Mitochondrial Superoxide Release Research Articles

Pitfalls of Mitochondrial Redox Signaling Research.

Redox signaling from mitochondria (mt) to the cytosol and plasma membrane (PM) has been scarcely reported, such as in the case of hypoxic cell adaptation or (2-oxo-) 2-keto-isocaproate (KIC) β-like-oxidation stimulating insulin secretion in pancreatic β-cells. Mutual redox state influence between mitochondrial major compartments, the matrix and the intracristal space, and the cytosol is therefore derived theoretically in this article to predict possible conditions, when mt-to-cytosol and mt-to-PM signals may occur, as well as conditions in which the cytosolic redox signaling is not overwhelmed by the mitochondrial antioxidant capacity. Possible peroxiredoxin 3 participation in mt-to-cytosol redox signaling is discussed, as well as another specific case, whereby mitochondrial superoxide release is diminished, whereas the matrix MnSOD is activated. As a result, the enhanced conversion to H2O2 allows H2O2 diffusion into the cytosol, where it could be a predominant component of the H2O2 release. In both of these ways, mt-to-cytosol and mt-to-PM signals may be realized. Finally, the use of redox-sensitive probes is discussed, which disturb redox equilibria, and hence add a surplus redox-buffering to the compartment, where they are localized. Specifically, when attempts to quantify net H2O2 fluxes are to be made, this should be taken into account.

SIRT3 activation promotes enteric neurons survival and differentiation

Enteric neuron degeneration has been observed during aging, and in individuals with metabolic dysfunction including obesity and diabetes. Honokiol, a naturally occurring compound, is an activator of Sirtuin-3 (SIRT3) that has antioxidant activity. Its role in modulating enteric neuron-specific neurodegeneration is unknown. We studied the effects of honokiol and its fluorinated analog, hexafluoro-honokiol, on enteric neuronal differentiation and survival. We used a previously established model of mouse primary enteric neuronal cells and an enteric neuronal cell line treated with palmitate (PA) and lipopolysaccharide (LPS) to induce mitochondrial dysfunction and enteric neuronal cell death. The effect of honokiol and hexafluoro-honokiol was assessed on neuronal phenotype, fiber density, differentiation, and pyroptosis. Honokiol and hexafluoro-honokiol significantly increased neuronal networks and fiber density in enteric neurons and increased levels of neuronal nitric oxide synthase and Choline acetyltransferase mRNA. Hexafluoro-honokiol and honokiol also significantly increased SIRT3 mRNA levels and suppressed palmitate and LPS-induced neuronal pyroptosis. SIRT3 knock-down prevented the hexafluoro-honokiol mediated suppression of mitochondrial superoxide release. Our data supports a neuroprotective effect of honokiol and its derivative and these could be used as prophylactic or therapeutic agents for treating enteric neurodegeneration and associated motility disorders.

The diabetic microenvironment causes mitochondrial oxidative stress in glomerular endothelial cells and pathological crosstalk with podocytes

