Site IIIQo Research Articles

Hypoxia decreases mitochondrial ROS production in cells

We re-examined the reported increase in mitochondrial ROS production during acute hypoxia in cells. Using the Amplex Ultrared/horseradish peroxidase assay we found a decrease, not increase, in hydrogen peroxide release from HEK293 cells under acute hypoxia, at times ranging from 1 min to 3 h. The rates of superoxide/hydrogen peroxide production from each of the three major sites (site IQ in complex I and site IIIQo in complex III in mitochondria, and NADH oxidases (NOX) in the cytosol) were decreased to the same extent by acute hypoxia, with no change in the cells’ ability to degrade added hydrogen peroxide. A similar decrease in ROS production under acute hypoxia was found using the diacetyldichlorofluorescein assay. Using a HIF1α reporter cell line we confirmed earlier observations that suppression of superoxide production by site IIIQo decreases HIF1α expression, and found similar effects of suppressing site IQ or NOX. We conclude that increased mitochondrial ROS do not drive the response of HIF1α to acute hypoxia, but suggest that cytosolic H2O2 derived from site IQ, site IIIQo and NOX in cells is necessary to permit HIF1α stabilization by other signals.

Electron Leak From the Mitochondrial Electron Transport Chain Complex I at Site IQ Is Crucial for Oxygen Sensing in Rabbit and Human Ductus Arteriosus.

Background As partial pressure of oxygen (pO2) rises with the first breath, the ductus arteriosus (DA) constricts, diverting blood flow to the pulmonary circulation. The DA's O2 sensor resides within smooth muscle cells. The DA smooth muscle cells' mitochondrial electron transport chain (ETC) produces reactive oxygen species (ROS) in proportion tooxygen tension, causing vasoconstriction by regulating redox-sensitive ion channels and enzymes. To identify which ETC complex contributes most to DA O2 sensing and determine whether ROS mediate O2 sensing independent of metabolism, we used electron leak suppressors, S1QEL (suppressor of site IQ electron leak) and S3QEL (suppressor of site IIIQo electron leak), which decrease ROS production by inhibiting electron leak from quinone sites IQ and IIIQo, respectively. Methods and Results The effects of S1QEL, S3QEL, and ETC inhibitors (rotenone and antimycin A) on DA tone, mitochondrial metabolism, O2-induced changes in intracellular calcium, and ROS were studied in rabbit DA rings, and human and rabbit DA smooth muscle cells. S1QEL's effects on DA patency were assessed in rabbit kits, using micro computed tomography. In DA rings, S1QEL, but not S3QEL, reversed O2-induced constriction (P=0.0034) without reducing phenylephrine-induced constriction. S1QEL did not inhibit mitochondrial metabolism or ETC-I activity. In human DA smooth muscle cells, S1QEL and rotenone inhibited O2-induced increases in intracellular calcium (P=0.02 and 0.001, respectively), a surrogate for DA constriction. S1QEL inhibited O2-induced ROS generation (P=0.02). In vivo, S1QEL prevented O2-induced DA closure (P<0.0001). Conclusions S1QEL, but not S3QEL, inhibited O2-induced rises in ROS and DA constriction ex vivo and in vivo. DA O2 sensing relies on pO2-dependent changes in electron leak at site IQ in ETC-I, independent of metabolism. S1QEL offers a therapeutic means to maintain DA patency.

Effects of sugars, fatty acids and amino acids on cytosolic and mitochondrial hydrogen peroxide release from liver cells

The rates of formation of superoxide and hydrogen peroxide at different electron-donating sites in isolated mitochondria are critically dependent on the substrates that are added, through their effects on the reduction level of each site and the components of the protonmotive force. However, in intact cells the acute effects of added substrates on different sites of cytosolic and mitochondrial hydrogen peroxide production are unclear. Here we tested the effects of substrate addition on cytosolic and mitochondrial hydrogen peroxide release from intact AML12 liver cells. In 30-min starved cells replete with endogenous substrates, addition of glucose, fructose, palmitate, alanine, leucine or glutamine had no effect on the rate or origin of cellular hydrogen peroxide release. However, following 150-min starvation of the cells to deplete endogenous glycogen (and other substrates), cellular hydrogen peroxide production, particularly from NADPH oxidases (NOXs), was decreased, GSH/GSSH ratio increased, and antioxidant gene expression was unchanged. Addition of glucose or glutamine (but not the other substrates) increased hydrogen peroxide release. There were similar relative increases from each of the three major sites of production: mitochondrial sites IQ and IIIQo, and cytosolic NOXs. Glucose supplementation also restored ATP production and mitochondrial NAD reduction level, suggesting that the increased rates of hydrogen peroxide release from the mitochondrial sites were driven by increases in the protonmotive force and the degree of reduction of the electron transport chain. Long-term (24 h) glucose or glutamine deprivation also diminished hydrogen peroxide release rate, ATP production rate and (for glucose deprivation) NAD reduction level. We conclude that the rates of superoxide and hydrogen peroxide production from mitochondrial sites in liver cells are insensitive to extra added substrates when endogenous substrates are not depleted, but these rates are decreased when endogenous substrates are lowered by 150 min of starvation, and can be enhanced by restoring glucose or glutamine supply through improvements in mitochondrial energetic state.

