Mitochondrial Peroxide Production Research Articles

Fatty acid oxidation drives mitochondrial hydrogen peroxide production by α-ketoglutarate dehydrogenase

In the present study, we examined the mitochondrial hydrogen peroxide (mH2O2) generating capacity of α-ketoglutarate dehydrogenase (KGDH) and compared it to components of the electron transport chain using liver mitochondria isolated from male and female C57BL6N mice. We show for the first time there are some sex dimorphisms in the production of mH2O2 by electron transport chain complexes I and III when mitochondria are fueled with different substrates. However, in our investigations into these sex effects, we made the unexpected and compelling discovery that 1) KGDH serves as a major mH2O2 supplier in male and female liver mitochondria and 2) KGDH can form mH2O2 when liver mitochondria are energized with fatty acids but only when malate is used to prime the Krebs cycle. Surprisingly, 2-keto-3-methylvaleric acid (KMV), a site-specific inhibitor for KGDH, nearly abolished mH2O2 generation in both male and female liver mitochondria oxidizing palmitoyl-carnitine. KMV inhibited mH2O2 production in liver mitochondria from male and female mice oxidizing myristoyl-, octanoyl-, or butyryl-carnitine as well. S1QEL 1.1 (S1) and S3QEL 2 (S3), compounds that inhibit reactive oxygen species generation by complexes I and III, respectively, without interfering with OxPhos and respiration, had a negligible effect on the rate of mH2O2 production when pyruvate or acyl-carnitines were used as fuels. However, inclusion of KMV in reaction mixtures containing S1 and/or S3 almost abolished mH2O2 generation. Together, our findings suggest KGDH is the main mH2O2 generator in liver mitochondria, even when fatty acids are used as fuel.

Cumulative Deleterious Effects of Tetrahydrocannabinoid (THC) and Ethanol on Mitochondrial Respiration and Reactive Oxygen Species Production Are Enhanced in Old Isolated Cardiac Mitochondria.

Delta 9 tetrahydrocannabinol (THC), the main component of cannabis, has adverse effects on the cardiovascular system, but whether concomitant ethanol (EtOH) and aging modulate its toxicity is unknown. We investigated dose responses of THC and its vehicle, EtOH, on mitochondrial respiration and reactive oxygen production in both young and old rat cardiac mitochondria (12 and 90 weeks). THC dose-dependently impaired mitochondrial respiration in both groups, and such impairment was enhanced in aged rats (-97.5 ± 1.4% vs. -75.6 ± 4.0% at 2 × 10-5 M, and IC50: 0.7 ± 0.05 vs. 1.3 ± 0.1 × 10-5 M, p < 0.01, for old and young rats, respectively). The EtOH-induced decrease in mitochondrial respiration was greater in old rats (-50.1 ± 2.4% vs. -19.8 ± 4.4% at 0.9 × 10-5 M, p < 0.0001). Further, mitochondrial hydrogen peroxide (H2O2) production was enhanced in old rats after THC injection (+46.6 ± 5.3 vs. + 17.9 ± 7.8%, p < 0.01, at 2 × 10-5 M). In conclusion, the deleterious cardiac effects of THC were enhanced with concomitant EtOH, particularly in old cardiac mitochondria, showing greater mitochondrial respiration impairment and ROS production. These data improve our knowledge of the mechanisms potentially involved in cannabis toxicity, and likely support additional caution when THC is used by elderly people who consume alcohol.

Necessary Role of Ceramides in the Human Microvascular Endothelium During Health and Disease.