BackgroundIn the setting of diabetes mellitus, mitochondrial dysfunction and oxidative stress are important pathogenic mechanisms causing end organ damage, including diabetic kidney disease (DKD), but mechanistic understanding at a cellular level remains obscure. In mouse models of DKD, glomerular endothelial cell (GEC) dysfunction precedes albuminuria and contributes to neighboring podocyte dysfunction, implicating GECs in breakdown of the glomerular filtration barrier. In the following studies we wished to explore the cellular mechanisms by which GECs become dysfunctional in the diabetic milieu, and the impact to neighboring podocytes.MethodsMouse GECs were exposed to high glucose media (HG) or 2.5% v/v serum from diabetic mice or serum from non-diabetic controls, and evaluated for mitochondrial function (oxygen consumption), structure (electron microscopy), morphology (mitotracker), mitochondrial superoxide (mitoSOX), as well as accumulation of oxidized products (DNA lesion frequency (8-oxoG, endo-G), double strand breaks (γ-H2AX), endothelial function (NOS activity), autophagy (LC3) and apoptotic cell death (Annexin/PI; caspase 3). Supernatant transfer experiments from GECs to podocytes were performed to establish the effects on podocyte survival and transwell experiments were performed to determine the effects in co-culture.ResultsDiabetic serum specifically causes mitochondrial dysfunction and mitochondrial superoxide release in GECs. There is a rapid oxidation of mitochondrial DNA and loss of mitochondrial biogenesis without cell death. Many of these effects are blocked by mitoTEMPO a selective mitochondrial anti-oxidant. Secreted factors from dysfunctional GECs were sufficient to cause podocyte apoptosis in supernatant transfer experiments, or in co-culture but this did not occur when GECs had been previously treated with mitoTEMPO.ConclusionDissecting the impact of the diabetic environment on individual cell-types from the kidney glomerulus indicates that GECs become dysfunctional and pathological to neighboring podocytes by increased levels of mitochondrial superoxide in GEC. These studies indicate that GEC-signaling to podocytes contributes to the loss of the glomerular filtration barrier in DKD.CcDz7iHMjDYmrcJBznsPoeVideo abstractGraphical abstract

The role of Mitochondrial Superoxide Release in Acute and Chronic Hypoxia Sensing and Signaling of the Pulmonary vasculature

The role of Mitochondrial Superoxide Release in Acute and Chronic Hypoxia Sensing and Signaling of the Pulmonary vasculature

A Topical Zinc Ionophore Blocks Tumorigenic Progression in UV‐exposed SKH‐1 High‐risk Mouse Skin

Nonmelanoma skin cancer (NMSC) is the most common malignancy in the United States representing a considerable public health burden. Pharmacological suppression of skin photocarcinogenesis has shown promise in preclinical and clinical studies, but more efficacious photochemopreventive agents are needed. Here, we tested feasibility of harnessing pharmacological disruption of intracellular zinc homeostasis for photochemoprevention in vitro and in vivo. Employing the zinc ionophore and FDA-approved microbicidal agent zinc pyrithione (ZnPT), used worldwide in over-the-counter (OTC) topical consumer products, we first demonstrated feasibility of achieving ZnPT-based intracellular Zn2+ overload in cultured malignant keratinocytes (HaCaT-ras II-4; SCC-25) employing membrane-permeable fluorescent probes. Zinc overload was accompanied by induction of intracellular oxidative stress, associated with mitochondrial superoxide release as substantiated by MitoSOX Red™ fluorescence microscopy. ZnPT-induced cell death observable in malignant keratinocytes was preceded by induction of metal (MT2A), proteotoxic (HSPA6, HSPA1A, DDIT3, HMOX1) and genotoxic stress response (GADD45A, XRCC2) gene expression at the mRNA and protein levels. Comet analysis revealed introduction of formamidopyrimidine-DNA glycosylase (Fpg)-sensitive oxidative DNA lesions. In a photocarcinogenesis model (UV-exposed SKH-1 high-risk mouse skin), topical ZnPT administration post-UV caused epidermal zinc overload and stress response gene expression with pronounced blockade of tumorigenesis. Taken together, these data suggest feasibility of repurposing a topical OTC drug for zinc-directed photochemoprevention of solar UV-induced NMSC.

Mitochondrial Complex IV Subunit 4 Isoform 2 Is Essential for Acute Pulmonary Oxygen Sensing.