Copper modulates heart mitochondrial H2O2 emission differently during fatty acid and pyruvate oxidation

Although the preferred cardiac metabolic fuels are fatty acids, glucose metabolism also plays an important role. However, irrespective of substrate type, energy generation results in mitochondrial reactive oxygen species (ROS) formation. To determine if the preference of fat over carbohydrates predisposes cardiomyocytes to oxidant production, we measured total and site-specific H2O2 emission in heart mitochondria oxidizing palmitoylcarnitine or pyruvate during copper (Cu) exposure. H2O2 emission was higher during oxidation of palmitoylcarnitine compared with pyruvate. Moreover, the bulk of the H2O2 emitted during palmitoylcarnitine oxidation originated from the outer ubiquinone binding site of complex III (site IIIQo) and the flavin site of electron transfer flavoprotein (site EF). We found no evidence of ROS production from complex I ubiquinone-binding site (site IQ) by reverse electron transport during oxidation of palmitoylcarnitine. Pyruvate oxidation also drove H2O2 emission primarily from sites IIIQo; however, the flavin sites of pyruvate dehydrogenase (site PF) and complex II (site IIF) contributed substantially. The effect of Cu depended on substrate and redox site, with effects at sites OF and IIIQo being more pronounced in mitochondria oxidizing pyruvate compared with palmitoylcarnitine. Cu imposed a concentration-saturable effect at site PF but concentration-dependently stimulated H2O2 emission at site EF. The substrate-dependent differences in H2O2 emission and effects of Cu suggest that fuel type and points of entry of electrons into the mitochondrial electron transport system determine the mitochondrial ROS production rate. Importantly, knowledge of sites of mitochondrial ROS production is crucial to the understanding of cardiac dysfunction associated with impaired substrate metabolism.

Anoxia-reoxygenation modulates cadmium-induced liver mitochondrial reactive oxygen species emission during oxidation of glycerol 3-phosphate

Aquatic organisms are frequently exposed to multiple stressors including low dissolved oxygen (O2) and metals such as cadmium (Cd). Reduced O2 concentration and Cd exposure alter cellular function in part by impairing energy metabolism and dysregulating reactive oxygen species (ROS) homeostasis. However, little is known about the role of mitochondrial glycerol 3-phosphate dehydrogenase (mGPDH) in ROS homeostasis in fish and its response to environmental stress. In this study, mGPDH activity and the effects of anoxia-reoxygenation (A-RO) and Cd on ROS (as hydrogen peroxide, H2O2) emission in rainbow trout liver mitochondria during oxidation of glycerol 3-phosphate (G3P) were probed. Trout liver mitochondria exhibited low mGPDH activity that supported a low respiratory rate but substantial H2O2 emission rate. Cd evoked a low concentration stimulatory-high concentration inhibitory H2O2 emission pattern that was blunted by A-RO. At specific redox centers, Cd suppressed H2O2 emission from site IQ, but stimulated emission from sites IIIQo and GQ. In contrast, A-RO stimulated H2O2 emission from site IQ following 15 min exposure and augmented Cd-stimulated emission from site IIF after 30 min exposure but did not alter the rate of H2O2 emission from sites IIIQo and GQ. Additionally, Cd neither altered the activities of catalase, glutathione peroxidase, or thioredoxin reductase nor the concentrations of total glutathione, reduced glutathione, or oxidized glutathione. Overall, this study indicates that oxidation of G3P drives ROS production from mGPDH and complexes I, II and III, whereas Cd directly modulates redox sites but not antioxidant defense systems to alter mitochondrial H2O2 emission.

S3QELs protect against diet-induced intestinal barrier dysfunction.

