Mitochondrial Oscillations and Waves in Cardiac Myocytes: Insights from Computational Models

Periodic cellwide depolarizations of mitochondrial membrane potential (Ψ_M) which are triggered by reactive oxygen species (ROS) and propagated by ROS-induced ROS release (RIRR) have been postulated to contribute to cardiac arrhythmogenesis and injury during ischemia/reperfusion. Two different modes of RIRR have been described: Ψ_M oscillations involving ROS-sensitive mitochondrial inner membrane anion channels (IMAC), and slow depolarization waves related to mitochondrial permeability transition pore (MPTP) opening. In this study, we developed a computational model of mitochondria exhibiting both IMAC-mediated RIRR and MPTP-mediated RIRR, diffusively coupled in a spatially extended network, to study the spatiotemporal dynamics of RIRR on Ψ_M. Our major findings are: 1), as the rate of ROS production increases, mitochondria can exhibit either oscillatory dynamics facilitated by IMAC opening, or bistable dynamics facilitated by MPTP opening; 2), in a diffusively-coupled mitochondrial network, the oscillatory dynamics of IMAC-mediated RIRR results in rapidly propagating (∼25 μm/s) cellwide Ψ_M oscillations, whereas the bistable dynamics of MPTP-mediated RIRR results in slow (0.1-2 μm/s) Ψ_M depolarization waves; and 3), the slow velocity of the MPTP-mediated depolarization wave is related to competition between ROS scavenging systems and ROS diffusion. Our observations provide mechanistic insights into the spatiotemporal dynamics underlying RIRR-induced Ψ_M oscillations and waves observed experimentally in cardiac myocytes.     

Mitochondrial Networks in Cardiac Myocytes Reveal Dynamic Coupling Behavior

Oscillatory behavior of mitochondrial inner membrane potential (ΔΨ_m) is commonly observed in cells subjected to oxidative or metabolic stress. In cardiac myocytes, the activation of inner membrane pores by reactive oxygen species (ROS) is a major factor mediating intermitochondrial coupling, and ROS-induced ROS release has been shown to underlie propagated waves of ΔΨ_m depolarization as well as synchronized limit cycle oscillations of ΔΨ_m in the network. The functional impact of ΔΨ_m instability on cardiac electrophysiology, Ca²⁺ handling, and even cell survival, is strongly affected by the extent of such intermitochondrial coupling. Here, we employ a recently developed wavelet-based analytical approach to examine how different substrates affect mitochondrial coupling in cardiac cells, and we also determine the oscillatory coupling properties of mitochondria in ventricular cells in intact perfused hearts. The results show that the frequency of ΔΨ_m oscillations varies inversely with the size of the oscillating mitochondrial cluster, and depends on the strength of local intermitochondrial coupling. Time-varying coupling constants could be quantitatively determined by applying a stochastic phase model based on extension of the well-known Kuramoto model for networks of coupled oscillators. Cluster size-frequency relationships varied with different substrates, as did mitochondrial coupling constants, which were significantly larger for glucose (7.78 × 10⁻² ± 0.98 × 10⁻² s⁻¹) and pyruvate (7.49 × 10⁻² ± 1.65 × 10⁻² s⁻¹) than lactate (4.83 × 10⁻² ± 1.25 × 10⁻² s⁻¹) or β-hydroxybutyrate (4.11 × 10⁻² ± 0.62 × 10⁻² s⁻¹). The findings indicate that mitochondrial spatiotemporal coupling and oscillatory behavior is influenced by substrate selection, perhaps through differing effects on ROS/redox balance. In particular, glucose-perfusion generates strong intermitochondrial coupling and temporal oscillatory stability. Pathological changes in specific catabolic pathways, which are known to occur during the progression of cardiovascular disease, could therefore contribute to altered sensitivity of the mitochondrial network to oxidative stress and emergent ΔΨ_m instability, ultimately scaling to produce organ level dysfunction.     