Elevated plasma ceramides and microvascular dysfunction both independently predict adverse cardiac events. Despite the known detrimental effects of ceramide on the microvasculature, evidence suggests that activation of the shear-sensitive, ceramide-forming enzyme NSmase (neutral sphingomyelinase) elicits formation of vasoprotective nitric oxide (NO). Here, we explore a novel hypothesis that acute ceramide formation through NSmase is necessary for maintaining NO signaling within the human microvascular endothelium. We further define the mechanism through which ceramide exerts beneficial effects and discern key mechanistic differences between arterioles from otherwise healthy adults (non-coronary artery disease [CAD]) and patients diagnosed with CAD. Human arterioles were dissected from discarded surgical adipose tissue (n=166), and vascular reactivity to flow and C2-ceramide was assessed. Shear-induced NO and mitochondrial hydrogen peroxide (H2O2) production were measured in arterioles using fluorescence microscopy. H2O2 fluorescence was assessed in isolated human umbilical vein endothelial cells. Inhibition of NSmase in arterioles from otherwise healthy adults induced a switch from NO to NOX-2 (NADPH-oxidase 2)-dependent H2O2-mediated flow-induced dilation. Endothelial dysfunction was prevented by treatment with sphingosine-1-phosphate (S1P) and partially prevented by C2-ceramide and an agonist of S1P-receptor 1 (S1PR1); the inhibition of the S1P/S1PR1 signaling axis induced endothelial dysfunction via NOX-2. Ceramide increased NO production in arterioles from non-CAD adults, an effect that was diminished with inhibition of S1P/S1PR1/S1P-receptor 3 signaling. In arterioles from patients with CAD, inhibition of NSmase impaired the overall ability to induce mitochondrial H2O2 production and subsequently dilate to flow, an effect not restored with exogenous S1P. Acute ceramide administration to arterioles from patients with CAD promoted H2O2 as opposed to NO production, an effect dependent on S1P-receptor 3 signaling. These data suggest that despite differential downstream signaling between health and disease, NSmase-mediated ceramide formation is necessary for proper functioning of the human microvascular endothelium. Therapeutic strategies that aim to significantly lower ceramide formation may prove detrimental to the microvasculature.

Redox Profile of Skeletal Muscles: Implications for Research Design and Interpretation.

Mammalian skeletal muscles contain varying proportions of Type I and II fibers, which feature different structural, metabolic and functional properties. According to these properties, skeletal muscles are labeled as 'red' or 'white', 'oxidative' or 'glycolytic', 'slow-twitch' or 'fast-twitch', respectively. Redox processes (i.e., redox signaling and oxidative stress) are increasingly recognized as a fundamental part of skeletal muscle metabolism at rest, during and after exercise. The aim of the present review was to investigate the potential redox differences between slow- (composed mainly of Type I fibers) and fast-twitch (composed mainly of Type IIa and IIb fibers) muscles at rest and after a training protocol. Slow-twitch muscles were almost exclusively represented in the literature by the soleus muscle, whereas a wide variety of fast-twitch muscles were used. Based on our analysis, we argue that slow-twitch muscles exhibit higher antioxidant enzyme activity compared to fast-twitch muscles in both pre- and post-exercise training. This is also the case between heads or regions of fast-twitch muscles that belong to different subcategories, namely Type IIa (oxidative) versus Type IIb (glycolytic), in favor of the former. No safe conclusion could be drawn regarding the mRNA levels of antioxidant enzymes either pre- or post-training. Moreover, slow-twitch skeletal muscles presented higher glutathione and thiol content as well as higher lipid peroxidation levels compared to fast-twitch. Finally, mitochondrial hydrogen peroxide production was higher in fast-twitch muscles compared to slow-twitch muscles at rest. This redox heterogeneity between different muscle types may have ramifications in the analysis of muscle function and health and should be taken into account when designing exercise studies using specific muscle groups (e.g., on an isokinetic dynamometer) or isolated muscle fibers (e.g., electrical stimulation) and may deliver a plausible explanation for the conflicting results about the ergogenic potential of antioxidant supplements.

Red blood cell trapping in the kidney vasculature promotes mitochondrial dysfunction independent of ischemia time

