Mitochondrial Superoxide Formation Research Articles

Sphingosine is involved in PAPTP-induced death of pancreas cancer cells by interfering with mitochondrial functions

Pancreas ductal adenocarcinoma belongs to the most common cancers, but also to the tumors with the poorest prognosis. Here, we pharmacologically targeted a mitochondrial potassium channel, namely mitochondrial Kv1.3, and investigated the role of sphingolipids and mutated Kirsten Rat Sarcoma Virus (KRAS) in Kv1.3-mediated cell death. We demonstrate that inhibition of Kv1.3 using the Kv1.3-inhibitor PAPTP results in an increase of sphingosine and superoxide in membranes and/or membranes associated with mitochondria, which is enhanced by KRAS mutation. The effect of PAPTP on sphingosine and mitochondrial superoxide formation as well as cell death is prevented by sh-RNA-mediated downregulation of Kv1.3. Induction of sphingosine in human pancreas cancer cells by PAPTP is mediated by activation of sphingosine-1-phosphate phosphatase and prevented by an inhibitor of sphingosine-1-phosphate phosphatase. A rapid depolarization of isolated mitochondria is triggered by binding of sphingosine to cardiolipin, which is neutralized by addition of exogenous cardiolipin. The significance of these findings is indicated by treatment of mutated KRAS-harboring metastasized pancreas cancer with PAPTP in combination with ABC294640, a blocker of sphingosine kinases. This treatment results in increased formation of sphingosine and death of pancreas cancer cells in vitro and, most importantly, prolongs in vivo survival of mice challenged with metastatic pancreas cancer.Key messagesPancreatic ductal adenocarcinoma (PDAC) is a common tumor with poor prognosis.The mitochondrial Kv1.3 ion channel blocker induced mitochondrial sphingosine.Sphingosine binds to cardiolipin thereby mediating mitochondrial depolarization.Sphingosine is formed by a PAPTP-mediated activation of S1P-Phosphatase.Inhibition of sphingosine-consumption amplifies PAPTP effects on PDAC in vivo.

Mitochondrial function and reactive oxygen species production during human sperm capacitation: Unraveling key players.

Sperm capacitation is a critical process for male fertility. It involves a series of biochemical and physiological changes that occur in the female reproductive tract, rendering the sperm competent for successful fertilization. The precise mechanisms and, specifically, the role of mitochondria, in sperm capacitation remain incompletely understood. Previously, we revealed that in mouse sperm mitochondrial activity (e.g., oxygen consumption, membrane potential, ATP/ADP exchange, and mitochondrial Ca2+ ) increases during capacitation. Herein, we studied mitochondrial function by high-resolution respirometry (HRR) and reactive oxygen species production in capacitated (CAP) and non-capacitated (NC) human spermatozoa. We found that in capacitated sperm from normozoospermic donors, the respiratory control ratio increased by 36%, accompanied by a double oxygen consumption rate (OCR) in the presence of antimycin A. Extracellular hydrogen peroxide (H2 O2 ) detection was three times higher in CAP than in NC sperm cells. To confirm that H2 O2 production depends on mitochondrial superoxide ( ) formation, we evaluated mitochondrial aconitase (ACO2) amount, activity, and role in the metabolic flux from the sperm tricarboxylic acid cycle. We estimated that CAP cells produce, on average by individual, (59 ± 22)% more in the steady-state compared to NC cells. Finally, we analyzed two targets of oxidative stress: lipid peroxidation by western blot against 4-hydroxynonenal and succinate dehydrogenase (SDH) activity by HRR. We did not observe modifications in lipoperoxidation nor the activity of SDH, suggesting that during capacitation, the increase in mitochondrial H2 O2 production does not damage sperm and it is necessary for the normal CAP process.

Clozapine suppresses NADPH oxidase activation, counteracts cytosolic H2O2, and triggers early onset mitochondrial dysfunction during adipogenesis of human liposarcoma SW872 cells

Long-term treatment of schizophrenia with clozapine (CLZ), an atypical antipsychotic drug, is associated with an increased incidence of metabolic disorders mediated by poorly understood mechanisms. We herein report that CLZ, while slowing down the morphological changes and lipid accumulation occurring during SW872 cell adipogenesis, also causes an early (day 3) inhibition of the expression/nuclear translocation of CAAT/enhancer-binding protein β and peroxisome proliferator-activated receptor γ. Under the same conditions, CLZ blunts NADPH oxidase-derived reactive oxygen species (ROS) by a dual mechanism involving enzyme inhibition and ROS scavenging. These effects were accompanied by hampered activation of the nuclear factor (erythroid-derived2)-like 2 (Nrf2)-dependent antioxidant responses compared to controls, and by an aggravated formation of mitochondrial superoxide. CLZ failed to exert ROS scavenging activities in the mitochondrial compartment but appeared to actively scavenge cytosolic H2O2 derived from mitochondrial superoxide. The early formation of mitochondrial ROS promoted by CLZ was also associated with signs of mitochondrial dysfunction. Some of the above findings were recapitulated using mouse embryonic fibroblasts.We conclude that the NADPH oxidase inhibitory and cytosolic ROS scavenging activities of CLZ slow down SW872 cell adipogenesis and suppress their Nrf2 activation, an event apparently connected with increased mitochondrial ROS formation, which is associated with insulin resistance and metabolic syndrome. Thus, the cellular events characterised herein may help to shed light on the more detailed molecular mechanisms explaining some of the adverse metabolic effects of CLZ.