Dissipation of the Membrane Potential in Susceptible Corn Mitochondria by the Toxin of Helminthosporium maydis, Race T, and Toxin Analogs

We have tested directly the effect of Helminthosporium maydis T (Hmt) toxin and various analogs on the membrane potential formed in mitochondria isolated from a Texas (T) cytoplasmic male-sterile and a normal (N) corn. ATP, malate or succinate generated a membrane potential (negative inside) as monitored by the absorbance change of a cationic dye, safranine. The relative membrane potential (Δψ) could also be detected indirectly as ⁴⁵Ca²⁺ uptake. Hmt toxin added to T mitochondria dissipated the steady state Δψ similar to addition of a protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP). Toxin analogs (Cpd XIII: C₄₁H₆₈O₁₂ and Cpd IV: C₂₅H₄₄O₆), reduced native toxin (RT2C: C₄₁H₈₄O₁₃) and Pm toxin (band A: C₃₃H₆₀O₈, produced by the fungus, Phyllosticta maydis) were effective in dissipating Δψ and decreasing Ca²⁺ uptake with the following order: Pm (100) » HmT (23-30) > Cpd XIII (11-25) » RT2C (0-4−1.8) > Cpd IV (0.2−1.0). In contrast, the toxins and analogs had no effect on Δψ formed in N mitochondria. The striking similarities of the HmT toxin (band 1: C₄₁H₆₈O₁₃) and Cpd XIII on T mitochondrial activities provide strong evidence supporting the correctness of the polyketol structure assigned to the native toxin. Since the Δψ in energized mitochondria is caused mainly by the electrogenic extrusion of H⁺, the results support the idea that HmT toxin increases membrane permeability of T mitochondria to H⁺. The host specificity of the toxin suggests that an interaction with unique target site(s) on the inner mitochondrial membrane of T corn causes H⁺ leakage.     

Coordinated behavior of mitochondria in both space and time: a reactive oxygen species-activated wave of mitochondrial depolarization

Reactive oxygen species (ROS) can trigger a transient burst of mitochondrial ROS production via ROS activation of the mitochondrial permeability transition pore (MPTP), a phenomenon termed ROS-induced ROS release (RIRR). The goal of this study was to investigate if the generation of ROS in a discrete region of a cardiomyocyte could serve to propagate RIRR-mediated mitochondrial depolarizations throughout a cell. Our experiments revealed that localized RIRR activated either RIRR-mediated fluctuations in mitochondrial membrane potential (time period: 3-10 min) or a traveling wave of depolarization of the cell's mitochondria (velocity: approximately 5 microm/min). Both phenomena appeared to be mediated by the mitochondrial permeability transition pore and eventually encompassed the majority of the mitochondrial population of both isolated rat and rabbit cardiomyocytes. Furthermore, depolarization was often reversible; the waves of depolarization were then followed by a rapid (approximately 40 microm/min) repolarization wave of the mitochondria. We show that the RIRR can function to communicate the mitochondrial permeability transition from one mitochondrion to another in the isolated adult cardiomyocyte.     

Targeting Cancer Cells with Reactive Oxygen and Nitrogen Species Generated by Atmospheric-Pressure Air Plasma

The plasma jet has been proposed as a novel therapeutic method for cancer. Anticancer activity of plasma has been reported to involve mitochondrial dysfunction. However, what constituents generated by plasma is linked to this anticancer process and its mechanism of action remain unclear. Here, we report that the therapeutic effects of air plasma result from generation of reactive oxygen/nitrogen species (ROS/RNS) including H₂O₂, Ox, OH⁻, •O_2, NOx, leading to depolarization of mitochondrial membrane potential and mitochondrial ROS accumulation. Simultaneously, ROS/RNS activate c-Jun NH₂-terminal kinase (JNK) and p38 kinase. As a consequence, treatment with air plasma jets induces apoptotic death in human cervical cancer HeLa cells. Pretreatment of the cells with antioxidants, JNK and p38 inhibitors, or JNK and p38 siRNA abrogates the depolarization of mitochondrial membrane potential and impairs the air plasma-induced apoptotic cell death, suggesting that the ROS/RNS generated by plasma trigger signaling pathways involving JNK and p38 and promote mitochondrial perturbation, leading to apoptosis. Therefore, administration of air plasma may be a feasible strategy to eliminate cancer cells.     

Intracellular ASIC1a regulates mitochondrial permeability transition-dependent neuronal death

