scholarly journals The intra-mitochondrial O-GlcNAcylation system acutely regulates OXPHOS capacity and ROS dynamics in the heart

2021 ◽  
Author(s):  
Justine Dontaine ◽  
Asma Bouali ◽  
Frederic Daussin ◽  
Laurent Bultot ◽  
Didier Vertommen ◽  
...  
Keyword(s):  
Mitochondrial Function ◽  
Complex I ◽  
Nadh Dehydrogenase ◽  
Regulatory Mechanism ◽  
Mitochondrial Proteins ◽  
Cardiac Mitochondria ◽  
Multiple Organs ◽  
Mitochondrial Ros ◽  
In Vitro Reconstitution

Abstract Protein O-GlcNAcylation is increasingly recognized as an important cellular regulatory mechanism, in multiple organs including the heart. However, the mechanisms leading to O-GlcNAcylation in mitochondria and the consequences on their function remain poorly understood. In this study, we used an in vitro reconstitution assay to characterize the intra-mitochondrial O- GlcNAc system without potential cytoplasmic confounding effects. We compared the O-GlcNAcylome of isolated cardiac mitochondria with that of mitochondria acutely exposed to NButGT, a specific O-GlcNAcylation inducer. Amongst the 409 O-GlcNAcylated mitochondrial proteins identified, 191 displayed increased O-GlcNAcylation in response to NButGT. This was associated with enhanced Complex I (CI) activity, increased maximal respiration in presence of CI substrates, and a striking reduction of mitochondrial ROS release, which could be related to O- GlcNAcylation of subunits within the NADH dehydrogenase module of CI. In conclusion, our work underlines the existence of a dynamic mitochondrial O-GlcNAcylation system capable of rapidly modifying mitochondrial function.

Abstract 355: Effect of Poloxamer 188 on Rat Brain Isolated Mitochondria After Exposure to Hydrogen Peroxide

Circulation ◽  
2019 ◽  
Vol 140 (Suppl_2) ◽  
Author(s):  
Johannes A Pille ◽  
Michele M Salzman ◽  
Anna A Sonju ◽  
Felicia P Lotze ◽  
Josephine E Hees ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Oxygen Consumption ◽  
Mitochondrial Function ◽  
Complex I ◽  
Room Temperature ◽  
In Vitro Model ◽  
Atp Synthesis ◽  
Complex Ii ◽  
Vitro Model

Introduction: In a pig model of myocardial infarction (MI), intracoronary delivered Poloxamer (P) 188 significantly reduces ischemia/reperfusion (IR) injury when given immediately upon reperfusion, with improved mitochondrial function as a predominant effect. As mitochondria are heavily damaged during IR, a direct effect of P188 on mitochondria may lead to better therapy options during reperfusion. To show not only a similar reduction of IR injury by P188 in the brain, but also a direct P188 effect on mitochondria, we established an in-vitro model of IR that consists of damaging isolated rat brain mitochondria with hydrogen peroxide (H 2 O 2 ), one component of ischemia, then applying P188, and analyzing mitochondrial function. Methods: Male Sprague-Dawley rat brains were removed, and the mitochondria isolated by differential centrifugation and Percoll gradients, then kept on ice to slow their bioenergetics prior to any experimental treatments. Mitochondria were exposed to 200 μM H 2 O 2 for 10 min at room temperature with slight agitation; controls received no H 2 O 2 . Samples were then diluted ½ with buffer ± P188 (250 μM after dilution) to simulate reperfusion and treatment, and kept at room temperature for 10 further minutes. ATP synthesis was measured in a luminometer using a luciferase enzymatic assay. Oxygen consumption was measured by closed cell respirometry with an oxygen meter. In both assays, Complex I and Complex II were examined; Complex I substrates glutamate and malate, Complex II substrate succinate plus the Complex I inhibitor rotenone. Statistics: Data are expressed as mean ± SEM. One-Way ANOVA, SNK-Test; Kruskal-Wallis-Test; α=0.05, * vs control. Results: In both Complex I and II, mitochondrial function was significantly impaired by H 2 O 2 , with ATP synthesis affected more at Complex I and oxygen consumption affected more at Complex II. Addition of P188 did not provide any significant improvement in mitochondrial function. Conclusions: Although P188 significantly reduced IR injury when given during reperfusion in a pig model of MI, it does not appear to provide direct protection to mitochondria in this in-vitro model. Whether the exposure to H 2 O 2 causes the appropriate injury for P188 to become effective remains to be elucidated.