Red blood cell (RBC) trapping presents as the expansion of- and obstruction, of the outer-medullary capillaries of the kidney with tightly packed RBCs. RBC trapping has been thought to promote hypoxic tubular injury by extending ischemia time to the outer-medulla. Recent evidence from our laboratory; however, indicates that RBC trapping may promote toxic tubular injury, secondary to the mitochondrial uptake of iron from hemoglobin released from damaged RBCs in nearby congested capillaries. In the current study, we tested the hypothesis that ‘RBC trapping promotes tubular mitochondrial dysfunction independent of ischemia’. We tested our hypothesis using 9–10-week-old male Wistar-Kyoto rats. In an initial study, rats underwent bilateral warm arterial ischemia for 45 minutes with 2 hours of reperfusion before harvesting the kidneys for analysis by transmission electron microscopy (n=3). In a second study, the left kidney from each of n=10 rats underwent either arterial, venous or sham control clamping for 45 minutes. An alternate procedure (either arterial, venous or sham clamping) was performed on the right kidney. To examine the effect of ischemia alone on mitochondrial function, renal arteries were clamped, and the kidney excised without releasing the clamp. To examine the effect of ischemia with RBC trapping on mitochondrial function, the renal vein was clamped. Renal venous clamping mimics the obstruction of the medullary venous vasculature that occurs during reperfusion from arterial clamping, resulting in both kidney ischemia and RBC trapping. In all animals body temperature was maintained using a servo-controlled heating table and heat lamp. At harvest the kidney medulla and cortex separated and mitochondria isolated from each region. Mitochondrial membrane potential (TMRM fluorescence) and hydrogen peroxide production (Amplex red) was then determined from n=6-7 kidneys for each intervention. Data were compared using ANOVA. Consistent with iron uptake into mitochondria, when viewed using transmission electron microscopy, following 2 hours of reperfusion from arterial clamping, tubular mitochondria near RBC congested vessels often appeared darkened. Both ischemia alone and ischemia with RBC trapping, resulted in a significant increase in mitochondrial H 2 0 2 production compared to control (7063±562 and 7519±803 vs 5688±389 A.U per mg of protein, respectively, p&lt;0.001). In contrast, ischemia alone resulted in a lower mitochondrial membrane potential (228±18 A.U) compared to control (253±18), whereas, mitochondrial membrane potential following ischemia with RBC trapping (308±26) was significantly greater than both ischemia alone and control (p&lt;0.001). Our data supports the hypothesis that congestion of the kidney vasculature with RBCs directly promotes mitochondrial dysfunction independent of ischemia. This likely occurs secondary to mitochondrial uptake of iron from hemoglobin released from damaged RBCs. NIH NIDDK ISAC 21AU4231, NIH NHLBI P01 HL134604 and an American Heart Association Transformational Project Award 970585 to P.O and NIH NIDDK F31 DK127683 to S.R.M. NIH R01 HL148114 D.I. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Mitochondrial Hydrogen Peroxide Activates PTEN and Inactivates Akt Leading to Autophagy Inhibition-Dependent Cell Death in Neuronal Models of Parkinson's Disease.

Defective autophagy relates to the pathogenesis of Parkinson's disease (PD), a typical neurodegenerative disease. Our recent study has demonstrated that PD toxins (6-OHDA, MPP+, or rotenone) induce neuronal apoptosis by impeding the AMPK/Akt-mTOR signaling. Here, we show that treatment with 6-OHDA, MPP+, or rotenone triggered decreases of ATG5/LC3-II and autophagosome formation with a concomitant increase of p62 in PC12, SH-SY5Y cells, and primary neurons, suggesting inhibition of autophagy. Interestingly, overexpression of wild-type ATG5 attenuated the inhibitory effect of PD toxins on autophagy, reducing neuronal apoptosis. The effects of PD toxins on autophagy and apoptosis were found to be associated with activation of PTEN and inactivation of Akt. Overexpression of dominant negative PTEN, constitutively active Akt and/or pretreatment with rapamycin rescued the cells from PD toxins-induced downregulation of ATG5/LC3-II and upregulation of p62, as well as consequential autophagosome diminishment and apoptosis in the cells. The effects of PD toxins on autophagy and apoptosis linked to excessive intracellular and mitochondrial hydrogen peroxide (H2O2) production, as evidenced by using a H2O2-scavenging enzyme catalase, a mitochondrial superoxide indicator MitoSOX and a mitochondria-selective superoxide scavenger Mito-TEMPO. Furthermore, we observed that treatment with PD toxins reduced the protein level of Parkin in the cells. Knockdown of Parkin alleviated the effects of PD toxins on H2O2 production, PTEN/Akt activity, autophagy, and apoptosis in the cells, whereas overexpression of wild-type Parkin exacerbated these effects of PD toxins, implying the involvement of Parkin in the PD toxins-induced oxidative stress. Taken together, the results indicate that PD toxins can elicit mitochondrial H2O2, which can activate PTEN and inactivate Akt leading to autophagy inhibition-dependent neuronal apoptosis, and Parkin plays a critical role in this process. Our findings suggest that co-manipulation of the PTEN/Akt/autophagy signaling by antioxidants may be exploited for the prevention of neuronal loss in PD.

Increasing plasma L-kynurenine impairs mitochondrial oxidative phosphorylation prior to the development of atrophy in murine skeletal muscle: A pilot study.