Acid-sensing ion channel 1a (ASIC1a) is the key proton receptor in nervous systems, mediating acidosis-induced neuronal injury in many neurological disorders, such as ischemic stroke. Up to now, functional ASIC1a has been found exclusively on the plasma membrane. Here, we show that ASIC1a proteins are also present in mitochondria of mouse cortical neurons where they are physically associated with adenine nucleotide translocase. Moreover, purified mitochondria from ASIC1a^−/− mice exhibit significantly enhanced Ca²⁺ retention capacity and accelerated Ca²⁺ uptake rate. When challenged with hydrogen peroxide (H₂O₂), ASIC1a^−/− neurons are resistant to cytochrome c release and inner mitochondrial membrane depolarization, suggesting an impairment of mitochondrial permeability transition (MPT) due to ASIC1a deletion. Consistently, H₂O₂-induced neuronal death, which is MPT dependent, is reduced in ASIC1a^−/− neurons. Additionally, significant increases in mitochondrial size and oxidative stress levels are detected in ASIC1a^−/− mouse brain, which also displays marked changes (>2-fold) in the expression of mitochondrial proteins closely related to reactive oxygen species signal pathways, as revealed by two-dimensional difference gel electrophoresis followed by mass spectrometry analysis. Our data suggest that mitochondrial ASIC1a may serve as an important regulator of MPT pores, which contributes to oxidative neuronal cell death.     

Accelerated Recovery of Mitochondrial Membrane Potential by GSK-3β Inactivation Affords Cardiomyocytes Protection from Oxidant-Induced Necrosis

Loss of mitochondrial membrane potential (ΔΨ_m) is known to be closely linked to cell death by various insults. However, whether acceleration of the ΔΨ_m recovery process prevents cell necrosis remains unclear. Here we examined the hypothesis that facilitated recovery of ΔΨ_m contributes to cytoprotection afforded by activation of the mitochondrial ATP-sensitive K⁺ (mK_ATP) channel or inactivation of glycogen synthase kinase-3β (GSK-3β). ΔΨ_m of H9c2 cells was determined by tetramethylrhodamine ethyl ester (TMRE) before or after 1-h exposure to antimycin A (AA), an inducer of reactive oxygen species (ROS) production at complex III. Opening of the mitochondrial permeability transition pore (mPTP) was determined by mitochondrial loading of calcein. AA reduced ΔΨ_m to 15±1% of the baseline and induced calcein leak from mitochondria. ΔΨ_m was recovered to 51±3% of the baseline and calcein-loadable mitochondria was 6±1% of the control at 1 h after washout of AA. mK_ATP channel openers improved the ΔΨ_m recovery and mitochondrial calcein to 73±2% and 30±7%, respectively, without change in ΔΨ_m during AA treatment. Activation of the mK_ATP channel induced inhibitory phosphorylation of GSK-3β and suppressed ROS production, LDH release and apoptosis after AA washout. Knockdown of GSK-3β and pharmacological inhibition of GSK-3β mimicked the effects of mK_ATP channel activation. ROS scavengers administered at the time of AA removal also improved recovery of ΔΨ_m. These results indicate that inactivation of GSK-3β directly or indirectly by mK_ATP channel activation facilitates recovery of ΔΨ_m by suppressing ROS production and mPTP opening, leading to cytoprotection from oxidant stress-induced cell death.     

KCNMA1 Encoded Cardiac BK Channels Afford Protection against Ischemia-Reperfusion Injury

Mitochondrial potassium channels have been implicated in myocardial protection mediated through pre-/postconditioning. Compounds that open the Ca²⁺- and voltage-activated potassium channel of big-conductance (BK) have a pre-conditioning-like effect on survival of cardiomyocytes after ischemia/reperfusion injury. Recently, mitochondrial BK channels (mitoBKs) in cardiomyocytes were implicated as infarct-limiting factors that derive directly from the KCNMA1 gene encoding for canonical BKs usually present at the plasma membrane of cells. However, some studies challenged these cardio-protective roles of mitoBKs. Herein, we present electrophysiological evidence for paxilline- and NS11021-sensitive BK-mediated currents of 190 pS conductance in mitoplasts from wild-type but not BK^−/− cardiomyocytes. Transmission electron microscopy of BK^−/− ventricular muscles fibres showed normal ultra-structures and matrix dimension, but oxidative phosphorylation capacities at normoxia and upon re-oxygenation after anoxia were significantly attenuated in BK^−/− permeabilized cardiomyocytes. In the absence of BK, post-anoxic reactive oxygen species (ROS) production from cardiomyocyte mitochondria was elevated indicating that mitoBK fine-tune the oxidative state at hypoxia and re-oxygenation. Because ROS and the capacity of the myocardium for oxidative metabolism are important determinants of cellular survival, we tested BK^−/− hearts for their response in an ex-vivo model of ischemia/reperfusion (I/R) injury. Infarct areas, coronary flow and heart rates were not different between wild-type and BK^−/− hearts upon I/R injury in the absence of ischemic pre-conditioning (IP), but differed upon IP. While the area of infarction comprised 28±3% of the area at risk in wild-type, it was increased to 58±5% in BK^−/− hearts suggesting that BK mediates the beneficial effects of IP. These findings suggest that cardiac BK channels are important for proper oxidative energy supply of cardiomyocytes at normoxia and upon re-oxygenation after prolonged anoxia and that IP might indeed favor survival of the myocardium upon I/R injury in a BK-dependent mode stemming from both mitochondrial post-anoxic ROS modulation and non-mitochondrial localizations.     