scholarly journals Superoxide Dismutase 2 is dispensable for platelet function

2017 ◽  
Vol 117 (10) ◽  
pp. 1859-1867 ◽  
Author(s):  
Trevor P. Fidler ◽  
Jesse W. Rowley ◽  
Claudia Araujo ◽  
Luc H. Boudreau ◽  
Alex Marti ◽  
...  
Keyword(s):  
Superoxide Dismutase ◽  
Platelet Activation ◽  
Platelet Function ◽  
Mitochondrial Function ◽  
Oxygen Species ◽  
Platelet Dysfunction ◽  
Mitochondrial Ros ◽  
Superoxide Dismutase 2

SummaryIncreased intracellular reactive oxygen species (ROS) promote platelet activation. The sources of platelet-derived ROS are diverse and whether or not mitochondrial derived ROS, modulates platelet function is incompletely understood. Studies of platelets from patients with sickle cell disease, and diabetes suggest a correlation between mitochondrial ROS and platelet dysfunction. Therefore, we generated mice with a platelet specific knockout of superoxide dismutase 2 (SOD2-KO) to determine if increased mitochondrial ROS increases platelet activation. SOD2-KO platelets demonstrated decreased SOD2 activity and increased mitochondrial ROS, however total platelet ROS was unchanged. Mitochondrial function and content were maintained in non-stimulated platelets. However SOD2-KO platelets demonstrated decreased mitochondrial function following thrombin stimulation. In vitro platelet activation and spreading was normal and in vivo, deletion of SOD2 did not change tail-bleeding or arterial thrombosis indices. In pathophysiological models mediated by platelet-dependent immune mechanisms such as sepsis and autoimmune inflammatory arthritis, SOD2-KO mice were phenotypically identical to wildtype controls. These data demonstrate that increased mitochondrial ROS does not result in platelet dysfunction.

scholarly journals Tumor growth of neurofibromin-deficient cells is driven by decreased respiration and hampered by NAD+ and SIRT3

2021 ◽  
Author(s):  
Ionica Masgras ◽  
Giuseppe Cannino ◽  
Francesco Ciscato ◽  
Carlos Sanchez-Martin ◽  
Marco Pizzi ◽  
...  
Keyword(s):  
Succinate Dehydrogenase ◽  
Complex I ◽  
Nadh Dehydrogenase ◽  
Chain Complex ◽  
Respiratory Chain Complex ◽  
Succinate Dehydrogenase Activity ◽  
Nadh Ratio ◽  
Shed Light

Neurofibromin loss drives neoplastic growth and a rewiring of mitochondrial metabolism. Here, we report that neurofibromin ablation dampens expression and activity of NADH dehydrogenase, the respiratory chain complex I, in an ERK-dependent fashion. This provides cells with resistance to pro-oxidants targeting complex I and decreases both respiration and intracellular NAD+. Expression of the alternative NADH dehydrogenase NDI1 raises NAD+/NADH ratio, enhances the activity of the mitochondrial NAD+-dependent deacetylase SIRT3 and interferes with tumorigenicity in neurofibromin-deficient cells. This anti-neoplastic effect is mimicked both in vitro and in vivo by administration of NAD+ precursors or by rising expression of the NAD+ deacetylase SIRT3, and is synergistic with ablation of the mitochondrial chaperone TRAP1, which augments succinate dehydrogenase activity further contributing to block pro-neoplastic metabolic changes of these cells. These findings shed light on chemotherapeutic resistance and on bioenergetic adaptations of tumors lacking neurofibromin, linking complex I inhibition to mitochondrial NAD+/NADH unbalance and SIRT3 inhibition, as well as to down-regulation of succinate dehydrogenase. This metabolic rewiring could unveil attractive therapeutic targets for neoplasms related to neurofibromin loss.

scholarly journals High temperature impairs mitochondrial function in rainbow trout cardiac mitochondria

2021 ◽  
pp. jeb.242382
Author(s):  
Jakob Michaelsen ◽  
Angela Fago ◽  
Amanda Bundgaard
Keyword(s):  
Rainbow Trout ◽  
High Temperature ◽  
Mitochondrial Function ◽  
Mitochondrial Respiration ◽  
Complex I ◽  
Effect Of Temperature ◽  
Cardiac Mitochondria ◽  
Aerobic Scope ◽  
Respiration Rates ◽  
Complex I Activity