The underlying causes of aging remain elusive, but may include decreased intestinal homeostasis followed by disruption of the intestinal barrier, which can be mimicked by nutrient‐rich diets. S3QELs are small‐molecule suppressors of site IIIQo electron leak; they suppress superoxide generation at complex III of the mitochondrial electron transport chain without inhibiting oxidative phosphorylation. Here we show that feeding different S3QELs to Drosophila on a high‐nutrient diet protects against greater intestinal permeability, greater enterocyte apoptotic cell number, and shorter median lifespan. Hif‐1α knockdown in enterocytes also protects, and blunts any further protection by S3QELs. Feeding S3QELs to mice on a high‐fat diet also protects against the diet‐induced increase in intestinal permeability. Our results demonstrate by inference of S3QEL use that superoxide produced by complex III in enterocytes contributes to diet‐induced intestinal barrier disruption in both flies and mice.

Anoxia-reoxygenation alters H2O2 efflux and sensitivity of redox centers to copper in heart mitochondria

Mitochondrial reactive oxygen species (ROS) have been implicated in organ damage caused by environmental stressors, prompting studies on the effect of oxygen deprivation and metal exposure on ROS metabolism. However, how anoxia and copper (Cu) jointly influence heart mitochondrial ROS metabolism is not understood. We used rainbow trout heart mitochondria to probe the effects of anoxia-reoxygenation and Cu on hydrogen peroxide (H2O2) emission during oxidation of palmitoylcarnitine (PC), succinate, or glutamate-malate. In addition, we examined the influence of anoxia-reoxygenation and Cu on site-specific H2O2 emission capacities and key antioxidant enzymes, glutathione peroxidase (GPx) and thioredoxin reductase (TrxR). Results showed that anoxia-reoxygenation suppressed H2O2 emission regardless of substrate type or duration of anoxia. Anoxia-reoxygenation reduced mitochondrial sensitivity to Cu during oxidation of succinate or glutamate-malate whereas high Cu concentration additively stimulated H2O2 emission in mitochondria oxidizing PC. Prolonged anoxia-reoxygenation stimulated H2O2 emission from sites OF and IF, inhibited emission from sites IQ, IIF and IIIQo, and disparately altered the sensitivity of the sites to Cu. Interestingly, anoxia-reoxygenation increased GPx and TrxR activities, more prominently when reoxygenation followed a short duration of anoxia. Cu did not alter GPx but reduced TrxR activity in normoxic and anoxic-reoxygenated mitochondria. Overall, our study revealed potential mechanisms that may reduce oxidative damage associated with anoxia-reoxygenation and Cu exposure in heart mitochondria. The increased and decreased H2O2 emission from NADH/NAD+ and QH2/Q isopotential sites, respectively, may represent a balance between H2O2 required for oxygen deprivation-induced signaling and prevention of ROS burst associated with anoxia-reoxygenation.

Superoxide produced by mitochondrial complex III plays a pivotal role in the execution of ferroptosis induced by cysteine starvation

Ferroptosis is a type of iron-dependent, non-apoptotic cell death, which is typically induced by cysteine starvation or by the inhibition of glutathione peroxidase 4 (GPX4) activity with the accompanying elevation in lipid peroxidation product levels. Despite the central role of mitochondria in oxidative metabolism and hence, as main sources of superoxide, the issue of whether mitochondrial superoxide participates in the execution of ferroptosis remains unclear. To gain additional insights into this issue, we employed suppressors of the site IQ electron leak (S1QEL) and suppressors of the site IIIQo electron leak (S3QEL), small molecules that suppress mitochondrial superoxide production from complex I and III, respectively. The findings indicate that S3QEL, but not S1QEL, significantly protected mouse hepatoma Hepa 1–6 cells from lipid peroxidation and the subsequent ferroptosis induced by cysteine (Cys) starvation (cystine deprivation from culture media or xCT inhibition by erastin). The intracellular levels of Cys and GSH remained low irrespective of life or death. Moreover, S3QEL also suppressed ferroptosis in xCT-knockout mouse-derived embryonic fibroblasts, which usually die under conventional cultivating conditions due to the absence of intracellular Cys and GSH. Although it has been reported that erastin induces the hyperpolarization of the mitochondrial membrane potential, no correlation was observed between hyperpolarization and cell death in xCT-knockout cells. Collectively, these results indicate that superoxide production from complex III plays a pivotal role in the ferroptosis that is induced by Cys starvation, suggesting that protecting mitochondria is a promising therapeutic strategy for the treatment of multiple diseases featuring ferroptosis.