Identification of Francisella tularensis Live Vaccine Strain CuZn Superoxide Dismutase as Critical for Resistance to Extracellularly Generated Reactive Oxygen Species

Francisella tularensis is an intracellular pathogen whose survival is in part dependent on its ability to resist the microbicidal activity of host-generated reactive oxygen species (ROS) and reactive nitrogen species (RNS). In numerous bacterial pathogens, CuZn-containing superoxide dismutases (SodC) are important virulence factors, localizing to the periplasm to offer protection from host-derived superoxide radicals (O₂⁻). In the present study, mutants of F. tularensis live vaccine strain (LVS) deficient in superoxide dismutases (SODs) were used to examine their role in defense against ROS/RNS-mediated microbicidal activity of infected macrophages. An in-frame deletion F. tularensis mutant of sodC (ΔsodC) and a F. tularensis ΔsodC mutant with attenuated Fe-superoxide dismutase (sodB) gene expression (sodB ΔsodC) were constructed and evaluated for susceptibility to ROS and RNS in gamma interferon (IFN-γ)-activated macrophages and a mouse model of respiratory tularemia. The F. tularensis ΔsodC and sodB ΔsodC mutants showed attenuated intramacrophage survival in IFN-γ-activated macrophages compared to the wild-type F. tularensis LVS. Transcomplementing the sodC gene in the ΔsodC mutant or inhibiting the IFN-γ-dependent production of O₂⁻ or nitric oxide (NO) enhanced intramacrophage survival of the sod mutants. The ΔsodC and sodB ΔsodC mutants were also significantly attenuated for virulence in intranasally challenged C57BL/6 mice compared to the wild-type F. tularensis LVS. As observed for macrophages, the virulence of the ΔsodC mutant was restored in ifn-γ^−/−, inos^−/−, and phox^−/− mice, indicating that SodC is required for resisting host-generated ROS. To conclude, this study demonstrates that SodB and SodC act to confer protection against host-derived oxidants and contribute to intramacrophage survival and virulence of F. tularensis in mice.Francisella tularensis is considered a potential biological threat due to its extreme infectivity, ease of artificial dissemination via aerosols, and substantial capacity to cause illness and death. A hallmark of all F. tularensis subspecies is their ability to survive and replicate within macrophages (18) and other cell types (6, 11, 25, 28). While recent work has furthered our understanding of F. tularensis virulence mechanisms, little is known with respect to its ability to resist the microbicidal production of reactive oxygen species (ROS) or reactive nitrogen species (RNS).Superoxide dismutases (SODs) are metalloproteins that are classified according to their coordinating active site metals. SODs catalyze the dismutation of the highly reactive superoxide (O₂⁻) anion to hydrogen peroxide (H₂O₂) and O₂ (26). The dismutation of O₂⁻ prevents accumulation of microbicidal ROS and RNS in infected macrophages. Three major categories of SODs have been identified in bacteria and include Mn-, Fe-, and CuZn-containing SODs (SodA, SodB, and SodC, respectively) and are required for aerobic survival (27). The F. tularensis genome encodes SodB (FTL_1791) and SodC (FTL_0380). In several intracellular bacterial pathogens, SodC is an important virulence factor, and its localization to the periplasmic space protects bacteria from host-derived O₂⁻ and NO radicals (8, 9, 21, 32). Moreover, many virulent bacteria possess two copies of the sodC gene (4). The evolutionary maintenance of an extra sodC gene copy suggests that it serves some essential function in survival (4). As an intracellular pathogen, F. tularensis is exposed to ROS and RNS generated by inflammatory cells during the macrophage activation process, which suggests that SODs may play an important role in its intracellular survival and pathogenesis. We have demonstrated that decreases in SodB activity render F. tularensis sensitive to ROS and attenuate virulence in mice (2). However, the contribution of F. tularensis SodC in virulence and intramacrophage survival has not been defined. In this study we have constructed a F. tularensis sodC mutant (ΔsodC) and a F. tularensis sodBC double mutant (sodB ΔsodC) and determined that SodC in conjunction with SodB primarily protects the pathogen from host-derived ROS and is required for intramacrophage survival and virulence of F. tularensis in mice.     