Acute pulmonary oxygen sensing is essential to avoid life-threatening hypoxemia via hypoxic pulmonary vasoconstriction (HPV) which matches perfusion to ventilation. Hypoxia-induced mitochondrial superoxide release has been suggested as a critical step in the signaling pathway underlying HPV. However, the identity of the primary oxygen sensor and the mechanism of superoxide release in acute hypoxia, as well as its relevance for chronic pulmonary oxygen sensing, remain unresolved. To investigate the role of the pulmonary-specific isoform 2 of subunit 4 of the mitochondrial complex IV (Cox4i2) and the subsequent mediators superoxide and hydrogen peroxide for pulmonary oxygen sensing and signaling. Isolated ventilated and perfused lungs from Cox4i2-/- mice lacked acute HPV. In parallel, pulmonary arterial smooth muscle cells (PASMCs) from Cox4i2-/- mice showed no hypoxia-induced increase of intracellular calcium. Hypoxia-induced superoxide release which was detected by electron spin resonance spectroscopy in wild-type PASMCs was absent in Cox4i2-/- PASMCs and was dependent on cysteine residues of Cox4i2. HPV could be inhibited by mitochondrial superoxide inhibitors proving the functional relevance of superoxide release for HPV. Mitochondrial hyperpolarization, which can promote mitochondrial superoxide release, was detected during acute hypoxia in wild-type but not Cox4i2-/- PASMCs. Downstream signaling determined by patch-clamp measurements showed decreased hypoxia-induced cellular membrane depolarization in Cox4i2-/- PASMCs compared with wild-type PASMCs, which could be normalized by the application of hydrogen peroxide. In contrast, chronic hypoxia-induced pulmonary hypertension and pulmonary vascular remodeling were not or only slightly affected by Cox4i2 deficiency, respectively. Cox4i2 is essential for acute but not chronic pulmonary oxygen sensing by triggering mitochondrial hyperpolarization and release of mitochondrial superoxide which, after conversion to hydrogen peroxide, contributes to cellular membrane depolarization and HPV. These findings provide a new model for oxygen-sensing processes in the lung and possibly also in other organs.

The antileishmanial activity of xanthohumol is mediated by mitochondrial inhibition.

Xanthohumol (Xan) is a natural constituent of human nutrition. Little is known about its actions on leishmanial parasites and their mitochondria as putative target. Therefore, we determined the antileishmanial activity of Xan and resveratrol (Res, as alternative compound with antileishmanial activity) with respect to mitochondria in Leishmania amazonensis promastigotes/amastigotes (LaP/LaA) in comparison with their activity in peritoneal macrophages from mouse (PMM) and macrophage cell line J774A.1 (J774). Mechanistic studies were conducted in Leishmania tarentolae promastigotes (LtP) and mitochondrial fractions isolated from LtP. Xan and Res demonstrated antileishmanial activity in LaA [half inhibitory concentration (IC50): Xan 7 µ m, Res 14 µ m]; while they had less influence on the viability of PMM (IC50: Xan 70 µ m, Res >438 µ m). In contrast to Res, Xan strongly inhibited oxygen consumption in Leishmania (LtP) but not in J774 cells. This was based on the inhibition of the mitochondrial electron transfer complex II/III by Xan, which was less pronounced with Res. Neither Xan nor Res increased mitochondrial superoxide release in LtP, while both decreased the mitochondrial membrane potential in LtP. Bioenergetic studies showed that LtP mitochondria have no spare respiratory capacity in contrast to mitochondria in J774 cells and can therefore much less adapt to stress by mitochondrial inhibitors, such as Xan. These data show that Xan may have antileishmanial activity, which is mediated by mitochondrial inhibition.

L‐type Ca2+ Current Increase by Rotenone, Stimulant of Mitochondrial Superoxide Release, in A7r5 Arterial Smooth Muscle Cells. Effect of Dithiothreitol, Hydrogen Peroxide, Tempol and Staurosporine