Introduction: L-Kynurenine (L-Kyn), a product of tryptophan (Trp) catabolism, has been linked with impairments in walking speed, muscle strength/size, and physical function. The purpose of this pilot study was to develop a dietary model that elevates plasma L-Kyn levels in mice and characterize its impact on muscle health and function. Methods: Four-month-old C57BL6J male mice were randomized to either a L-Kyn supplemented (150 mg/kg) or chow diet for 10 weeks. Plasma L-Kyn and Trp levels were measured via mass spectrometry. Primary outcomes included assessments of muscle weights, myofiber cross-sectional area (CSA), nerve-stimulated contractile performance, and mitochondrial oxidative phosphorylation (OXPHOS) and hydrogen peroxide (H2O2) production. Additional experiments in cultured myotubes explored the impact of enhancing L-Kyn metabolism. Results: Mice randomized to the L-Kyn diet displayed significant increases in plasma L-Kyn levels (p = 0.0028) and the L-Kyn/Trp ratio (p = 0.011) when compared to chow fed mice. Food intake and body weights were not different between groups. There were no detectable differences in muscle weights, myofiber CSA, or contractile performance. L-Kyn fed mice displayed reductions in mitochondrial OXPHOS (p = 0.05) and maximal ADP-stimulated respiration (p = 0.0498). In cultured myotubes, overexpression of peroxisome proliferator-activated receptor-gamma coactivator 1 alpha prevented atrophy and proteolysis, as well as deficits in mitochondrial respiration with L-Kyn treatment. Conclusion: Dietary feeding of L-Kyn increases plasma L-Kyn levels and the L-Kyn/Trp ratio in healthy male mice. Mitochondrial impairments in muscle were observed in mice with elevated L-Kyn without changes in muscle size or function. Enhancing L-Kyn metabolism can protect against these effects in culture myotubes.

Short-term effect of molecular hydrogen on mitochondrial respiration and hydrogen peroxide production in permeabilized HEK 293T cells

Short-term effect of molecular hydrogen on mitochondrial respiration and hydrogen peroxide production in permeabilized HEK 293T cells

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.

Sexual dimorphisms in renal mitochondrial bioenergetics

BackgroundStudies have shown that men have higher incidences of end‐stage renal disease than women at every point across the lifespan. Animal models for various renal diseases have similar occurrence with males being affected more frequently. Recent efforts have been heavily focused on delineating the mechanisms behind these sex‐based differences from various angles, with the mitochondria being highlighted as a target for sex difference‐related renal pathologies. The goal of this study is to investigate the sexual dimorphisms of renal mitochondria and their dependency on renal hemodynamics before disease onset.MethodsMitochondria were isolated from PBS‐flushed kidneys of Sprague Dawley male (SDM) and female (SDF) rats at 11 weeks of age using differential centrifugation. Renal cortex (SDMC vs SDFC) and medulla (SDMMvs SDFM) were separated for individual measurements. Mitochondrial membrane potential and hydrogen peroxide production were measured in spectrofluorimetry using the fluorescent dyes TMRM and Amplex Red, respectively. A portion of mitochondria were submitted to a histology core for electron microscopy. Additionally, mitochondrial oxygen consumption rate (OCR) was also assessed using Seahorse assay. Lipid peroxide radical formation was detected using electron paramagnetic resonance (EPR) with in vivo spin trapping. As a measure of renal function, GFR was assessed using FITC‐Inulin in conscious animals.ResultsElectron microscopy revealed similar yield of mainly round‐shaped isolated mitochondria with retained cristae and membrane structures in all groups. SDFM showed a higher membrane potential than SDMM (238.1±12.4 vs 162.0±8.8 au, p&lt;0.001; respectively). Both SDFC and SDFM had higher hydrogen peroxide production in comparison to their male counterparts (cortex: 99443.7±3061.7 vs 69943.4±1708.6 au/protein, p&lt;0.001; respectively. medulla: 113366.7±5430.7 vs 60613±2297.1 au/protein, p&lt;0.001; respectively). Overall female mitochondria exhibited decreases in their basal, ATP‐linked, leaked OCRs as well as spare capacity compared to males (spare capacity: cortex: 5.9±0.6 vs 14.3±1.3, p&lt;0.001; respectively. medulla: 4.9±0.6 vs 11.6±0.7 pmol/min/protein, p&lt;0.001; respectively). EPR showed similar lipid peroxide radical levels in both sexes and sections of kidney. In the next part of the study, we explored if renal mitochondrial bioenergetics correlated with the GFR of the animal. Our data show the following GFR values for male and female rats as normalized to two kidney weights (2KW) and 2KW/ total body weight (2KW/TBW), respectively: 1.4±0.2 vs 0.8±0.1 mL/min/2KW, p=0.1416 and 0.5±0.1 vs 0.2±0.02 mL/min/2KW/TBW, p=0.0143. We did not observe any correlation between mitochondrial membrane potential and ROS production (measured in the isolated renal cortical mitochondria) with GFR in male or female rats (n= 5 in each group).ConclusionsOverall, we show here that female renal mitochondria exhibit lower oxygen consumption, higher ROS emission, and membrane potential vs males; however, lipid peroxidation is similar. Currently, we do not see a correlation between mitochondrial bioenergetics and GFR. More studies designed with higher statistical power are required to further elucidate the relationship between renal blood flow and mitochondrial bioenergetics.