Mitochondrial network determines intracellular ROS dynamics and sensitivity to oxidative stress through switching inter-mitochondrial messengers

Oxidative stresses caused by reactive oxygen species (ROS) can induce rapid depolarization of inner mitochondrial membrane potential and subsequent impairment of oxidative phosphorylation. Damaged mitochondria produce more ROS, especially the superoxide anion (O(2)(-)) and hydrogen peroxide (H(2)O(2)), which potentiate mitochondria-driven ROS propagation, so-called ROS-induced ROS release (RIRR), via activation of an inter-mitochondria signaling network. Therefore, loss of function in only a fraction of mitochondria might eventually affect cell viability through this positive feedback loop. Since ROS are very short-lived molecules in the biological milieu, mitochondrial network dynamics, such as density, number, and spatial distribution, can affect mitochondria-driven ROS propagation. To address this issue, we developed a mathematical model using an agent-based modeling approach, and tested the effect of mitochondrial network dynamics on RIRR for mitochondria under various conditions. Simulation results show that the intracellular ROS signaling pattern, such as ROS propagation speed and oxidative stress vulnerability, are critically affected by mitochondrial network dynamics. Mitochondrial network dynamics of mitochondrial distribution, density, activity, and size can mediate inter-mitochondrial signaling under certain conditions and determine the identity of the ROS signaling pattern. We further elucidated the potential mechanism of these actions, i.e., conversion of major messenger molecules involved in ROS signaling. If the average distance between neighboring mitochondria is large or mitochondrial distribution becomes randomized, messenger molecule of the ROS signaling network can be switched from O(2)(-) to H(2)O(2). In this case, mitochondria-driven ROS propagation is efficiently blocked by introduction of excess cytosolic glutathione peroxidase 1, while introduction of cytosolic superoxide dismutase has no effect. Together, these results suggest that mitochondrial network dynamics is a major determinant for cellular responses to RIRR through changing the key messenger molecules.     

Electrogenic transport of protons driven by the plasma membrane ATPase in membrane vesicles from radish : biochemical characterization

Mg:ATP-dependent H⁺ pumping has been studied in microsomal vesicles from 24-hour-old radish (Raphanus sativus L.) seedlings by monitoring both intravesicular acidification and the building up of an inside positive membrane potential difference (Δ ψ). ΔpH was measured as the decrease of absorbance of Acridine orange and Δ ψ as the shift of absorbance of bis(3-propyl-5-oxoisoxazol-4-yl)pentamethine oxonol. Both Mg:ATP-dependent Δ pH and Δ ψ generation are completely inhibited by vanadate and insensitive to oligomycin; moreover, Δ pH generation is not inhibited by NO₃⁻. These findings indicate that this membrane preparation is virtually devoid of mitochondrial and tonoplast H⁺-ATPases. Both intravesicular acidification and Δ ψ generation are influenced by anions: Δ pH increases and Δ ψ decreases following the sequence SO₄²⁻, Cl⁻, Br⁻, NO₃⁻. ATP-dependent H⁺ pumping strictly requires Mg²⁺. It is very specific for ATP (apparent K_m 0.76 millimolar) compared to GTP, UTP, CTP, ITP. Δ pH generation is inhibited by CuSO₄ and diethylstilbestrol as well as vanadate. Δ pH generation is specificially stimulated by K⁺ (+ 80%) and to a lesser extent by Na⁺ and choline (+28% and +14%, respectively). The characteristics of H⁺ pumping in these microsomal vesicles closely resemble those described for the plasma membrane ATPase partially purified from several plant materials.     

Membrane potential-related effect of calcium on reactive oxygen species generation in isolated brain mitochondria