Superoxide produced by mitochondrial site IQ inactivates cardiac succinate dehydrogenase and induces hepatic steatosis in Sod2 knockout mice

Superoxide produced by mitochondria has been implicated in numerous physiologies and pathologies. Eleven different mitochondrial sites that can produce superoxide and/or hydrogen peroxide (O2.-/H2O2) have been identified in vitro, but little is known about their contributions in vivo. We introduce novel variants of S1QELs and S3QELs (small molecules that suppress O2.-/H2O2 production specifically from mitochondrial sites IQ and IIIQo, respectively, without compromising bioenergetics), that are suitable for use in vivo. When administered by intraperitoneal injection, they achieve total tissue concentrations exceeding those that are effective in vitro. We use them to study the engagement of sites IQ and IIIQo in mice lacking functional manganese-superoxide dismutase (SOD2). Lack of SOD2 is expected to elevate superoxide levels in the mitochondrial matrix, and leads to severe pathologies and death about 8 days after birth. Compared to littermate wild-type mice, 6-day-old Sod2−/− mice had significantly lower body weight, lower heart succinate dehydrogenase activity, and greater hepatic lipid accumulation. These pathologies were ameliorated by treatment with a SOD/catalase mimetic, EUK189, confirming previous observations. A 3-day treatment with S1QEL352 decreased the inactivation of cardiac succinate dehydrogenase and hepatic steatosis in Sod2−/− mice. S1QEL712, which has a distinct chemical structure, also decreased hepatic steatosis, confirming that O2.- derived specifically from mitochondrial site IQ is a significant driver of hepatic steatosis in Sod2−/− mice. These findings also demonstrate the ability of these new S1QELs to suppress O2.- production in the mitochondrial matrix in vivo. In contrast, suppressing site IIIQo using S3QEL941 did not protect, suggesting that site IIIQo does not contribute significantly to mitochondrial O2.- production in the hearts or livers of Sod2−/− mice. We conclude that the novel S1QELs are effective in vivo, and that site IQ runs in vivo and is a significant driver of pathology in Sod2−/− mice.

Production of superoxide and hydrogen peroxide in the mitochondrial matrix is dominated by site IQ of complex I in diverse cell lines

Understanding how mitochondria contribute to cellular oxidative stress and drive signaling and disease is critical, but quantitative assessment is difficult. Our previous studies of cultured C2C12 cells used inhibitors of specific sites of superoxide and hydrogen peroxide production to show that mitochondria generate about half of the hydrogen peroxide released by the cells, and site IQ of respiratory complex I produces up to two thirds of the superoxide and hydrogen peroxide generated in the mitochondrial matrix. Here, we used the same approach to measure the engagement of these sites in seven diverse cell lines to determine whether this pattern is specific to C2C12 cells, or more general. These diverse cell lines covered primary, immortalized, and cancerous cells, from seven tissues (liver, cervix, lung, skin, neuron, heart, bone) of three species (human, rat, mouse). The rate of appearance of hydrogen peroxide in the extracellular medium spanned a 30-fold range from HeLa cancer cells (3 pmol/min/mg protein) to AML12 liver cells (84 pmol/min/mg protein). The mean contribution of identified mitochondrial sites to this extracellular hydrogen peroxide signal was 30 ± 7% SD; the mean contribution of NADPH oxidases was 60 ± 14%. The relative contributions of different sites in the mitochondrial electron transport chain were broadly similar in all seven cell types (and similar to published results for C2C12 cells). 70 ± 4% of identified superoxide/hydrogen peroxide generation in the mitochondrial matrix was from site IQ; 30 ± 4% was from site IIIQo. We conclude that although absolute rates vary considerably, the relative contributions of different sources of hydrogen peroxide production are similar in nine diverse cell types under unstressed conditions in vitro. Identified mitochondrial sites account for one third of total cellular hydrogen peroxide production (half each from sites IQ and IIIQo); in the mitochondrial matrix the majority (two thirds) of superoxide/hydrogen peroxide is from site IQ.

