permeability transition pore in low conductance state. The observed decrease of state 3 and DNP-stimulated respiration in the nitrate media resulted from the mitochondrial swelling rather than from an inhibition of respiratory enzymes as is the case with the bivalent heavy metals.     

The role of thiol antioxidants in restoring mitochondrial functions modified by microbial metabolites

The effects of phenolic acids of microbial origin on mitochondrial functions and the possibility of removing their effects by thiol antioxidants dithiotreitol and N-acetylcysteine were studied. The action of some phenolic acids on the redox state of NADH, the membrane potential and calcium capacity of mitochondria is due to their interaction with thiol groups. Partial restoration of mitochondrial functions occurred in the presence of dithiotreitol and N-acetylcysteine, full recovery (short-term duration) was promoted by the combined action of dithiotreitol and menadione (vitamin K₃). It was found that the protective effect of thiol antioxidants became prooxidant if the medium contained free iron and compounds with quinone structure, capable of entering into a redox cycle with thiols. It is shown that the interaction of thiols with iron and menadione is accompanied by absorption of oxygen to form superoxide anion. Prooxidant effect of thiol antioxidants may explain the absence of the protective effect at the later stages of sepsis and systemic inflammatory syndrome.     

Mitochondrial Ca2+-activated K+ channels and their role in cell life and death pathways

Ca²⁺-activated K⁺ channels (K_Ca) are expressed at the plasma membrane and in cellular organelles. Expression of all K_Ca channel subtypes (BK, IK and SK) has been detected at the inner mitochondrial membrane of several cell types. Primary functions of these mitochondrial K_Ca channels include the regulation of mitochondrial ROS production, maintenance of the mitochondrial membrane potential and preservation of mitochondrial calcium homeostasis. These channels are therefore thought to contribute to cellular protection against oxidative stress through mitochondrial mechanisms of preconditioning. In this review, we summarize the current knowledge on mitochondrial KCa channels, and their role in mitochondrial function in relation to cell death and survival pathways. More specifically, we systematically discuss studies on the role of these mitochondrial K_Ca channels in pharmacological preconditioning, and according protective effects on ischemic insults to the brain and the heart.     

Mitochondria impairment correlates with increased sensitivity of aging RPE cells to oxidative stress

Impairment of mitochondria function and cellular antioxidant systems are linked to aging and neurodegenerative diseases. In the eye, the retinal pigment epithelium (RPE) is exposed to a highly oxidative environment that contributes to age-related visual dysfunction. Here, we examined changes in mitochondrial function in human RPE cells and sensitivity to oxidative stress with increased chronological age. Primary RPE cells from young (9–20)-, mid-age (48–60)-, and >60 (62–76)-year-old donors were grown to confluency and examined by electron microscopy and flow cytometry using several mitochondrial functional assessment tools. Susceptibility of RPE cells to H₂O₂ toxicity was determined by lactate dehydrogenase and cytochrome c release, as well as propidium iodide staining. Reactive oxygen species, cytoplasmic Ca²⁺ [Ca²⁺]_c, and mitochondrial Ca²⁺ [Ca²⁺]_m levels were measured using 2′,7′-dichlorodihydrofluorescein diacetate, fluo-3/AM, and Rhod-2/AM, respectively, adenosine triphosphate (ATP) levels were measured by a luciferin/luciferase-based assay and mitochondrial membrane potential (ΔΨm) estimated using 5,5′,6,6′-tetrachloro 1,1′3,3′-tetraethylbenzimid azolocarbocyanine iodide. Expression of mitochondrial and antioxidant genes was determined by real-time polymerase chain reaction. RPE cells show greater sensitivity to oxidative stress, reduction in expression of mitochondrial heat shock protein 70, uncoupling protein 2, and superoxide dismutase 3, and greater expression of superoxide dismutase 2 levels with increased chronological age. Changes in mitochondrial number, size, shape, matrix density, cristae architecture, and membrane integrity were more prominent in samples obtained from >60 years old compared to mid-age and younger donors. These mitochondria abnormalities correlated with lower ATP levels, reduced ΔΨm, decreased [Ca²⁺]_c, and increased sequestration of [Ca²⁺]_m in cells with advanced aging. Our study provides evidence for mitochondrial decay, bioenergetic deficiency, weakened antioxidant defenses, and increased sensitivity of RPE cells to oxidative stress with advanced aging. Our findings suggest that with increased severity of mitochondrial decay and oxidative stress, RPE function may be altered in some individuals in a way that makes the retina more susceptible to age-related injury.