On 'Production of superoxide radicals and hydrogen peroxide by NADH-ubiquinone reductase and ubiquinol-cytochrome c reductase from beef-heart mitochondria' by Enrique Cadenas, Alberto Boveris, C. Ian Ragan and Andres O.M.Stoppani

On Production of superoxide radicals and hydrogen peroxide by NADH-ubiquinone reductase and ubiquinol-cytochrome c reductase from beef-heart mitochondria (Arch Biochem Biophys (1977) 180, 248–257) reviews early work that influenced future studies on the mitochondrial production of superoxide anion and hydrogen peroxide.

Nitrate consumption preserves HFD-induced skeletal muscle mitochondrial ADP sensitivity and lysine acetylation: A potential role for SIRT1

Dietary nitrate supplementation, and the subsequent serial reduction to nitric oxide, has been shown to improve glucose homeostasis in several pre-clinical models of obesity and insulin resistance. While the mechanisms remain poorly defined, the beneficial effects of nitrate appear to be partially dependent on AMPK-mediated signaling events, a central regulator of metabolism and mitochondrial bioenergetics. Since AMPK can activate SIRT1, we aimed to determine if nitrate supplementation (4 mM sodium nitrate via drinking water) improved skeletal muscle mitochondrial bioenergetics and acetylation status in mice fed a high-fat diet (HFD: 60% fat). Consumption of HFD induced whole-body glucose intolerance, and within muscle attenuated insulin-induced Akt phosphorylation, mitochondrial ADP sensitivity (higher apparent Km), submaximal ADP-supported respiration, mitochondrial hydrogen peroxide (mtH2O2) production in the presence of ADP and increased cellular protein carbonylation alongside mitochondrial-specific acetylation. Consumption of nitrate partially preserved glucose tolerance and, within skeletal muscle, normalized insulin-induced Akt phosphorylation, mitochondrial ADP sensitivity, mtH2O2, protein carbonylation and global mitochondrial acetylation status. Nitrate also prevented the HFD-mediated reduction in SIRT1 protein, and interestingly, the positive effects of nitrate ingestion on glucose homeostasis and mitochondrial acetylation levels were abolished in SIRT1 inducible knock-out mice, suggesting SIRT1 is required for the beneficial effects of dietary nitrate. Altogether, dietary nitrate preserves mitochondrial ADP sensitivity and global lysine acetylation in HFD-fed mice, while in the absence of SIRT1, the effects of nitrate on glucose tolerance and mitochondrial acetylation were abrogated.

Publisher Notice

On Production of superoxide radicals and hydrogen peroxide by NADH-ubiquinone reductase and ubiquinol-cytochrome c reductase from beef-heart mitochondria (Arch Biochem Biophys (1977) 180, 248-257) reviews early work that influenced future studies on the mitochondrial production of superoxide anion and hydrogen peroxide.

Airway epithelial Paraoxonase-2 in obese asthma.