L‐type Ca current (ICaL) is primary Ca2+ current to initiate contraction in arterial smooth muscle cells. Mitochondrial respiratory chain (MRC) generates and releases superoxide (O2−) in energy metabolism. O2− is converted to H2O2 by superoxide dismutase (SOD). Rotenone (Rot), phytogenic toxin, inhibits complex I of MRC and stimulates the release of O2−. We recently found that Rot increases ICaL in A7r5 cultured aortic smooth muscle cells. Here, we performed further pharmacological study of the increase by applying several redox‐related agents. Rot (0.01 mM) and other drugs were introduced during repetitive depolarization at 1/20s or recording I–V relationship with 2 min interval. Rot increased ICaL by 25% in 1–2 minute. It increased macroscopic conductance and shifted V0.5 of activation curve to the hyperpolarizing direction. Dithiothreitol (DTT, 2 mM), thiol‐reducing agent, increased ICaL by 20% without producing the negative shift and, in the presence of DTT, Rot increased ICaL with the negative shift. H2O2 (0.3 mM) inhibited ICaL by 13% in 5 min without shifting I–V and, in the presence of H2O2, Rot induced 17% increase of ICaL with negative shift. Pretreatment by Tempol, SOD mimetic scavenger of O2−, markedly depressed Rot‐induced increase of ICaL. Staurosporine (100 nM), broad‐spectrum protein‐kinase inhibitor, inhibited ICaL with slow time course and completely suppressed Rot‐induced increase of ICaL. Rotenone increases ICaL by increasing the release of O2− from mitochondrial complex I and the increase is independent of thiol‐oxidation but is mediated by phosphorylation of proteins that regulates ICaL.

Rotenone-stimulated superoxide release from mitochondrial complex I acutely augments L-type Ca2+ current in A7r5 aortic smooth muscle cells.

Voltage-gated L-type Ca(2+) current (ICa,L) induces contraction of arterial smooth muscle cells (ASMCs), and ICa,L is increased by H2O2 in ASMCs. Superoxide released from the mitochondrial respiratory chain (MRC) is dismutated to H2O2 We studied whether superoxide per se acutely modulates ICa,L in ASMCs using cultured A7r5 cells derived from rat aorta. Rotenone is a toxin that inhibits complex I of the MRC and increases mitochondrial superoxide release. The superoxide content of mitochondria was estimated using mitochondrial-specific MitoSOX and HPLC methods, and was shown to be increased by a brief exposure to 10 μM rotenone. ICa,L was recorded with 5 mM BAPTA in the pipette solution. Rotenone administration (10 nM to 10 μM) resulted in a greater ICa,L increase in a dose-dependent manner to a maximum of 22.1% at 10 μM for 1 min, which gradually decreased to 9% after 5 min. The rotenone-induced ICa,L increase was associated with a shift in the current-voltage relationship (I-V) to a hyperpolarizing direction. DTT administration resulted in a 17.9% increase in ICa,L without a negative shift in I-V, and rotenone produced an additional increase with a shift. H2O2 (0.3 mM) inhibited ICa,L by 13%, and additional rotenone induced an increase with a negative shift. Sustained treatment with Tempol (4-hydroxy tempo) led to a significant ICa,L increase but it inhibited the rotenone-induced increase. Staurosporine, a broad-spectrum protein kinase inhibitor, partially inhibited ICa,L and completely suppressed the rotenone-induced increase. Superoxide released from mitochondria affected protein kinases and resulted in stronger ICa,L preceding its dismutation to H2O2 The removal of nitric oxide is a likely mechanism for the increase in ICa,L.

Pinacidil protects osteoblastic cells against antimycin A-induced oxidative damage.

The present study aimed to investigate the protective effect of a non-selective mitochondrial adenosine triphosphate (ATP)-sensitive potassium channel (mito-KATP) opener, pinacidil, on antimycin A-induced oxidative damage in osteoblastic MC3T3-E1 cells. Antimycin A inhibits mitochondrial electron transport by binding to complex III. Osteoblastic MC3T3-E1 cells were treated with antimycin A in the presence or absence of pinacidil and markers of mitochondrial function and oxidative stress were subsequently examined. The effects of pinacidil on the activation of phosphoinositide 3-kinase (PI3K), Akt and cyclic adenosine monophosphate‑responsive element-binding protein (CREB) were also examined. In osteoblastic MC3T3-E1 cells exposed to antimycin A, pinacidil inhibited antimycin A-induced cell death. The protective effects of pinacidil on cell survival were prevented by the addition of LY294002 (a PI3K inhibitor), an Akt inhibitor or auranofin [a thioredoxin reductase (TrxR) inhibitor], but not by KATP channel inhibitor glibenclamide. Pinacidil inhibited antimycin A-induced inactivation of PI3K and Akt as well as phosphorylation of CREB and TrxR. Furthermore, pinacidil prevented antimycin A-induced mitochondrial superoxide release, mitochondrial membrane potential dissipation, reduced ATP synthesis and intracellular [Ca2+] elevation. In conclusion, these results suggested that pinacidil may rescue osteoblastic cells from antimycin A-induced cellular damage, potentially via antioxidant activity and restoration of mitochondrial function, which are mediated in part by the PI3K/Akt/CREB signaling pathway.