Mitochondria provides cellular energy through oxidative phosphorylation, and thus temperature-induced constraints on mitochondrial function may be crucial to animal aerobic scope and thermal tolerance. Here, we report the effect of temperature in the range 5-30°C on respiration rates of isolated cardiac mitochondria from rainbow trout (Oncorhyncus mykiss) studied by high-resolution respirometry and spectrophotometric enzyme activity assays. Arrhenius breakpoint temperature analysis indicated that mitochondrial respiration rates under phosphorylating and fully uncoupled conditions increased exponentially up to 20°C, but stopped increasing at higher temperatures. In contrast, respiration rates measured under non-phosphorylating leak conditions continued to increase up to 30°C. The decrease in the ratio between phosphorylating and uncoupled respiration at high temperature indicated that phosphorylation was gradually impaired with increasing temperature, possibly because of the steadily increasing proton leak across the membrane. In addition, we found that complex I (NADH dehydrogenase) activity decreased above 20°C, similarly to mitochondrial respiration, and that complex I was unstable in the presence of detergents, suggesting that it may be particularly sensitive to changes in its interaction with membrane phospholipids. In contrast, complex II (succinate dehydrogenase) maintained activity at temperatures above 20°C, although succinate oxidation was insufficient to compensate for the loss of complex I activity in intact mitochondria. Together, these results indicate that the temperature-induced decrease in cardiac mitochondrial function coincides with the temperature at which trout aerobic scope peaks, and is largely due to impaired phosphorylation and complex I activity.

scholarly journals An antibody toolbox to track complex I assembly defines AIF’s mitochondrial function

2020 ◽  
Vol 219 (10) ◽  
Author(s):  
Anjaneyulu Murari ◽  
Shauna-Kay Rhooms ◽  
Naga Sri Goparaju ◽  
Maximino Villanueva ◽  
Edward Owusu-Ansah
Keyword(s):  
Mitochondrial Function ◽  
Complex I ◽  
Biosynthetic Pathway ◽  
Mitochondrial Proteins ◽  
Contact Site ◽  
Apoptosis Inducing Factor ◽  
Complex I Assembly

An ability to comprehensively track the assembly intermediates (AIs) of complex I (CI) biogenesis in Drosophila will enable the characterization of the precise mechanism(s) by which various CI regulators modulate CI assembly. Accordingly, we generated 21 novel antibodies to various mitochondrial proteins and used this resource to characterize the mechanism by which apoptosis-inducing factor (AIF) regulates CI biogenesis by tracking the AI profile observed when AIF expression is impaired. We find that when the AIF–Mia40 translocation complex is disrupted, the part of CI that transfers electrons to ubiquinone is synthesized but fails to progress in the CI biosynthetic pathway. This is associated with a reduction in intramitochondrial accumulation of the Mia40 substrate, MIC19. Importantly, knockdown of either MIC19 or MIC60, components of the mitochondrial contact site and cristae organizing system (MICOS), fully recapitulates the AI profile observed when AIF is inhibited. Thus, AIF’s effect on CI assembly is principally due to compromised intramitochondrial transport of the MICOS complex.

scholarly journals A yeast-based drug discovery platform to identify Plasmodium falciparum type II NADH dehydrogenase inhibitors

2021 ◽  
Author(s):  
Yu Cao ◽  
Chen Sun ◽  
Han Wen ◽  
Mengfei Wang ◽  
Pan Zhu ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Complex I ◽  
Nadh Dehydrogenase ◽  
Specific Inhibitor ◽  
Type I ◽  
Type Ii ◽  
Protein Activity ◽  
Asexual Blood Stage

Conventional methods utilizing in vitro protein activity assay or in vivo parasite survival to screen for malaria inhibitors suffer from high experimental background and/or inconvenience. Here we introduce a yeast-based system to facilitate chemical screen for specific protein or pathway inhibitors. The platform comprises several isogeneic Pichia strains that only differ in the target of interest, so that a compound which inhibits one strain but not the other is implicated in working specifically against the target. We used Plasmodium falciparum NDH2(PfNDH2), a type II NADH dehydrogenase, as a proof of principle to show how well this works. Three isogenic Pichia strains harboring respectively exogeneously introduced PfNDH2, its own complex I (a type I NADH dehydrogenase), and PfNDH2 with its own complex I were constructed. In a pilot screen of more than2000 compounds, we identified a highly specific inhibitor that acts on PfNDH2. This compound poorly inhibit the parasites at the asexual blood stage, however, is highly effective in repressing oocyst maturation in the mosquito stage. Our results demonstrate that the yeast cell based screen platform is feasible, efficient, economical and with very low background noise. Similar strategies could be extended to the functional screen for interacting molecules of other targets.