The effect of Ca²⁺ applied in high concentrations (50 and 300 µM) was addressed on the generation of reactive oxygen species in isolated mitochondria from guinea-pig brain. The experiments were performed in the presence of ADP, a very effective inhibitor of mitochondrial permeability transition. Moderate increase in H₂O₂ release from mitochondria was induced by Ca²⁺ applied in 50 µM, but not in 300 µM concentration as measured with Amplex red fluorescent assay starting with a delay of 100-150 sec after exposure to Ca²⁺. Parallel measurements of membrane potential (ΔΨm) by safranine fluorescence showed a transient depolarization by Ca²⁺ followed by the recovery of ΔΨm to a value, which was more negative than that observed before addition of Ca²⁺ indicating a relative hyperpolarization. NAD(P)H fluorescence was also increased by Ca²⁺ given in 50 µM concentration. In mitochondria having high ΔΨm in the presence of oligomycin or ATP, the basal rate of release of H₂O₂ was significantly higher than that observed in a medium containing ADP and Ca²⁺ no longer increased but rather decreased the rate of H₂O₂ release. With 300 µM Ca²⁺ only a loss but no tendency of a recovery of ΔΨm was detected and H₂O₂ release was unchanged. It is suggested that in the presence of nucleotides the effect of Ca²⁺ on mitochondrial ROS release is related to changes in ΔΨm; in depolarized mitochondria, in the presence of ADP, moderate increase in H₂O₂ release is induced by calcium, but only in ≤ 100 µM concentration, when after a transient Ca²⁺-induced depolarization mitochondria became more polarized. In highly polarized mitochondria, in the presence of ATP or oligomycin, where no hyperpolarization follows the Ca²⁺-induced depolarization, Ca²⁺ fails to stimulate mitochondrial ROS generation. These effects of calcium (≤ 300 µM) are unrelated to mitochondrial permeability transition.     

Calcium Movement in Cardiac Mitochondria

Existing theory suggests that mitochondria act as significant, dynamic buffers of cytosolic calcium ([Ca²⁺]_i) in heart. These buffers can remove up to one-third of the Ca²⁺ that enters the cytosol during the [Ca²⁺]_i transients that underlie contractions. However, few quantitative experiments have been presented to test this hypothesis. Here, we investigate the influence of Ca²⁺ movement across the inner mitochondrial membrane during both subcellular and global cellular cytosolic Ca²⁺ signals (i.e., Ca²⁺ sparks and [Ca²⁺]_i transients, respectively) in isolated rat cardiomyocytes. By rapidly turning off the mitochondria using depolarization of the inner mitochondrial membrane potential (ΔΨ_m), the role of the mitochondria in buffering cytosolic Ca²⁺ signals was investigated. We show here that rapid loss of ΔΨ_m leads to no significant changes in cytosolic Ca²⁺ signals. Second, we make direct measurements of mitochondrial [Ca²⁺] ([Ca²⁺]_m) using a mitochondrially targeted Ca²⁺ probe (MityCam) and these data suggest that [Ca²⁺]_m is near the [Ca²⁺]_i level (∼100 nM) under quiescent conditions. These two findings indicate that although the mitochondrial matrix is fully buffer-capable under quiescent conditions, it does not function as a significant dynamic buffer during physiological Ca²⁺ signaling. Finally, quantitative analysis using a computational model of mitochondrial Ca²⁺ cycling suggests that mitochondrial Ca²⁺ uptake would need to be at least ∼100-fold greater than the current estimates of Ca²⁺ influx for mitochondria to influence measurably cytosolic [Ca²⁺] signals under physiological conditions. Combined, these experiments and computational investigations show that mitochondrial Ca²⁺ uptake does not significantly alter cytosolic Ca²⁺ signals under normal conditions and indicates that mitochondria do not act as important dynamic buffers of [Ca²⁺]_i under physiological conditions in heart.     

Enhancement of hydrogen peroxide formation by protophores and ionophores in antimycin-supplemented mitochondria

Rat and pigeon heart mitochondria supplemented with antimycin produce 0.3–1.0nmol of H₂O₂/min per mg of protein. These rates are stimulated up to 13-fold by addition of protophores (carbonyl cyanide p-trifluoromethoxyphenylhydrazone, carbonyl cyanide m-chloromethoxyphenylhydrazone and pentachlorophenol). Ionophores, such as valinomycin and gramicidin, and Ca²⁺ also markedly stimulated H₂O₂ production by rat heart mitochondria. The enhancement of H₂O₂ generation in antimycin-supplemented mitochondria and the increased O₂ uptake of the State 4-to-State 3 transition showed similar protophore, ionophore and Ca²⁺ concentration dependencies. Thenoyltrifluoroacetone and N-bromosuccinimide, which inhibit succinate–ubiquinone reductase activity, also decreased mitochondrial H₂O₂ production. Addition of cyanide to antimycin-supplemented beef heart submitochondrial particles inhibited the generation of O₂⁻, the precursor of mitochondrial H₂O₂. This effect was parallel to the increase in cytochrome c reduction and it is interpreted as indicating the necessity of cytochrome c₁³⁺ to oxidize ubiquinol to ubisemiquinone, whose autoxidation yields O₂⁻. The effect of protophores, ionophores and Ca²⁺ is analysed in relation to the propositions of a cyclic mechanism for the interaction of ubiquinone with succinate dehydrogenase and cytochromes b and c₁ [Wikstrom & Berden (1972) Biochim. Biophys. Acta 283, 403–420; Mitchell (1976) J. Theor. Biol. 62, 337–367]. A collapse in membrane potential, increasing the rate of ubisemiquinone formation and O₂⁻ production, is proposed as the molecular mechanism for the enhancement of H₂O₂ formation rates observed on addition of protophores, ionophores and Ca²⁺.     