Complex I generated, mitochondrial matrix-directed superoxide is released from the mitochondria through voltage dependent anion channels

Mitochondrial complex I has previously been shown to release superoxide exclusively towards the mitochondrial matrix, whereas complex III releases superoxide to both the matrix and the cytosol. Superoxide produced at complex III has been shown to exit the mitochondria through voltage dependent anion channels (VDAC). To test whether complex I-derived, mitochondrial matrix-directed superoxide can be released to the cytosol, we measured superoxide generation in mitochondria isolated from wild type and from mice genetically altered to be deficient in MnSOD activity (TnIFastCreSod2fl/fl). Under experimental conditions that produce superoxide primarily by complex I (glutamate/malate plus rotenone, GM+R), MnSOD-deficient mitochondria release ∼4-fold more superoxide than mitochondria isolated from wild type mice. Exogenous CuZnSOD completely abolished the EPR-derived GM+R signal in mitochondria isolated from both genotypes, evidence that confirms mitochondrial superoxide release. Addition of the VDAC inhibitor DIDS significantly reduced mitochondrial superoxide release (∼75%) in mitochondria from either genotype respiring on GM+R. Conversely, inhibition of potential inner membrane sites of superoxide exit, including the matrix face of the mitochondrial permeability transition pore and the inner membrane anion channel did not reduce mitochondrial superoxide release in the presence of GM+R in mitochondria isolated from either genotype. These data support the concept that complex I-derived mitochondrial superoxide release does indeed occur and that the majority of this release occurs through VDACs.

MnSOD deficiency results in elevated oxidative stress and decreased mitochondrial function but does not lead to muscle atrophy during aging

In a previous study, we reported that a deficiency in MnSOD activity (approximately 80% reduction) targeted to type IIB skeletal muscle fibers was sufficient to elevate oxidative stress and to reduce muscle function in young adult mice (TnIFastCreSod2(fl/fl) mice). In this study, we used TnIFastCreSod2(fl/fl) mice to examine the effect of elevated oxidative stress on mitochondrial function and to test the hypothesis that elevated oxidative stress and decreased mitochondrial function over the lifespan of the TnIFastCreSod2(fl/fl) mice would be sufficient to accelerate muscle atrophy associated with aging. We found that mitochondrial function is reduced in both young and old TnIFastCreSod2(fl/fl) mice, when compared with control mice. Complex II activity is reduced by 47% in young and by approximately 90% in old TnIFastCreSod2(fl/fl) mice, and was found to be associated with reduced levels of the catalytic subunits for complex II, SDHA and SDHB. Complex II-linked mitochondrial respiration is reduced by approximately 70% in young TnIFastCreSod2(fl/fl) mice. Complex II-linked mitochondrial Adenosine-Tri-Phosphate (ATP) production is reduced by 39% in young and was found to be almost completely absent in old TnIFastCreSod2(fl/fl) mice. Furthermore, in old TnIFastCreSod2(fl/fl) mice, aconitase activity is almost completely abolished; mitochondrial superoxide release remains > 2-fold elevated; and oxidative damage (measured as F(2) - isoprostanes) is increased by 30% relative to age-matched controls. These data show that despite elevated skeletal muscle-specific mitochondrial oxidative stress, oxidative damage, and complex II-linked mitochondrial dysfunction, age-related muscle atrophy was not accelerated in old TnIFastCreSod2(fl/fl) mice, suggesting mitochondrial oxidative stress may not be causal for age-related muscle atrophy.