BackgroundObesity in asthmatics has been associated with higher airway oxidative stress in which dysfunctional mitochondria are a potential contributing source of excess free radicals. Paraoxonase 2 (PON2) plays an important role in reducing mitochondrial-derived oxidative stress and could, therefore, have therapeutic potential in these patients.ObjectivesWe used primary human bronchial epithelial cells (HBECs) from asthmatics and healthy controls to evaluate: a) protein levels of Paraoxonase 2 and b) to test the potential protective effect of quercetin supplementation in cells under oxidative stress conditions.ResultsCompared to lean controls, obese asthmatics had significantly lower PON2 airway epithelial levels (respectively, 1.08 vs. 0.47 relative units normalized by GAPDH) (p-value < 0.006). Treating HBECs in vitro for 24 hrs. with 25μM quercetin significantly increased PON2 protein levels: 15.5 treated cells vs. 9.8 untreated cells (relative units normalized by GAPDH) (p value = 0.004). Notably, compared to untreated cells, quercetin supplementation reduces mitochondrial superoxide and hydrogen peroxide production on HBECs cells exposed to different oxidative stress triggers such as 1–2 Naphthoquinone (1–2 NQ) and hydrogen peroxide, suggesting that PON2 might play a protective role ameliorating oxidative injury on human airway epithelium.ConclusionCompared to lean controls, obese asthmatics have significantly reduced PON2 levels in airway epithelial cells. Treatment with quercetin in vitro increased PON2 protein levels and prevented oxidative stress from different types of stimuli. Hence, quercetin supplementation may be a potential therapeutic strategy to prevent obesity-mediated airway oxidative stress in obese asthmatics.

P66Shc signaling does not contribute to tubular damage induced by renal ischemia-reperfusion injury in rat

Renal ischemia-reperfusion (IR) injury is one of the major causes of acute kidney injury and represents a significant risk factor for renal transplantation. The level of renal damage is influenced by the ischemic duration and is caused by excessive amounts of produced reactive oxygen species (ROS). Adaptor protein p66Shc is known to regulate cellular and organ's sensitivity to oxidative stress and to contribute significantly to mitochondrial production of hydrogen peroxide in stress conditions. Studies carried out in cultured renal cells suggest that p66Shc-mediated mitochondrial dysfunction and ROS production are responsible for renal ischemic injury. We used our genetically modified rats, which either lack p66Shc expression, or express p66Shc variant, which constitutively generates increased quantities of hydrogen peroxide, to evaluate potential contribution of p66Shc signaling to renal damage in ischemia reperfusion rat model. Analysis of outer medulla tubule damage revealed the lack of contribution of either p66Shc expression or its constitutive signaling to IR injury in rat model.

S100A8 and S100A9 are elevated in chronically threatened ischemic limb muscle and induce ischemic mitochondrial pathology in mice

ObjectiveThe objective of the present study was to determine whether elevated levels of S100A8 and S100A9 (S100A8/A9) alarmins contribute to ischemic limb pathology. MethodsGastrocnemius muscle was collected from control patients without peripheral arterial disease (PAD; n = 14) and patients with chronic limb threatening limb ischemia (CLTI; n = 14). Mitochondrial function was assessed in permeabilized muscle fibers, and RNA and protein analyses were used to quantify the S100A8/A9 levels. Additionally, a mouse model of hindlimb ischemia with and without exogenous delivery of S100A8/A9 was used. ResultsCompared with the non-PAD control muscles, CLTI muscles displayed significant increases in the abundance of S100A8 and S100A9 at both mRNA and protein levels (P < .01). The CLTI muscles also displayed significant impairment in mitochondrial oxidative phosphorylation and increased mitochondrial hydrogen peroxide production compared with the non-PAD controls. The S100A8/A9 levels correlated significantly with the degree of muscle mitochondrial dysfunction (P < .05 for all). C57BL6J mice treated with recombinant S100A8/A9 displayed impaired perfusion recovery and muscle mitochondrial impairment compared with the placebo-treated mice after hindlimb ischemia surgery. These mitochondrial deficits observed after S100A8/A9 treatment were confirmed in the muscle cell culture system under normoxic conditions. ConclusionsThe S100A8/A9 levels were increased in CLTI limb muscle specimens compared with the non-PAD control muscle specimens, and the level of accumulation was associated with muscle mitochondrial impairment. Elevated S100A8/A9 levels in mice subjected to hindlimb ischemia impaired perfusion recovery and mitochondrial function. Together, these findings suggest that the inflammatory mediators S100A8/A9 might be directly involved in ischemic limb pathology.