Central Mechanisms of Acetaminophen Hepatotoxicity: Mitochondrial Dysfunction by Protein Adducts and Oxidant Stress.

Acetaminophen (APAP) is an analgesic and antipyretic drug used worldwide, which is safe at therapeutic doses. However, an overdose can induce liver injury and even liver failure. Mechanistic studies in mice beginning with the seminal papers published by B.B. Brodie's group in the 1970s have resulted in important insight into the pathophysiology. Although the metabolic activation of APAP with generation of a reactive metabolite, glutathione depletion, and protein adduct formation are critical initiating events, more recently, mitochondria have come into focus as an important target and decision point of cell death. This review provides a comprehensive overview of the induction of mitochondrial superoxide and peroxynitrite formation and its propagation through a mitogen-activated protein kinase cascade, the mitochondrial permeability transition pore opening caused by iron-catalyzed protein nitration, and the mitochondria-dependent nuclear DNA fragmentation. In addition, the role of adaptive mechanisms that can modulate the pathophysiology, including autophagy, mitophagy, nuclear erythroid 2 p45-related factor 2 activation, and mitochondrial biogenesis, are discussed. Importantly, it is outlined how the mechanisms elucidated in mice translate to human hepatocytes and APAP overdose patients, and how this mechanistic insight explains the mechanism of action of the clinically approved antidote N-acetylcysteine and led to the recent discovery of a novel compound, fomepizole, which is currently under clinical development. SIGNIFICANCE STATEMENT: Acetaminophen (APAP)-induced liver injury is the most frequent cause of acute liver failure in western countries. Extensive mechanistic research over the last several decades has revealed a central role of mitochondria in the pathophysiology of APAP hepatotoxicity. This review article provides a comprehensive discussion of a) mitochondrial protein adducts and oxidative/nitrosative stress, b) mitochondria-regulated nuclear DNA fragmentation, c) adaptive mechanisms to APAP-induced cellular stress, d) translation of cell death mechanisms to overdose patients, and e) mechanism-based antidotes against APAP-induced liver injury.

Quercetin induces MGMT+ glioblastoma cells apoptosis via dual inhibition of Wnt3a/β-Catenin and Akt/NF-κB signaling pathways

BackgroundSurgical resection combined with radiotherapy and chemotherapy remains a common clinical treatment for glioblastoma multiforme (GBM). However, the therapeutic outcomes have not been satisfying due to drug resistance and other factors. Quercetin, a phytoingredient capable of crossing the blood-brain barrier, has shown effectiveness in the treatment of various solid tumors. Nevertheless, the potential of quercetin in GBM treatment has not been adequately explored. PurposeThis study aims to investigate the effects and mechanisms of quercetin on MGMT+GBM cells. MethodsThe potential targets and mechanisms of quercetin in glioma treatment were predicted based on network pharmacology and molecular docking. The effects of quercetin on cell inhibition rate, cell migration ability, cell cycle arrest, mitochondrial membrane potential (MMP), reactive oxygen species (ROS), Mitochondrial superoxide formation and apoptosis were measured by the CCK8 assay, wound healing assay, PI/RNase staining, JC-1 assay, DCFH-DA assay, MitoSOX staining and Annexin V-FITC/PI double staining, respectively. The methylation status of the MGMT promoter was assessed through methylation-specific polymerase chain reaction (MS-PCR). DNA damage was quantified by alkaline/neutral comet assay and TUNEL assay. The intracellular localization and expression of NF-κB and MGMT were revealed by immunofluorescence. The expression of migration-related proteins, matrix metalloproteinases, apoptosis-related proteins, cyclins, DNA damage/repair enzymes and related pathway proteins was detected by Western blot. ResultsNetwork pharmacology identified 96 targets and potential molecular mechanisms of quercetin in glioma treatment. Subsequent experiments confirmed the synergistic effect of quercetin in combination with temozolomide (TMZ) on T98G cells. Quercetin significantly suppressed the growth and migration of human GBM T98G cells, induced apoptosis, and arrested cells in the S-phase cell cycle. The collapse of mitochondrial membrane potential, ROS generation, enhanced Bax/Bcl-2 ratio, and strengthened cleaved-Caspase 9 and cleaved-Caspase 3 suggested the involvement of ROS-mediated mitochondria-dependent apoptosis in the process of quercetin-induced apoptosis. In addition, quercetin-induced apoptosis was accompanied by intense DNA double-strand breaks (DSBs), γH2AX foci formation, methylation of MGMT promoter, increased cleaved-PARP, and reduced MGMT expression. Quercetin may influence the expression of the key DNA repair enzyme, MGMT, by dual suppression of the Wnt3a/β-Catenin and the Akt/NF-κB signaling pathways, thereby promoting apoptosis. Inhibition of Wnt3a and Akt using specific inhibitors hindered MGMT expression. ConclusionOur study provides the first evidence that quercetin may induce apoptosis in MGMT+GBM cells via dual inhibition of the Wnt3a/β-Catenin pathway and the Akt/NF-κB signaling pathway. These findings suggest that quercetin could be a novel agent for improving GBM treatment, especially in TMZ-resistant GBM with high MGMT expression.

