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 to oxygen 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.

Suppression of superoxide/hydrogen peroxide production at mitochondrial site IQ decreases fat accumulation, improves glucose tolerance and normalizes fasting insulin concentration in mice fed a high-fat diet.

We developed S1QEL1.719, a novel bioavailable S1QEL (suppressor of site IQ electron leak). S1QEL1.719 prevented superoxide/hydrogen peroxide production at site IQ of mitochondrial complex I in vitro. The free concentration giving half-maximal suppression (IC50) was 52 nM. Even at 50-fold higher concentrations S1QEL1.719 did not inhibit superoxide/hydrogen peroxide production from other sites. The IC50 for inhibition of complex I electron flow was 500-fold higher than the IC50 for suppression of superoxide/hydrogen peroxide production from site IQ. S1QEL1.719 was used to test the metabolic effects of suppressing superoxide/hydrogen peroxide production from site IQin vivo. C57BL/6J male mice fed a high-fat chow for one, two or eight weeks had increased body fat, decreased glucose tolerance, and increased fasting insulin concentrations, classic symptoms of metabolic syndrome. Daily prophylactic or therapeutic oral treatment of high-fat-fed animals with S1QEL1.719 decreased fat accumulation, strongly protected against decreased glucose tolerance and prevented or reversed the increase in fasting insulin level. Free exposures in plasma and liver at Cmax were 1-4 fold the IC50 for suppression of superoxide/hydrogen peroxide production at site IQ and substantially below levels that inhibit electron flow through complex I. These results show that the production of superoxide/hydrogen peroxide from mitochondrial site IQin vivo is necessary for the induction and maintenance of glucose intolerance caused by a high-fat diet in mice. They raise the possibility that oral administration of S1QELs may be beneficial in metabolic syndrome.

Site IQ in mitochondrial complex I generates S1QEL-sensitive superoxide/hydrogen peroxide in both the reverse and forward reactions.

Superoxide/hydrogen peroxide production by site IQ in complex I of the electron transport chain is conventionally assayed during reverse electron transport (RET) from ubiquinol to NAD. However, S1QELs (specific suppressors of superoxide/hydrogen peroxide production by site IQ) have potent effects in cells and in vivo during presumed forward electron transport (FET). Therefore, we tested whether site IQ generates S1QEL-sensitive superoxide/hydrogen peroxide during FET (site IQf), or alternatively, whether RET and associated S1QEL-sensitive superoxide/hydrogen peroxide production (site IQr) occurs in cells under normal conditions. We introduce an assay to determine if electron flow through complex I is thermodynamically forward or reverse: on blocking electron flow through complex I, the endogenous matrix NAD pool will become more reduced if flow before the challenge was forward, but more oxidised if flow was reverse. Using this assay we show in the model system of isolated rat skeletal muscle mitochondria that superoxide/hydrogen peroxide production by site IQ can be equally great whether RET or FET is running. We show that sites IQr and IQf are equally sensitive to S1QELs, and to rotenone and piericidin A, inhibitors that block the Q-site of complex I. We exclude the possibility that some sub-fraction of the mitochondrial population running site IQr during FET is responsible for S1QEL-sensitive superoxide/hydrogen peroxide production by site IQ. Finally, we show that superoxide/hydrogen peroxide production by site IQ in cells occurs during FET, and is S1QEL-sensitive.

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.

Riding the tiger - physiological and pathological effects of superoxide and hydrogen peroxide generated in the mitochondrial matrix.

Elevated mitochondrial matrix superoxide and/or hydrogen peroxide concentrations drive a wide range of physiological responses and pathologies. Concentrations of superoxide and hydrogen peroxide in the mitochondrial matrix are set mainly by rates of production, the activities of superoxide dismutase-2 (SOD2) and peroxiredoxin-3 (PRDX3), and by diffusion of hydrogen peroxide to the cytosol. These considerations can be used to generate criteria for assessing whether changes in matrix superoxide or hydrogen peroxide are both necessary and sufficient to drive redox signaling and pathology: is a phenotype affected by suppressing superoxide and hydrogen peroxide production; by manipulating the levels of SOD2, PRDX3 or mitochondria-targeted catalase; and by adding mitochondria-targeted SOD/catalase mimetics or mitochondria-targeted antioxidants? Is the pathology associated with variants in SOD2 and PRDX3 genes? Filtering the large literature on mitochondrial redox signaling using these criteria highlights considerable evidence that mitochondrial superoxide and hydrogen peroxide drive physiological responses involved in cellular stress management, including apoptosis, autophagy, propagation of endoplasmic reticulum stress, cellular senescence, HIF1α signaling, and immune responses. They also affect cell proliferation, migration, differentiation, and the cell cycle. Filtering the huge literature on pathologies highlights strong experimental evidence that 30-40 pathologies may be driven by mitochondrial matrix superoxide or hydrogen peroxide. These can be grouped into overlapping and interacting categories: metabolic, cardiovascular, inflammatory, and neurological diseases; cancer; ischemia/reperfusion injury; aging and its diseases; external insults, and genetic diseases. Understanding the involvement of mitochondrial matrix superoxide and hydrogen peroxide concentrations in these diseases can facilitate the rational development of appropriate therapies.