Effects of copper and temperature on heart mitochondrial hydrogen peroxide production

High energy demand for continuous mechanical work and large number of mitochondria predispose the heart to excessive reactive oxygen species (ROS) production that may precipitate oxidative stress and heart failure. While mitochondria have been proposed as a unifying cellular target and driver of adverse effects induced by diverse stressful states, there is limited understanding of how heart mitochondrial ROS homeostasis is affected by combinations of stress factors. Thus, we probed the effect of copper (Cu) and thermal stress on ROS (as hydrogen peroxide, H2O2) emission and elucidated the effects of Cu on ROS production sites in rainbow trout heart mitochondria using the Amplex UltraRed-horseradish peroxidase detection system optimized for our model. Mitochondria oxidizing malate-glutamate or succinate were incubated at 4, 11 (control) and 23 °C and exposed to a range (1–100 μM) of Cu concentrations. We found that the rates and patterns of H2O2 emission depended on substrate type, Cu concentration and temperature. In mitochondria oxidizing malate-glutamate, Cu increased the rate of H2O2 emission with a spike at 1 μM while temperature had no effect. In contrast, both temperature and Cu increased the rate of H2O2 emission in mitochondria oxidizing succinate with a prominent spike at 25 μM Cu. The rates of H2O2 emission at the three temperatures during the spike imposed by 25 μM Cu were of the order 11 > 23 > 4 °C. Interestingly, 5 μM Cu supressed H2O2 emission in mitochondria oxidizing succinate or malate-glutamate suggesting a common mechanism of action independent of substrate type. In the absence of Cu, the site-specific capacities of H2O2 emission were: complex III outer ubiquinone binding site (site IIIQo) > complex II flavin site (site IIF) ≥ complex I flavin site (site IF) > complex I ubiquinone-binding site (site IQ). Rotenone marginally increased succinate-driven H2O2 emission suggesting either the absence of reverse electron transport (RET)-driven ROS production at site IQ or masking of the expected rotenone response (reduction) by H2O2 produced from other sites. Cu acted at multiple sites in the electron transport system resulting in different site-specific H2O2 emission responses depending on the concentration. Specifically, site IF H2O2 emission was suppressed by Cu concentration-dependently while H2O2 emission by site IIF was inhibited and stimulated by low and high concentrations of Cu, respectively. Additionally, emission from site IIIQo was stimulated by low and inhibited by high Cu concentrations. Overall, our study unveiled distinctive effects and sites of modulation of mitochondrial ROS production by Cu with implications for cardiac redox signaling networks and development of mitochondria-targeted Cu-based drugs.

JNK activation and translocation to mitochondria mediates mitochondrial dysfunction and cell death induced by VDAC opening and sorafenib in hepatocarcinoma cells

The multikinase inhibitor sorafenib, and opening of voltage dependent anion channels (VDAC) by the erastin-like compound X1 promotes oxidative stress and mitochondrial dysfunction in hepatocarcinoma cells. Here, we hypothesized that X1 and sorafenib induce mitochondrial dysfunction by increasing reactive oxygen species (ROS) formation and activating c-Jun N-terminal kinases (JNKs), leading to translocation of activated JNK to mitochondria. Both X1 and sorafenib increased production of ROS and activated JNK. X1 and sorafenib caused a drop in mitochondrial membrane potential (ΔΨ), a readout of mitochondrial metabolism, after 60 min. Mitochondrial depolarization after X1 and sorafenib occurred in parallel with JNK activation, increased superoxide (O2•−) production, decreased basal and oligomycin sensitive respiration, and decreased maximal respiratory capacity. Increased production of O2•− after X1 or sorafenib was abrogated by JNK inhibition and antioxidants. S3QEL 2, a specific inhibitor of site IIIQo, at Complex III, prevented depolarization induced by X1. JNK inhibition by JNK inhibitors VIII and SP600125 also prevented mitochondrial depolarization. After X1, activated JNK translocated to mitochondria as assessed by proximity ligation assays. Tat-Sab KIM1, a peptide selectively preventing the binding of JNK to the outer mitochondrial membrane protein Sab, blocked the depolarization induced by X1 and sorafenib. X1 promoted cell death mostly by necroptosis that was partially prevented by JNK inhibition. These results indicate that JNK activation and translocation to mitochondria is a common mechanism of mitochondrial dysfunction induced by both VDAC opening and sorafenib.

The use of site-specific suppressors to measure the relative contributions of different mitochondrial sites to skeletal muscle superoxide and hydrogen peroxide production