A novel role of KEAP1/PGAM5 complex: ROS sensor for inducing mitophagy

When ROS production exceeds the cellular antioxidant capacity, the cell needs to eliminate the defective mitochondria responsible for excessive ROS production. It has been proposed that the removal of these defective mitochondria involves mitophagy, but the mechanism of this regulation remains unclear. Here, we demonstrate that moderate mitochondrial superoxide and hydrogen peroxide production oxidates KEAP1, thus breaking the interaction between this protein and PGAM5, leading to the inhibition of its proteasomal degradation. Accumulated PGAM5 interferes with the processing of the PINK1 in the mitochondria leading to the accumulation of PINK1 on the outer mitochondrial membrane. In turn, PINK1 promotes Parkin recruitment to mitochondria and sensitizes mitochondria for autophagic removal. We also demonstrate that inhibitors of the KEAP1-PGAM5 protein-protein interaction (including CPUY192018) mimic the effect of mitochondrial ROS and sensitize mitophagy machinery, suggesting that these inhibitors could be used as pharmacological regulators of mitophagy. Together, our results show that KEAP1/PGAM5 complex senses mitochondrially generated superoxide/hydrogen peroxide to induce mitophagy.

The localization of the photosensitizer determines the dynamics of the secondary production of hydrogen peroxide in cell cytoplasm and mitochondria

Photodynamic therapy (PDT) is based on the production of the cytotoxic reactive oxygen species (ROS) by light irradiation of a photosensitizer dye in the presence of molecular oxygen. Along with photochemical ROS production, it becomes evident that PDT induces massive secondary production of ROS which is registered long after the irradiation is completed. We created cell lines of human epidermoid carcinoma with the cytoplasmic and mitochondrial localization of protein sensor HyPer sensitive to hydrogen peroxide to compare its concentration in two cellular compartments. The lag-period between irradiation and accumulation of hydrogen peroxide in cells was registered; its duration was dose-dependent and increased up to 80 min when lowering the exposition dose from 50 to 15 J/cm2. We have shown that localization of the photosensitizer determines the spatiotemporal pattern of the cell response to PDT: secondary hydrogen peroxide accumulation in cell cytoplasm induced by photodynamic treatment with lysosome-localized phtalocyianine Photosens occurs several minutes prior to that in mitochondria; on the contrary, membranotropic arylcyanoporphyrazine dye leads to massive mitochondrial hydrogen peroxide production followed by its cytoplasmic accumulation. We hypothesize that photosensitizers with various physicochemical properties and intracellular localization can trigger different patterns not only of primary but also secondary ROS production leading to different cell fate outcomes.

Does the mitochondrial import and assembly (MIA) pathway contribute to the hydrogen peroxide production in mitochondria?

Does the mitochondrial import and assembly (MIA) pathway contribute to the hydrogen peroxide production in mitochondria?

Nrf2 is activated by disruption of mitochondrial thiol homeostasis but not by enhanced mitochondrial superoxide production

The transcription factor nuclear factor erythroid 2–related factor 2 (Nrf2) regulates the expression of genes involved in antioxidant defenses to modulate fundamental cellular processes such as mitochondrial function and GSH metabolism. Previous reports proposed that mitochondrial reactive oxygen species production and disruption of the GSH pool activate the Nrf2 pathway, suggesting that Nrf2 senses mitochondrial redox signals and/or oxidative damage and signals to the nucleus to respond appropriately. However, until now, it has not been possible to disentangle the overlapping effects of mitochondrial superoxide/hydrogen peroxide production as a redox signal from changes to mitochondrial thiol homeostasis on Nrf2. Recently, we developed mitochondria-targeted reagents that can independently induce mitochondrial superoxide and hydrogen peroxide production mitoParaquat (MitoPQ) or selectively disrupt mitochondrial thiol homeostasis MitoChlorodinitrobenzoic acid (MitoCDNB). Using these reagents, here we have determined how enhanced generation of mitochondrial superoxide and hydrogen peroxide or disruption of mitochondrial thiol homeostasis affects activation of the Nrf2 system in cells, which was assessed by the Nrf2 protein level, nuclear translocation, and expression of its target genes. We found that selective disruption of the mitochondrial GSH pool and inhibition of its thioredoxin system by MitoCDNB led to Nrf2 activation, whereas using MitoPQ to enhance the production of mitochondrial superoxide and hydrogen peroxide alone did not. We further showed that Nrf2 activation by MitoCDNB requires cysteine sensors of Kelch-like ECH-associated protein 1 (Keap1). These findings provide important information on how disruption to mitochondrial redox homeostasis is sensed in the cytoplasm and signaled to the nucleus.