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.

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.

S1QELs suppress mitochondrial superoxide/hydrogen peroxide production from site IQ without inhibiting reverse electron flow through Complex I.

Mitochondria are important sources of superoxide and hydrogen peroxide in cell signaling and disease. In particular, superoxide/hydrogen peroxide production during reverse electron transport from ubiquinol to NAD+ though Complex I is implicated in several physiological and pathological processes. S1QELs are small molecules that suppress superoxide/hydrogen peroxide production at Complex I without affecting forward electron transport. Their mechanism of action is disputed. To test different mechanistic models, we compared the effects of two inhibitors of Complex I electron transport (piericidin A and rotenone) and two S1QELs from different chemical families on superoxide/hydrogen peroxide production and electron transport by Complex I in isolated mitochondria. Piericidin A and rotenone (and S1QEL1.1 at higher concentrations) prevented superoxide/hydrogen peroxide production from sites IQ and IF in Complex I by inhibiting reverse electron transport into the complex. S1QELs decreased the potency of electron transport inhibition by piericidin A and rotenone, suggesting that S1QELs bind directly to Complex I. S1QEL2.1 (and S1QEL1.1 at lower concentrations) suppressed site IQ without affecting reverse electron transport or site IF, showing that sites IQ and IF are distinct, and that S1QELs do not work simply by decreasing reverse electron transport to site IF (or site IQ). S1QELs did not affect the reduction of NAD+ or the rate of site IF driven by reverse electron transport, therefore they do not alter the driving forces for reverse electron transport and that is not how they suppress site IQ. We conclude that S1QELs bind to Complex I to influence the conformation of the piericidin A and rotenone binding sites and directly suppress superoxide/hydrogen peroxide production at site IQ, which is a separate site from site IF.

Modulation of complex II-energized respiration in muscle, heart, and brown adipose mitochondria by oxaloacetate and complex I electron flow.

We recently reported that membrane potential (ΔΨ) primarily determines the relationship of complex II-supported respiration by isolated skeletal muscle mitochondria to ADP concentrations. We observed that O2 flux peaked at low ADP concentration ([ADP]) (high ΔΨ) before declining at higher [ADP] (low ΔΨ). The decline resulted from oxaloacetate (OAA) accumulation and inhibition of succinate dehydrogenase. This prompted us to question the effect of incremental [ADP] on respiration in interscapular brown adipose tissue (IBAT) mitochondria, wherein ΔΨ is intrinsically low because of uncoupling protein 1 (UCP1). We found that succinate-energized IBAT mitochondria, even in the absence of ADP, accumulate OAA and manifest limited respiration, similar to muscle mitochondria at high [ADP]. This could be prevented by guanosine 5'-diphosphate inhibition of UCP1. NAD+ cycling with NADH requires complex I electron flow and is needed to form OAA. Therefore, to assess the role of electron transit, we perturbed flow using a small molecule, N1-(3-acetamidophenyl)-N2-(2-(4-methyl-2-(p-tolyl)thiazol-5-yl)ethyl)oxalamide. We observed decreased OAA, increased NADH/NAD+, and increased succinate-supported mitochondrial respiration under conditions of low ΔΨ (IBAT) but not high ΔΨ (heart). In summary, complex II-energized respiration in IBAT mitochondria is tempered by complex I-derived OAA in a manner dependent on UCP1. These dynamics depend on electron transit in complex I.-Fink, B. D., Yu, L., Sivitz, W. I. Modulation of complex II-energized respiration in muscle, heart, and brown adipose mitochondria by oxaloacetate and complex I electron flow.

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.