Mitochondrial Cristae Morphology Reflecting Metabolism, Superoxide Formation, Redox Homeostasis, and Pathology.

Significance: Mitochondrial (mt) reticulum network in the cell possesses amazing ultramorphology of parallel lamellar cristae, formed by the invaginated inner mitochondrial membrane. Its non-invaginated part, the inner boundary membrane (IBM) forms a cylindrical sandwich with the outer mitochondrial membrane (OMM). Crista membranes (CMs) meet IBM at crista junctions (CJs) of mt cristae organizing system (MICOS) complexes connected to OMM sorting and assembly machinery (SAM). Cristae dimensions, shape, and CJs have characteristic patterns for different metabolic regimes, physiological and pathological situations. Recent Advances: Cristae-shaping proteins were characterized, namely rows of ATP-synthase dimers forming the crista lamella edges, MICOS subunits, optic atrophy 1 (OPA1) isoforms and mitochondrial genome maintenance 1 (MGM1) filaments, prohibitins, and others. Detailed cristae ultramorphology changes were imaged by focused-ion beam/scanning electron microscopy. Dynamics of crista lamellae and mobile CJs were demonstrated by nanoscopy in living cells. With tBID-induced apoptosis a single entirely fused cristae reticulum was observed in a mitochondrial spheroid. Critical Issues: The mobility and composition of MICOS, OPA1, and ATP-synthase dimeric rows regulated by post-translational modifications might be exclusively responsible for cristae morphology changes, but ion fluxes across CM and resulting osmotic forces might be also involved. Inevitably, cristae ultramorphology should reflect also mitochondrial redox homeostasis, but details are unknown. Disordered cristae typically reflect higher superoxide formation. Future Directions: To link redox homeostasis to cristae ultramorphology and define markers, recent progress will help in uncovering mechanisms involved in proton-coupled electron transfer via the respiratory chain and in regulation of cristae architecture, leading to structural determination of superoxide formation sites and cristae ultramorphology changes in diseases.

Arsenite enhances ERO1α expression via ryanodine receptor dependent and independent mechanisms

Arsenite is a potent carcinogen and toxic compound inducing an array of deleterious effects via different mechanisms, which include the Ca2+-dependent formation of reactive oxygen species. The mechanism whereby the metalloid affects Ca2+ homeostasis involves an initial stimulation of the inositol 1, 4, 5-triphosphate receptor, an event associated with an endoplasmic reticulum (ER) stress leading to increased ERO1α expression, and ERO1α dependent activation of the ryanodine receptor (RyR). Ca2+ release from the RyR is then critically connected with the mitochondrial accumulation of Ca2+. We now report that the resulting formation of mitochondrial superoxide triggers a second mechanism of ER stress dependent ERO1α expression, which however fails to impact on Ca2+ release from the RyR or, more generally, on Ca2+ homeostasis. Our results therefore demonstrate that arsenite stimulates two different and sequential mechanisms leading to increased ERO1α expression with different functions, possibly due to their different subcellular compartmentalization.

Notoginsenoside Fc ameliorates renal tubular injury and mitochondrial damage in acetaminophen-induced acute kidney injury partly by regulating SIRT3/SOD2 pathway.