scholarly journals Adaptation of mitochondrial expression and ATP production in dedifferentiating vascular smooth muscle cells

2017 ◽  
Vol 95 (12) ◽  
pp. 1473-1479 ◽  
Author(s):  
Celena Scheede-Bergdahl ◽  
Andreas Bergdahl
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Mitochondrial Function ◽  
Complex I ◽  
Clinical Manifestations ◽  
Western World ◽  
Aortic Tissue ◽  
Atp Production

Atherosclerosis is one of the leading causes of morbidity and mortality in the Western world. Although the clinical manifestations of this disease are well documented, the etiology and progression remain to be fully understood. Recently, the mitochondria have been implicated in important cellular processes involved in development of atherosclerosis. Despite the link between mitochondria and atherosclerosis, early-phase mechanisms of the disease have yet to be elucidated. The aim of this project was to explore the role of mitochondria in vascular smooth muscle (VSMC) dedifferentiation. A murine in vitro model, involving organ culture of aortic tissue in serum-free media, was used. Mitochondrial function was measured by high-resolution respirometry. Proteins associated with the VSMC phenotype switch, as well as mitochondrial density, were assessed by immunoblotting. The findings show that intrinsic mitochondrial Complex I activity is significantly upregulated during VSMC dedifferentiation. Diminished coupling between phosphorylation and oxidation was also found, indicating a greater ADP:ATP ratio. This data suggests increased leak in the electron transport chain and altered mitochondrial function specifically at Complex I. This project provides important information regarding the role of mitochondria in the early atherosclerotic process and that detectable changes in mitochondrial function and expression are related to VSMC dedifferentiation.

scholarly journals Differential Effects of Yeast NADH Dehydrogenase (Ndi1) Expression on Mitochondrial Function and Inclusion Formation in a Cell Culture Model of Sporadic Parkinson’s Disease

Biomolecules ◽  
2019 ◽  
Vol 9 (4) ◽  
pp. 119 ◽  
Author(s):  
Emily N. Cronin-Furman ◽  
Jennifer Barber-Singh ◽  
Kristen E. Bergquist ◽  
Takao Yagi ◽  
Patricia A. Trimmer
Keyword(s):  
Gene Expression ◽  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Mitochondrial Dysfunction ◽  
Mitochondrial Function ◽  
Lewy Body ◽  
Complex I ◽  
Nadh Dehydrogenase ◽  
Lewy Bodies ◽  
Body Formation

Parkinson’s disease (PD) is a neurodegenerative disorder that exhibits aberrant protein aggregation and mitochondrial dysfunction. Ndi1, the yeast mitochondrial NADH dehydrogenase (complex I) enzyme, is a single subunit, internal matrix-facing protein. Previous studies have shown that Ndi1 expression leads to improved mitochondrial function in models of complex I-mediated mitochondrial dysfunction. The trans-mitochondrial cybrid cell model of PD was created by fusing mitochondrial DNA-depleted SH-SY5Y cells with platelets from a sporadic PD patient. PD cybrid cells reproduce the mitochondrial dysfunction observed in a patient’s brain and periphery and form intracellular, cybrid Lewy bodies comparable to Lewy bodies in PD brain. To improve mitochondrial function and alter the formation of protein aggregates, Ndi1 was expressed in PD cybrid cells and parent SH-SY5Y cells. We observed a dramatic increase in mitochondrial respiration, increased mitochondrial gene expression, and increased PGC-1α gene expression in PD cybrid cells expressing Ndi1. Total cellular aggregated protein content was decreased but Ndi1 expression was insufficient to prevent cybrid Lewy body formation. Ndi1 expression leads to improved mitochondrial function and biogenesis signaling, both processes that could improve neuron survival during disease. However, other aspects of PD pathology such as cybrid Lewy body formation were not reduced. Consequently, resolution of mitochondrial dysfunction alone may not be sufficient to overcome other aspects of PD-related cellular pathology.