Reactive oxygen species are important signaling molecules crucial for muscle differentiation and adaptation to exercise. However, their uncontrolled generation is associated with an array of pathological conditions. To identify and quantify the sources of superoxide and hydrogen peroxide in skeletal muscle we used site-specific suppressors (S1QELs, S3QELs and NADPH oxidase inhibitors). We measured the rates of hydrogen peroxide release from isolated rat muscle mitochondria incubated in media mimicking the cytosol of intact muscle. By measuring the extent of inhibition caused by the addition of different site-specific suppressors of mitochondrial superoxide/hydrogen peroxide production (S1QELs for site IQ and S3QELs for site IIIQo), we determined the contributions of these sites to the total signal. In media mimicking resting muscle, their contributions were each 12–18%, consistent with a previous method. In C2C12 myoblasts, site IQ contributed 12% of cellular hydrogen peroxide production and site IIIQo contributed about 30%. When C2C12 myoblasts were differentiated to myotubes, hydrogen peroxide release increased five-fold, and the proportional contribution of site IQ doubled. The use of S1QELs and S3QELs is a powerful new way to measure the relative contributions of different mitochondrial sites to muscle hydrogen peroxide production under different conditions. Our results show that mitochondrial sites IQ and IIIQo make a substantial contribution to superoxide/hydrogen peroxide production in muscle mitochondria and C2C12 myoblasts. The total hydrogen peroxide release rate and the relative contribution of site IQ both increase substantially upon differentiation to myotubes.

P4607A specific complex I-induced ROS modulator, OP2113, is a new cardioproctective agent against acute myocardial infarction injuries during reperfusion

Abstract Introduction Hospital death rates due to myocardial infarction are still a concern and heart failure occurs in nearly a quarter of older patients who present with ST elevation myocardial infarction. Novel therapies are thus desperately needed to reduce infarct size to preserve left ventricular function and to prevent the onset of heart failure. Purpose OP2113, trithio-p-methoxyphenylpropene (anethole trithione, 5-(4-methoxyphenyl)-3H-1,2-dithiole-3-thione, anethole dithionethiol), a potential suppressor of complex I superoxide/hydrogen peroxide (ROS) production was evaluated in a sheep model of regional ischemia in order to demonstrate cardioprotective properties against infarct size and left ventricular function remodeling. Methods A series of experiments was performed in vitro on isolated mitochondria and on C2C12 cells to clarify the mechanism of action and to test whether OP2113 impacts biological variables of mitochondria. In vivo, anesthetized sheep underwent 60 min ischemia and 120 min reperfusion and either received OP2113 (1–5 μg/kg, i.v at start of and for the duration of the reperfusion) or the vehicle. The animals underwent then a 3-day follow-up period until euthanasia. Ischemic biomarkers, troponin I, CPK, and ventricular function through echocardiography were quantified during reperfusion and infarct size through histological analysis. Results OP2113 reduced ROS production from site IQ in mitochondria isolated from rat skeletal muscle, with IC50 of 26±1.4 μM (n=3). It is specific; IC50 values for other sites are at least 15-fold higher (site IIIQo – 414 μM; site IIF – 582 μM; sites IF/DH, PF, GQ &gt;5 mM). Furthermore, OP2113 did not affect oxidative phosphorylation, respiration or growth of C2C12 cells. In the sheep model, OP2113 (1–10 μg/kg) dose-dependently prevented reperfusion-induced ST segment increase during the first minutes. When OP2113 plasmatic concentration was superior to 1 ng/mL (generally associated with a threshold dose of 5 μg/kg), troponin Ic levels measured over the experiment was strongly decreased and the cardiac function significantly improved (+86%), when compared to the vehicle group (left panel). Finally, the infarct size was diminished by 57% comparing to the vehicle group (10.3% vs 23.8%, respectively, right panel). Conclusion In a large animal model of ischemia-reperfusion, OP2113 with very low infused dose, reduced ST segment elevation, reduced troponin release, improved left ejection fraction and reduced infarct size. Collectively, these results demonstrate that OP2113, through a new mechanism of action, is a potent cardioprotective compound.

Use of S1QELs and S3QELs to link mitochondrial sites of superoxide and hydrogen peroxide generation to physiological and pathological outcomes.

Changes in mitochondrial superoxide and hydrogen peroxide production may contribute to various pathologies, and even aging, given that over time and in certain conditions, they damage macromolecules and disrupt normal redox signalling. Mitochondria-targeted antioxidants such as mitoQ, mitoVitE, and mitoTEMPO have opened up the study of the importance of altered mitochondrial matrix superoxide/hydrogen peroxide in disease. However, the use of such tools has caveats and they are unable to distinguish precise sites of production within the reactions of substrate oxidation and the electron transport chain. S1QELs are specific small-molecule Suppressors of site IQElectron Leak and S3QELs are specific small-molecule Suppressors of site IIIQoElectron Leak; they prevent superoxide/hydrogen production at specific sites without affecting electron transport or oxidative phosphorylation. We discuss the benefits of using S1QELs and S3QELs as opposed to mitochondria-targeted antioxidants, mitochondrial poisons, and genetic manipulation. We summarise pathologies in which site IQ in mitochondrial complex I and site IIIQo in mitochondrial complex III have been implicated using S1QELs and S3QELs.