Mitochondria dysfunction is one of the primary causes of tubular injury in acute kidney injury (AKI). Notoginsenoside Fc (Fc), a new saponin isolated from Panax notoginseng, exhibited numerous pharmacological actions. However, the beneficial effects of Fc on renal tubular impairment and mitochondrial dysfunction in AKI have not been fully studied. In this study, we established acetaminophen (APAP)-induced AKI model in mice to examine the therapeutic impacts of Fc on AKI. Our results showed that Fc could decrease the levels of the serum creatinine (Scr), blood urea nitrogen (BUN) and Cystatin C in mice with AKI. Fc also ameliorated renal histopathology, renal tubular cells apoptosis and restored expression of apoptosis-related proteins such as Bax, Bcl-2 and caspase3 (C-caspase3). Additionally, Fc increased the protein expression of SIRT3 and SOD2 in kidneys from mice with AKI. In vitro studies further showed Fc reduced the apoptosis of HK-2 cells exposure to APAP, attenuated the loss of mitochondrial membrane potential and decreased the formation of mitochondrial superoxide. Fc also partly restored the protein expression of Bax, Bcl-2, C-Caspase3, SIRT3, and SOD2 in HK-2 cells exposure to APAP. In summary, Fc might reduce renal tubular injury and mitochondrial dysfunction in AKI partly through the regulation of SIRT3/SOD2 pathway.

The role of Iron in lipid peroxidation and protein nitration during acetaminophen-induced liver injury in mice

Acetaminophen (APAP) hepatotoxicity, a leading cause of acute liver failure in western countries, is characterized by mitochondrial superoxide and peroxynitrite formation. However, the role of iron, especially as facilitator of lipid peroxidation (LPO), has been controversial. Our aim was to determine the mechanism by which iron promotes cell death in this context. Fasted male C57BL/6J mice were treated with the iron chelator deferoxamine, minocycline (inhibitor of the mitochondrial calcium uniporter) or vehicle 1 h before 300 mg/kg APAP. Deferoxamine and minocycline significantly attenuated APAP-induced elevations in serum alanine amino transferase levels and hepatic necrosis at 6 h. This protection correlated with reduced 3-nitro-tyrosine protein adducts; LPO (malondialdehyde, 4-hydroxynonenal) was not detected. Activation of c-jun N-terminal kinase (JNK) was not affected but mitochondrial release of intermembrane proteins was reduced suggesting that the effect of iron was at the level of mitochondria. Co-treatment of APAP with FeSO4 exacerbated liver injury and protein nitration and triggered significant LPO; all effects were reversed by deferoxamine. Thus, after APAP overdose, iron imported into mitochondria facilitates protein nitration by peroxynitrite triggering mitochondrial dysfunction and cell death. Under these conditions, endogenous defense mechanisms largely prevent LPO. However, after iron overload, protein nitration and LPO contribute to APAP hepatotoxicity.

Crosstalk between ERO1α and ryanodine receptor in arsenite-dependent mitochondrial ROS formation

Arsenite, a well-established human carcinogen and toxic compound, promotes the formation of mitochondrial superoxide (mitoO2−) via a Ca2+-dependent mechanism, in which an initial stimulation of the inositol 1, 4, 5-trisphosphate receptor (IP3R) is followed by the activation of the ryanodine receptor (RyR), critical for providing Ca2+ to the mitochondria. We now report that, under the same conditions, arsenite triggers endoplasmic reticulum (ER) stress and a threefold increase in ER oxidoreductin 1α (ERO1 α) levels in proliferating U937 cells. EN460, an inhibitor of ERO1 α, recapitulated all the effects associated with RyR inhibition or downregulation, including prevention of RyR-induced Ca2+ accumulation in mitochondria and the resulting O2–. formation. Quantitatively similar results were obtained in inhibitor studies performed in terminally differentiated wild type C2C12 cells. Moreover, ERO1 α knockout C2C12 myotubes responded to arsenite as their wild type counterpart supplemented with EN460. As a final note, arsenite enhanced the expression of ERO1 α via a mechanism mediated by Ca2+ release from both the IP3R and RyR.We therefore conclude that arsenite activates a positive feedback amplification cycle between Ca2+ levels and ERO1 α in the ER, by which IP3R-dependent Ca2+ induces ERO1 α and ERO1 α promotes Ca2+ release via RyR, thereby amplifying the initial Ca2+ load and causing the mitochondrial accumulation of the cation, critical for mitoO2− formation.