Conditional knockout of Mn-SOD targeted to type IIB skeletal muscle fibers increases oxidative stress and is sufficient to alter aerobic exercise capacity.

In vitro studies of isolated skeletal muscle have shown that oxidative stress is limiting with respect to contractile function. Mitochondria are a potential source of muscle function-limiting oxidants. To test the hypothesis that skeletal muscle-specific mitochondrial oxidative stress is sufficient to limit muscle function, we bred mice expressing Cre recombinase driven by the promoter for the inhibitory subunit of troponin (TnIFast-iCre) with mice containing a floxed Sod2 (Sod2(fl/fl)) allele. Mn-SOD activity was reduced by 82% in glycolytic (mainly type II) muscle fiber homogenates from young TnIFastCreSod2(fl/fl) mice. Furthermore, Mn-SOD content was reduced by 70% only in type IIB muscle fibers. Aconitase activity was decreased by 56%, which suggests an increase in mitochondrial matrix superoxide. Mitochondrial superoxide release was elevated more than twofold by mitochondria isolated from glycolytic skeletal muscle in TnIFastCreSod2(fl/fl) mice. In contrast, the rate of mitochondrial H(2)O(2) production was reduced by 33%, and only during respiration with complex II substrate. F(2)-isoprostanes were increased by 36% in tibialis anterior muscles isolated from TnIFastCreSod2(fl/fl) mice. Elevated glycolytic muscle-specific mitochondrial oxidative stress and damage in TnIFastCreSod2(fl/fl) mice were associated with a decreased ability of the extensor digitorum longus and gastrocnemius muscles to produce contractile force as a function of time, whereas force production by the soleus muscle was unaffected. TnIFastCreSod2(fl/fl) mice ran 55% less distance on a treadmill than wild-type mice. Collectively, these data suggest that elevated mitochondrial oxidative stress and damage in glycolytic muscle fibers are sufficient to reduce contractile muscle function and aerobic exercise capacity.

Sirolimus-Induced Vascular Dysfunction: Increased Mitochondrial and Nicotinamide Adenosine Dinucleotide Phosphate Oxidase-Dependent Superoxide Production and Decreased Vascular Nitric Oxide Formation

This study sought to analyze mechanisms that mediate vascular dysfunction induced by sirolimus. Despite excellent antirestenotic capacity, sirolimus-eluting stents have been found to trigger coronary endothelial dysfunction and impaired re-endothelialization. To mimic the continuous sirolimus exposure of a stented vessel, Wistar rats underwent drug infusion with an osmotic pump for 7 days. Sirolimus treatment caused a marked degree of endothelial dysfunction as well as a desensitization of the vasculature to the endothelium-independent vasodilator nitroglycerin. Also, sirolimus stimulated intense transmural superoxide formation as detected by dihydroethidine fluorescence in aortae. Increased superoxide production was mediated in part by the vascular nicotinamide adenosine dinucleotide phosphate (NADPH) oxidase as indicated by a marked stimulation of p67(phox)/rac1 NADPH oxidase subunit expression and by increased rac1 membrane association. In addition, superoxide production in rat heart mitochondria was up-regulated by sirolimus, as measured by L012-enhanced chemiluminescence. As a consequence, electron spin resonance measurements showed a 40% reduction in vascular nitric oxide bioavailability, which was further supported by decreased serum nitrite levels. Sirolimus causes marked vascular dysfunction and nitrate resistance after continuous treatment for 7 days. This impaired vasorelaxation may, in part, be induced by up-regulated mitochondrial superoxide release as well as by an up-regulation of NADPH oxidase-driven superoxide production. Both processes could contribute to endothelial dysfunction observed after coronary vascular interventions with sirolimus-coated stents.