Effects of bioenergetics, temperature and cadmium on liver mitochondria reactive oxygen species production and consumption

A by-product of mitochondrial substrate oxidation and electron transfer to generate cellular energy (ATP) is reactive oxygen species (ROS). Superoxide anion radical and hydrogen peroxide (H2O2) are the proximal ROS produced by the mitochondria. Because low levels of ROS serve critical regulatory roles in cell physiology while excessive levels or inappropriately localized ROS result in aberrant physiological states, mitochondrial ROS need to be tightly regulated. While it is known that regulation of mitochondrial ROS involves balancing the rates of production and removal, the effects of stressors on these processes remain largely unknown. To illuminate how stressors modulate mitochondrial ROS homeostasis, we investigated the effects of temperature and cadmium (Cd) on H2O2 emission and consumption in rainbow trout liver mitochondria. We show that H2O2 emission rates increase with temperature and Cd exposure. Energizing mitochondria with malate-glutamate or succinate increased the rate of H2O2 emission; however, Cd exposure imposed different patterns of H2O2 emission depending on the concentration and substrate. Specifically, mitochondria respiring on malate-glutamate exhibited a saturable graded concentration-response curve that plateaued at 5 μM while mitochondria respiring on succinate had a biphasic concentration-response curve characterized by a spike in the emission rate at 1 μM Cd followed by gradual diminution at higher Cd concentrations. To explain the observed substrate- and concentration-dependent effects of Cd, we sequestered specific mitochondrial ROS-emitting sites using blockers of electron transfer and then tested the effect of the metal. The results indicate that the biphasic H2O2 emission response imposed by succinate is due to site IIF but is further modified at sites IQ and IIIQo. Moreover, the saturable graded H2O2 emission response in mitochondria energized with malate-glutamate is consistent with effect of Cd on site IF. Additionally, Cd and temperature acted cooperatively to increase mitochondrial H2O2 emission suggesting that increased toxicity of Cd at high temperature may be due to increased oxidative insult. Surprisingly, despite their clear stimulatory effect on H2O2 emission, Cd, temperature and bioenergetic status did not affect the kinetics of mitochondrial H2O2 consumption; the rate constants and half-lives for all the conditions tested were similar. Overall, our study indicates that the production processes of rainbow trout liver mitochondrial H2O2 metabolism are highly responsive to stressors and bioenergetics while the consumption processes are recalcitrant. The latter denotes the presence of a robust H2O2 scavenging system in liver mitochondria that would maintain H2O2 homeostasis in the face of increased production and reduced scavenging capacity.

Mitochondrial electron transport chain, ROS generation and uncoupling (Review).

The mammalian mitochondrial electron transport chain (ETC) includes complexes I-IV, as well as the electron transporters ubiquinone and cytochrome c. There are two electron transport pathways in the ETC: Complex I/III/IV, with NADH as the substrate and complex II/III/IV, with succinic acid as the substrate. The electron flow is coupled with the generation of a proton gradient across the inner membrane and the energy accumulated in the proton gradient is used by complex V (ATP synthase) to produce ATP. The first part of this review briefly introduces the structure and function of complexes I-IV and ATP synthase, including the specific electron transfer process in each complex. Some electrons are directly transferred to O2 to generate reactive oxygen species (ROS) in the ETC. The second part of this review discusses the sites of ROS generation in each ETC complex, including sites IF and IQ in complex I, site IIF in complex II and site IIIQo in complex III, and the physiological and pathological regulation of ROS. As signaling molecules, ROS play an important role in cell proliferation, hypoxia adaptation and cell fate determination, but excessive ROS can cause irreversible cell damage and even cell death. The occurrence and development of a number of diseases are closely related to ROS overproduction. Finally, proton leak and uncoupling proteins (UCPS) are discussed. Proton leak consists of basal proton leak and induced proton leak. Induced proton leak is precisely regulated and induced by UCPs. A total of five UCPs (UCP1-5) have been identified in mammalian cells. UCP1 mainly plays a role in the maintenance of body temperature in a cold environment through non-shivering thermogenesis. The core role of UCP2-5 is to reduce oxidative stress under certain conditions, therefore exerting cytoprotective effects. All diseases involving oxidative stress are associated with UCPs.