Mitochondrial Dysfunction in Trauma-Related Coagulopathy: Is There Causality? Study Protocol for a Prospective Observational Study

Hemorrhage control often poses a great challenge for clinicians due to trauma-induced coagulopathy (TIC). The pathogenesis of TIC is not completely revealed; however, growing evidence attributes a central role to altered platelet biology. The activation of thrombocytes and subsequent clot formation are highly energetic processes being tied to mitochondrial activity, and the inhibition of the electron transport chain (ETC) impedes on thrombogenesis, suggesting the potential role of mitochondria in TIC. Our present study protocol provides a guide to quantitatively characterize the derangements of mitochondrial functions in TIC. One hundred eleven severely injured (injury severity score ≥16), bleeding trauma patients with an age of 18 or greater will be included in this prospective observational study. Patients receiving oral antiplatelet agents including cyclooxygenase-1 or adenosine diphosphate receptor inhibitors (aspirin, clopidogrel, prasugrel, and ticagrelor) will be excluded from the final analysis. Hemorrhage will be confirmed and assessed with computer tomography. Conventional laboratory markers of hemostasis such as prothrombin time and international normalized ratio will be measured and rotational thromboelastometry (ROTEM) will be performed directly upon patient arrival. Platelets will be isolated from venous blood samples and subjected to high-resolution fluororespirometry (Oxygraph-2k, Oroboros Instruments, Innsbruck, Austria) to evaluate the efficacy of mitochondrial respiration. Oxidative phosphorylation (OxPhos), coupling of the ETC, mitochondrial superoxide formation, mitochondrial membrane potential changes, and extramitochondrial Ca²⁺-movement will be recorded. The association between OxPhos capacity of platelet mitochondria and numerical parameters of ROTEM aggregometry will constitute our primary outcome. The relation between OxPhos capacity and results of viscoelastic assays and conventional markers of hemostasis will serve as secondary outcomes. The association of the OxPhos capacity of platelet mitochondria upon patient arrival to the need for massive blood transfusion and 24-h mortality will constitute our tertiary outcomes. Mitochondrial dysfunction and its importance in TIC are yet to be assessed for the deeper understanding of this common, life-threatening condition. Disclosure of mitochondria-mediated processes in thrombocytes may reveal new therapeutic targets in the management of hemorrhaging trauma patients, thereby leading to a reduction of potentially preventable mortality. The present protocol was registered to ClinicalTrials.gov on 12 August 2021, under the reference number NCT05004844.

Functional organization of the endoplasmic reticulum dictates the susceptibility of target cells to arsenite-induced mitochondrial superoxide formation, mitochondrial dysfunction and apoptosis

Arsenite induces many critical effects associated with the formation of reactive oxygen species (ROS) through different mechanisms. We focused on Ca2+-dependent mitochondrial superoxide (mitoO2-.) formation and addressed questions on the effects of low concentrations of arsenite on the mobilization of the cation from the endoplasmic reticulum and the resulting mitochondrial accumulation. Using various differentiated and undifferentiated cell types uniquely expressing the inositol-1, 4, 5-triphosphate receptor (IP3R), or both the IP3R and the ryanodine receptor (RyR), we determined that expression of this second Ca2+ channel is an absolute requirement for mitoO2-. formation and for the ensuing mitochondrial dysfunction and downstream apoptosis. In arsenite-treated cells, RyR was recruited after IP3R stimulation and agonist studies provided an indirect indication for a close apposition between RyR and mitochondria. It was also interesting to observe that arsenite fails to promote mitochondrial Ca2+ accumulation, mitoO2-. formation and mitochondrial toxicity in RyR-devoid cells, in which the IP3R is in close contact with the mitochondria. We therefore conclude that low dose arsenite-induced mitoO2- formation, and the resulting mitochondrial dysfunction and toxicity, are prerequisite of cell types expressing the RyR in close apposition with mitochondria.

Antioxidant Synergy of Mitochondrial Phospholipase PNPLA8/iPLA2γ with Fatty Acid-Conducting SLC25 Gene Family Transporters.

Patatin-like phospholipase domain-containing protein PNPLA8, also termed Ca2+-independent phospholipase A2γ (iPLA2γ), is addressed to the mitochondrial matrix (or peroxisomes), where it may manifest its unique activity to cleave phospholipid side-chains from both sn-1 and sn-2 positions, consequently releasing either saturated or unsaturated fatty acids (FAs), including oxidized FAs. Moreover, iPLA2γ is directly stimulated by H2O2 and, hence, is activated by redox signaling or oxidative stress. This redox activation permits the antioxidant synergy with mitochondrial uncoupling proteins (UCPs) or other SLC25 mitochondrial carrier family members by FA-mediated protonophoretic activity, termed mild uncoupling, that leads to diminishing of mitochondrial superoxide formation. This mechanism allows for the maintenance of the steady-state redox status of the cell. Besides the antioxidant role, we review the relations of iPLA2γ to lipid peroxidation since iPLA2γ is alternatively activated by cardiolipin hydroperoxides and hypothetically by structural alterations of lipid bilayer due to lipid peroxidation. Other iPLA2γ roles include the remodeling of mitochondrial (or peroxisomal) membranes and the generation of specific lipid second messengers. Thus, for example, during FA β-oxidation in pancreatic β-cells, H2O2-activated iPLA2γ supplies the GPR40 metabotropic FA receptor to amplify FA-stimulated insulin secretion. Cytoprotective roles of iPLA2γ in the heart and brain are also discussed.