scholarly journals Capsaicin Alleviates the Deteriorative Mitochondrial Function by Upregulating 14-3-3η in Anoxic or Anoxic/Reoxygenated Cardiomyocytes

2020 ◽  
Vol 2020 ◽  
pp. 1-16 ◽  
Author(s):  
Yang Qiao ◽  
Tianhong Hu ◽  
Bin Yang ◽  
Hongwei Li ◽  
Tianpeng Chen ◽  
...  
Keyword(s):  
Mitochondrial Function ◽  
Complex I ◽  
Redox Homeostasis ◽  
Redox Status ◽  
Neonatal Rat ◽  
Ros Generation ◽  
Consumption Rates ◽  
Vitro Model ◽  
The Impact

Reactive oxygen species (ROS) are byproducts of a defective electron transport chain (ETC). The redox couples, GSH/GSSG and NAD+/NADH, play an essential role in physiology as internal defenses against excessive ROS generation by facilitating intracellular/mitochondrial (mt) redox homeostasis. Anoxia alone and anoxia/reoxygenation (A/R) are dissimilar pathological processes. In this study, we measured the impact of capsaicin (Cap) on these pathological processes using a primary cultured neonatal rat cardiomyocyte in vitro model. The results showed that overproduction of ROS was tightly associated with disturbed GSH/GSSG and NAD+/NADH suppressed mt complex I and III activities, decreased oxygen consumption rates, and elevated extracellular acidification rates. During anoxia or A/R period, these indices interact with each other causing the mitochondrial function to worsen. Cap protected cardiomyocytes against the different stages of A/R injury by rescuing NAD+/NADH, GSH/GSSG, and mt complex I/III activities and cellular energy metabolism. Importantly, Cap-mediated upregulation of 14-3-3η, a protective phosphoserine-binding protein in cardiomyocytes, ameliorated mt function caused by a disruptive redox status and an impaired ETC. In conclusion, redox pair, mt complex I/III, and metabolic equilibrium were significantly different in anoxia alone and A/R injury; Cap through upregulating 14-3-3η plays a protection against the above injury in cardiomyocyte.

Mitochondrial complex I, aconitase, and succinate dehydrogenase during hypoxia-reoxygenation: modulation of enzyme activities by MnSOD

2003 ◽  
Vol 285 (1) ◽  
pp. L189-L198 ◽  
Author(s):  
Charles S. Powell ◽  
Robert M. Jackson
Keyword(s):  
Epithelial Cell ◽  
Succinate Dehydrogenase ◽  
Enzyme Activities ◽  
Complex I ◽  
Nadh Dehydrogenase ◽  
Alveolar Epithelial Cell ◽  
A549 Cells ◽  
Enzyme Inactivation ◽  
Hypoxia Reoxygenation

Both NADH dehydrogenase (complex I) and aconitase are inactivated partially in vitro by superoxide ([Formula: see text]) and other oxidants that cause loss of iron from enzyme cubane (4Fe-4S) centers. We tested whether hypoxia-reoxygenation (H-R) by itself would decrease lung epithelial cell NADH dehydrogenase, aconitase, and succinate dehydrogenase (SDH) activities and whether transfection with adenoviral vectors expressing MnSOD (Ad.MnSOD) would inhibit oxidative enzyme inactivation and thus confirm a mechanism involving [Formula: see text]. Human lung carcinoma cells with alveolar epithelial cell characteristics (A549 cells) were exposed to <1% O2-5% CO2(hypoxia) for 24 h followed by air-5% CO2for 24 h (reoxygenation). NADH dehydrogenase activity was assayed in submitochondrial particles; aconitase and SDH activities were measured in cell lysates. H-R significantly decreased NADH dehydrogenase, aconitase, and SDH activities. Ad.MnSOD increased mitochondrial MnSOD substantially and prevented the inhibitory effects of H-R on enzyme activities. Addition of α-ketoglutarate plus aspartate, but not succinate, to medium prevented cytotoxicity due to 2,3-dimethoxy-1,4-naphthoquinone. After hypoxia, cells displayed significantly increased dihydrorhodamine fluorescence, indicating increased mitochondrial oxidant production. Inhibition of NADH dehydrogenase, aconitase, and SDH activities during reoxygenation are due to excess [Formula: see text] produced in mitochondria, because enzyme inactivation can be prevented by overexpression of MnSOD.

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