From mitochondrial dynamics to arrhythmias

The reactive oxygen species (ROS)-dependent mitochondrial oscillator described in cardiac cells exhibits at least two modes of function under physiological conditions or in response to metabolic and oxidative stress. Both modes depend upon network behavior of mitochondria. Under physiological conditions cardiac mitochondria behave as a network of coupled oscillators with a broad range of frequencies. ROS weakly couples mitochondria under normal conditions but becomes a strong coupling messenger when, under oxidative stress, the mitochondrial network attains criticality. Mitochondrial criticality is achieved when a threshold of ROS is overcome and a certain density of mitochondria forms a cluster that spans the whole cell. Under these conditions, the slightest perturbation triggers a cell-wide collapse of the mitochondrial membrane potential, Δψ_m, visualized as a depolarization wave throughout the cell which is followed by whole cell synchronized oscillations in Δψ_m, NADH, ROS, and GSH. This dynamic behavior scales from the mitochondrion to the cell by driving cellular excitability and the whole heart into catastrophic arrhythmias. A network collapse of Δψ_m under criticality leads to: (i) energetic failure, (ii) temporal and regional alterations in action potential (AP), (iii) development of zones of impaired conduction in the myocardium, and, ultimately, (iv) a fatal ventricular arrhythmia.     

Mitochondrial transmembrane potential is diminished in phorbol myristate acetate-stimulated peritoneal resident macrophages isolated from wild-type mice,but not in those from gp91-phox-deficient mice

Macrophages produce superoxide (O₂^–) during phagocytosis or upon stimulation with a variety of agents including phorbol myristate acetate (PMA) through the activation of NADPH oxidase, and the formed O₂^– is converted to other reactive oxygen species (ROS) such as hydrogen peroxide (H₂O₂). The aim of the present study was to elucidate the effect of the intracellularly produced ROS on mitochondrial transmembrane potential (MTP) in mouse (C57BL/6) peritoneal resident macrophages stimulated with PMA. Using a fluorescent dye, succinimidyl ester of dichlorodihydrofluorescein (H₂DCFDA), O₂^– was visualized in intracellular compartments in a certain subpopulation of macrophages isolated from wild-type mice. Cells deficient in gp91-phox, one of the membrane components of NADPH oxidase, were negative for the fluorescence. When cells were loaded with both H₂DCFDA and MitoCapture, a fluorescent dye for mitochondria, mitochondrial fluorescence was diminished in O₂^–-producing cells, but not in O₂^–-deficient cells. Flow cytometry also revealed the decrease of mitochondrial fluorescence in wild-type cells, but not in gp91-phox-deficient cells. The loss of mitochondrial fluorescence was prevented by microinjection of catalase into cells. The present findings demonstrate that MTP is diminished by ROS, including the H₂O₂ dismutated from O₂^–, produced intracellularly by activation of the NADPH oxidase in mouse peritoneal resident macrophages stimulated with PMA.     

Production of Superoxide Anions by Keratinocytes Initiates P. acnes-Induced Inflammation of the Skin

Acne vulgaris is a chronic inflammatory disorder of the sebaceous follicles. Propionibacterium acnes (P. acnes), a gram-positive anareobic bacterium, plays a critical role in the development of these inflammatory lesions. This study aimed at determining whether reactive oxygen species (ROS) are produced by keratinocytes upon P. acnes infection, dissecting the mechanism of this production, and investigating how this phenomenon integrates in the general inflammatory response induced by P. acnes. In our hands, ROS, and especially superoxide anions (O₂ ^•−), were rapidly produced by keratinocytes upon stimulation by P. acnes surface proteins. In P. acnes-stimulated keratinocytes, O₂ ^•− was produced by NAD(P)H oxidase through activation of the scavenger receptor CD36. O₂ ^•− was dismuted by superoxide dismutase to form hydrogen peroxide which was further detoxified into water by the GSH/GPx system. In addition, P. acnes-induced O₂ ^•− abrogated P. acnes growth and was involved in keratinocyte lysis through the combination of O₂ ^•− with nitric oxide to form peroxynitrites. Finally, retinoic acid derivates, the most efficient anti-acneic drugs, prevent O₂ ^•− production, IL-8 release and keratinocyte apoptosis, suggesting the relevance of this pathway in humans.     