Dihydronicotinamide riboside promotes cell-specific cytotoxicity by tipping the balance between metabolic regulation and oxidative stress.

Nicotinamide adenine dinucleotide (NAD+), the essential cofactor derived from vitamin B3, is both a coenzyme in redox enzymatic processes and substrate in non-redox events; processes that are intimately implicated in all essential bioenergetics. A decrease in intracellular NAD+ levels is known to cause multiple metabolic complications and age-related disorders. One NAD+ precursor is dihydronicotinamide riboside (NRH), which increases NAD+ levels more potently in both cultured cells and mice than current supplementation strategies with nicotinamide riboside (NR), nicotinamide mononucleotide (NMN) or vitamin B3 (nicotinamide and niacin). However, the consequences of extreme boosts in NAD+ levels are not fully understood. Here, we demonstrate the cell-specific effects of acute NRH exposure in mammalian cells. Hepatocellular carcinoma (HepG3) cells show dose-dependent cytotoxicity when supplemented with 100–1000 μM NRH. Cytotoxicity was not observed in human embryonic kidney (HEK293T) cells over the same dose range of NRH. PUMA and BAX mediate the cell-specific cytotoxicity of NRH in HepG3. When supplementing HepG3 with 100 μM NRH, a significant increase in ROS was observed concurrent with changes in the NAD(P)H and GSH/GSSG pools. NRH altered mitochondrial membrane potential, increased mitochondrial superoxide formation, and induced mitochondrial DNA damage in those cells. NRH also caused metabolic dysregulation, altering mitochondrial respiration. Altogether, we demonstrated the detrimental consequences of an extreme boost of the total NAD (NAD+ + NADH) pool through NRH supplementation in HepG3. The cell-specific effects are likely mediated through the different metabolic fate of NRH in these cells, which warrants further study in other systemic models.

Mitochondrial Superoxide Production Decreases on Glucose-Stimulated Insulin Secretion in Pancreatic β Cells Due to Decreasing Mitochondrial Matrix NADH/NAD+ Ratio.

Aims: Glucose-stimulated insulin secretion (GSIS) in pancreatic β cells was expected to enhance mitochondrial superoxide formation. Hence, we elucidated relevant redox equilibria.Results: Unexpectedly, INS-1E cells at transitions from 3 (11 mM; pancreatic islets from 5 mM) to 25 mM glucose decreased matrix superoxide release rates (MitoSOX Red monitoring validated by MitoB) and H2O2 (mitoHyPer, subtracting mitoSypHer emission). Novel double-channel fluorescence lifetime imaging, approximating free mitochondrial matrix NADHF, indicated its ∼20% decrease. Matrix NAD+F increased on GSIS, indicated by the FAD-emission lifetime decrease, reflecting higher quenching of FAD by NAD+F. The participation of pyruvate/malate and pyruvate/citrate redox shuttles, elevating cytosolic NADPHF (iNAP1 fluorescence monitoring) at the expense of matrix NADHF, was indicated, using citrate (2-oxoglutarate) carrier inhibitors and cytosolic malic enzyme silencing: All changes vanished on these manipulations. 13C-incorporation from 13C-L-glutamine into 13C-citrate reflected the pyruvate/isocitrate shuttle. Matrix NADPHF (iNAP3 monitored) decreased. With decreasing glucose, the suppressor of Complex III site Q electron leak (S3QEL) suppressor caused a higher Complex I IF site contribution, but a lower superoxide fraction ascribed to the Complex III site IIIQo. Thus, the diminished matrix NADHF/NAD+F decreased Complex I flavin site IF superoxide formation on GSIS.Innovation: Mutually validated methods showed decreasing superoxide release into the mitochondrial matrix in pancreatic β cells on GSIS, due to the decreasing matrix NADHF/NAD+F (NADPHF/NADP+F) at increasing cytosolic NADPHF levels. The developed innovative methods enable real-time NADH/NAD+ and NADPH/NADP+ monitoring in any distinct cell compartment.Conclusion: The export of reducing equivalents from mitochondria adjusts lower mitochondrial superoxide production on GSIS, but it does not prevent oxidative stress in pancreatic β cells.