45 - Suppression of site IQ electron leak attenuates lung epithelial cell proliferation

Hyperoxia contributes to the development of acute lung injury in neonates and adults. Hyperoxia enhances cellular oxidative stress, yet mitochondrially derived reactive oxygen species (ROS) are poorly defined. Mitochondrial superoxide (O2•–) is produced as a consequence of electron leak during the reduction of molecular oxygen by oxidative phosphorylation. The primary sites electron leak include complex I (IQ) and III (IIIQo). Suppressors of site IQ electron leak (S1QEL) and site IIIQo electron leak (S3QEL) decrease O2•– production without affecting oxidative phosphorylation. These studies tested the hypothesis that S1QEL and S3QEL will attenuate the effects of hyperoxia on cell proliferation. Murine airway epithelial cells (mtCC) were cultured in room air (21% O2; RA) or hyperoxia (85% O2; HO) for 24 hours in the presence or absence of DMSO, 1 µM S1QEL or 1 µM S3QEL. Cell numbers were determined by Bio-Rad TC20 cell counter and cell viability assessed by trypan blue exclusion. Data (n=3) were analyzed by 2-way ANOVA followed by Tukey’s post-hoc analyses. Our data indicated independent effects of hyperoxia and S1QEL on total cell number. Hyperoxic exposure decreased cell numbers by 57% vs RA exposed cells (p=0.003). In RA exposed cells, S1QEL treatment decreased cell numbers by 52% (p=0.006). There were no differences between DMSO and S1QEL-treated HO-exposed groups. There was no effect of S3QEL on cell numbers. There were no independent effects of hyperoxia, S1QEL, or S3QEL on viable cell numbers. Hyperoxic exposure decreased mtCC numbers. Though neither compound prevented hyperoxia-induced decreases in mtCC numbers, S1QEL treatment decreased mtCC numbers but not viability in RA. Given the lack of a comparable effect of S3QEL, we interpret these novel data to indicate that complex I derived O2•- contributes to mtCC proliferation. This unique contribution of mitochondrially derived oxidants toward lung epithelial proliferation has not been reported.

Mitochondrial and cytosolic sources of hydrogen peroxide in resting C2C12 myoblasts

The relative contributions of different mitochondrial and cytosolic sources of superoxide and hydrogen peroxide in cells are not well established because of a lack of suitable quantitative assays. To address this problem using resting C2C12 myoblasts we measured the effects of specific inhibitors that do not affect other pathways on the rate of appearance of hydrogen peroxide in the extracellular medium. We used inhibitors of NADPH oxidases (NOXs), suppressors of site IQ electron leak (S1QELs) at mitochondrial Complex I, and suppressors of site IIIQo electron leak (S3QELs) at mitochondrial Complex III. Around 40% of net cellular hydrogen peroxide release was from NOXs and approximately 45% was from the two mitochondrial sites; 30% from site IIIQo and 15% from site IQ. As expected, decreasing cytosolic antioxidant capacity by lowering glutathione levels increased the absolute rates from all sites without changing their proportions, whereas decreasing antioxidant defenses in the mitochondrial matrix increased only the absolute and relative contributions of the two mitochondrial sites. These results show directly that mitochondria are a major contributor to cytosolic hydrogen peroxide in resting C2C12 myoblasts, and provide the first direct evidence of superoxide/hydrogen peroxide production from site IQ in unstressed cells.

Tunicamycin Exposure Triggers Apopotosis by Superoxide Formation from Site III Qo of Mitochondrial Complex III

Tunicamycin is a model inducer of endoplasmic reticulum stress and cell death. Its primary effect is inhibition of N-linked protein glycosylation, which leads to accumulation of misfolded proteins and the endoplasmic reticulum unfolded protein response. This then leads to alteration of mitochondrial function and ultimately to caspase activation and cell death. Recently, we reported the identification of small molecules that prevent superoxide formation at site IIIQo of mitochondrial Complex III without impairing oxidative phosphorylation (S3QELs, suppressors of site IIIQo electron leak), and that S3QELs decreased tunicamycin-induced caspase activation in INS-1 insulinoma cells (Orr et al. (2015) Nature Chem Biol 11, 834-836). Here we aimed to find whether this phenomenon also occurs in murine C2C12 myoblasts. C2C12 myoblasts, which are commonly used for analysis of bioenergetics and metabolism were exposed to 1 µM tunicamycin for 24 h with or without different chemical classes of S3QELs. Cells were then stained for activated caspases 3 and 7 (CellEvent), cell death (propidium iodide) and nuclei (Hoechst 33342). Exposure to tunicamycin led to apoptotic cell death in 10% and necrotic cell death in 5% of cells. Addition of 10 µM S3QEL2 or S3QEL3 decreased apoptosis by 30% (p