Erythropoietin Protects Cardiomyocytes from Cell Death during Hypoxia/Reperfusion Injury through Activation of Survival Signaling Pathways

Hypoxia/Reoxygenation (H/R) cardiac injury is of great importance in understanding Myocardial Infarctions, which affect a major part of the working population causing debilitating side effects and often-premature mortality. H/R injury primarily consists of apoptotic and necrotic death of cardiomyocytes due to a compromise in the integrity of the mitochondrial membrane. Major factors associated in the deregulation of the membrane include fluctuating reactive oxygen species (ROS), deregulation of mitochondrial permeability transport pore (MPTP), uncontrolled calcium (Ca²⁺) fluxes, and abnormal caspase-3 activity. Erythropoietin (EPO) is strongly inferred to be cardioprotective and acts by inhibiting the above-mentioned processes. Surprisingly, the underlying mechanism of EPO''s action and H/R injury is yet to be fully investigated and elucidated. This study examined whether EPO maintains Ca²⁺ homeostasis and the mitochondrial membrane potential (ΔΨ_m) in cardiomyocytes when subjected to H/R injury and further explored the underlying mechanisms involved. H9C2 cells were exposed to different concentrations of EPO post-H/R, and 20 U/ml EPO was found to significantly increase cell viability by inhibiting the intracellular production of ROS and caspase-3 activity. The protective effect of EPO was abolished when H/R-induced H9C2 cells were treated with Wortmannin, an inhibitor of Akt, suggesting the mechanism of action through the activation Akt, a major survival pathway.     

Dysfunction of mitochondria Ca uptake in cystic fibrosis airway epithelial cells

In the genetic disease cystic fibrosis (CF), the most common mutation F508del promotes the endoplasmic reticulum (ER) retention of misfolded CF proteins. Furthermore, in homozygous F508del-CFTR airway epithelial cells, the histamine Ca²⁺ mobilization is abnormally increased. Because the uptake of Ca²⁺ by mitochondria during Ca²⁺ influx or Ca²⁺ release from ER stores may be crucial for maintaining a normal Ca²⁺ homeostasis, we compared the mitochondria morphology and distribution by transmission electron microscopy technique and the mitochondria membrane potential variation (ΔΨ_mit) using a fluorescent probe (TMRE) on human CF (CF-KM4) and non-CF (MM39) tracheal serous gland cell lines. Confocal imaging of Rhod-2–AM-loaded or of the mitochondrial targeted cameleon 4mtD3cpv-transfected human CF and non-CF cells, were used to examine the ability of mitochondria to sequester intracellular Ca²⁺. The present study reveals that (i) the mitochondria network is fragmented in F508del-CFTR cells, (ii) the ΔΨ_mit of CF mitochondria is depolarized compared non-CF mitochondria, and (iii) the CF mitochondria Ca²⁺ uptake is reduced compared non-CF cells. We propose that these defects in airway epithelial F508del-CFTR cells are the consequence of mitochondrial membrane depolarization leading to a deficient mitochondrial Ca²⁺ uptake.     

The uniqueness of the plant mitochondrial potassium channel

The ATP-inhibited Plant Mitochondrial K⁺ Channel (PmitoK_ATP) was discovered about fifteen years ago in Durum Wheat Mitochondria (DWM). PmitoKATP catalyses the electrophoretic K⁺ uniport through the inner mitochondrial membrane; moreover, the co-operation between PmitoK_ATP and ⁺/H⁺ antiporter allows such a great operation of a K⁺ cycle to collapse mitochondrial membrane potential (ΔΨ) and ΔpH, thus impairing protonmotive force (Δp). A possible physiological role of such ΔΨ control is the restriction of harmful reactive oxygen species (ROS) production under environmental/oxidative stress conditions. Interestingly, DWM lacking Δp were found to be nevertheless fully coupled and able to regularly accomplish ATP synthesis; this unexpected behaviour makes necessary to recast in some way the classical chemiosmotic model. In the whole, PmitoK_ATP may oppose to large scale ROS production by lowering ΔΨ under environmental/oxidative stress, but, when stress is moderate, this occurs without impairing ATP synthesis in a crucial moment for cell and mitochondrial bioenergetics. [BMB Reports 2013; 46(8): 391-397]