Trastuzumab-induced cardiotoxicity and role of mitochondrial connexin43 in the adaptive response

Trastuzumab, the humanized monoclonal antibody specific for HER2 receptor, is the gold standard in the treatment of HER2+ breast cancer. Despite its high therapeutic efficacy, cardiotoxicity has emerged as a significant side effect. The molecular mechanisms involved are not well understood, but all converge on mitochondria. Mitochondrial Cx43 can confer cardioprotection by regulating mitochondrial calcium homeostasis, ROS production and propagation of apoptotic signals, and studies report that it is overexpressed both in ischemic preconditioning and in Doxorubicin-induced cardiotoxicity. This study was designed to evaluate whether mitochondrial Cx43 (mCx43) is also involved in Trastuzumab-induced cardiotoxicity. Here we demonstrated that mCx43 is overexpressed in Trastuzumab-treated H9c2 cells. Our data showed that inhibition of Cx43 translocation to mitochondria, obtained by radicicol pre-treatment, significantly increases cytosolic and mitochondrial superoxide formation, mitochondrial membrane depolarization and the consequent apoptosis induced by Trastuzumab. Our results support the hypothesis that disruption of mitochondrial function is the principal mechanism by which Trastuzumab elicits its cardiotoxicity and mCx43 appears to counteract the Trastuzumab-induced mitochondrial damage.

SVCT2-Dependent plasma and mitochondrial membrane transport of ascorbic acid in differentiating myoblasts

The Na+-dependent Vitamin C transporter 2 (SVCT2) is expressed in the plasma and mitochondrial membranes of various cell types. This notion was also established in proliferating C2C12 myoblasts (Mb), in which the transporter was characterised by a high and low affinity in the plasma and mitochondrial membranes, respectively. In addition, the mitochondrial expression of SVCT2 appeared particularly elevated and, consistently, a brief pre-exposure to low concentrations of Ascorbic Acid (AA) abolished mitochondrial superoxide formation selectively induced by the cocktail arsenite/ATP. Early myotubes (Mt) derived from these cells after 4 days of differentiation presented evidence of slightly increased SVCT2 expression, and were characterised by kinetic parameters for plasma membrane transport of AA in line with those detected in Mb. Confocal microscopy studies indicated that the mitochondrial expression of SVCT2 is well preserved in Mt with one or two nuclei, but progressively reduced in Mt with three or more nuclei. Cellular and mitochondrial expression of SVCT2 was found reduced in day 7 Mt. While the uptake studies were compromised by the poor purity of the mitochondrial preparations obtained from day 4 Mt, we nevertheless obtained evidence of poor transport of the vitamin using the same functional studies successfully employed with Mb. Indeed, even greater concentrations of/longer pre-exposure to AA failed to induce scavenging of mitochondrial superoxide in Mt. These results are therefore indicative of a severely reduced mitochondrial uptake of the vitamin in early Mt, attributable to decreased expression as well as impaired activity of mitochondrial SVCT2.

MitoTEMPO provides an antiarrhythmic effect in aged-rats through attenuation of mitochondrial reactive oxygen species.

The death prevalence from cardiovascular disease is significantly high in elderly-populations, while mitochondrial-aging plays an important in abnormal function of vital organs through high mitochondrial ROS production. Mitochondria have a unique mode of action by providing ATP production and modulating the cytosolic Ca2+-signaling and maintain the redox status of cardiomyocytes. There is an aging-associated impairment in oxidative phosphorylation which causes a marked dysregulation of mitochondrial biogenesis. Therefore, we aimed to examine whether a mitochondria-targeting antioxidant, MitoTEMPO, can directly provide a cardioprotective effect on ventricular cardiomyocyte function under in vitro conditions. The MitoTEMPO-treatment (0.1μM for 4-h) of aged-ventricular cardiomyocytes (from 24-mo-old rats), compared to those of the adults (from 8-mo-old rats) markedly augmented not only the depressed biochemical parameters but also the ultrastructure of mitochondria. It also provided marked protective action against increased mitochondrial superoxide formation and Bnip3 overexpression, which both markedly induce depolarized mitochondrial potential, increase reactive oxygen species, mitochondrial swelling and fission, and accelerate mitochondrial turnover via autophagy. Furthermore, it provided marked protection against spontaneous action potentials, via shortening the prolonged action potential duration, at most, through recovery in depressed K+-channel currents. Moreover, we determined significant recovery in the depressed intracellular Ca2+-changes under electrical stimulation in MitoTEMPO-treated the aged-cardiomyocytes. Overall, we provided important information associated with an antiarrhythmic action, thereby controlling cytosolic and mitochondrial Ca2+-handling, implying its possible protective role of mitochondria-targeting antioxidant-treatment during aging.

New and classical methods to compare oxidative stress levels and parameters of vascular function in rat models of hypertension, diabetes and nitrate tolerance

BackgroundOxidative stress was identified as a hallmark of almost all cardiovascular and neurodegenerative diseases. The term oxidative stress describes a condition that is either characterized by increased generation of reactive oxygen and nitrogen species (RONS) and/or dysregulated cellular antioxidant defense mechanisms (e.g. decreased expression of central antioxidant enzymes). Since the transition between oxidative stress and redox signaling is a thin line, the exact knowledge of the identity of formed reactive species, the cellular and subcellular localization of their formation as well as their time duration and concentration are of high clinical and pharmacological importance.Methods and ResultsWith the present studies, we evaluated the specific probe for mitochondrial superoxide formation, mitochondria‐targeted, triphenylphosphonium‐linked hydroethidium (mitoSOX), for its usefulness in the detection of mitochondrial superoxide formation ex vivo/in vivo and compared this HPLC‐based assay with alternative/traditional methods. For this purpose, we used three different animal models with well‐established oxidative stress burden, namely diabetic, hypertensive and nitrate tolerant rats. With our previous work we have stressed that the traditional or old RONS assays are not necessarily inferior as compared to up‐to‐date or even cutting‐edge assays and at least have their specific features and application spectra. Vascular function was assessed by isometric tension methodology and was impaired in the rat models of oxidative stress. Vascular dysfunction correlated with increased mitoSOX oxidation but also classical RONS detection assays as well as typical markers of oxidative stress. Additionally, we successfully applied this method for the mitochondrial superoxide detection in hypertensive (AT‐II induced), aircraft noise exposure mouse model as well as in the combination of these two stressors. The application of this method was also effective in the wild type and Nox2 knockout mice exposed to e‐cigarette vapor over 3 days for 2 h/d.ConclusionIn conclusion, we report that mitoSOX/HPLC indeed represents a suitable method for quantification of mitochondrial superoxide formation in biological samples of animals with cardiometabolic disease, pharmacologically induced cardiovascular complications and cardiovascular diseases induced by novel (environmental/behavioral) stressors. The knowledge on the spatial distribution of RONS formation may be of specific interest for drug development and when it comes to therapy.Support or Funding InformationOur experimental studies were supported by vascular biology research grants from the Boehringer Ingelheim Foundation for the collaborative research group „Novel and neglected cardiovascular risk factors: molecular mechanisms and therapeutic implications”.

Development and validation of an analytical assay for electrochemical detection and quantification of protein‐bound 3‐nitrotyrosine in biological samples and comparison with classical, antibody‐based methods

BackgroundReactive oxygen species (ROS) cause oxidative damage which is associated with endothelial dysfunction and cardiovascular disease. Majority of ROS are formed by mitochondria and NADPH oxidase. Overproduction of ROS leads to oxidative damage and disease progression, therefore, accurate determination of ROS and reactive nitrogen species (like nitric oxide, superoxide and peroxynitrite) is of great importance but also represents a great challenge, since these species are short‐lived and methods that are usually used relay on indirect detection. Here we compared 3 different methods for the detection of 3‐nitrotyrosine, a footprint of peroxynitrite formation in vivo, in biological samples.Methods and ResultsNitrated proteins were generated by incubation with peroxynitrite or 3‐morpholino sydnonimine (SIN‐1) and digested using pronase, a mixture of different proteases. The free 3‐nitrotyrosine was then separated by HPLC and quantified by a coulometric method using the CoulArray system. Concentration‐response‐curves of 3‐nitrotyrosine standards were highly linear (detection limit 50 nM). Concentration‐response‐curves of free 3‐nitrotyrosine obtained from digested nitrated bovine serum albumin standards were also highly linear before reaching a plateau, probably representing nitration of all available tyrosine residues. The presence of 3‐nitrotyrosine in nitrated bovine serum albumin standards was validated by other methods (ELISA and dot blot analysis using specific 3‐nitrotyrosine antibodies), showing a similar pattern as compared to HPLC/ECD detection. Ex vivo nitration of isolated mitochondrial or membranous fractions as well as serum/plasma yielded a concentration‐dependent 3‐nitrotyrosine signal using the HPLC/ECD method. Ongoing studies are dedicated to the quantification of 3‐nitrotyrosine in tissue samples of nitrate‐tolerant, diabetic, hypertensive and septic mice and rats. A major draw‐back seems to be the separation of the 3‐nitrotyrosine peak in complex biological samples, which is currently our main focus.ConclusionsBased on our ex vivo data, the CoulArray quantification method for 3‐nitrotyrosine seems to have some advantages regarding sensitivity and selectivity. In the future, we hope to be able to establish a reliable automated HPLC assay for the routine quantification of 3‐nitrotyrosine in biological samples of cell culture, animal and human origin. Our most recent data show that protein‐bound 3‐nitrotyrosine not only represents a footprint of peroxynitrite formation and marker of oxidative stress but also correlates with increased mitochondrial superoxide formation rates pointing towards self‐propagating oxidative stress vicious circles.Support or Funding InformationVascular biology research grant from the Boehringer Ingelheim Foundation for the collaborative research group „Novel and neglected cardiovascular risk factors: molecular mechanisms and therapeutic implications”