Reactive Oxygen Species Emission Research Articles

3143Excessive mitochondrial reactive oxygen species emission from circulating blood cells is associated with severity of heart failure and exercise intolerance

3143Excessive mitochondrial reactive oxygen species emission from circulating blood cells is associated with severity of heart failure and exercise intolerance

Molecular and Functional Characterization of Fcγ Receptor IIIb-Ligand Interaction: Implications for Neutrophil-Mediated Immune Mechanisms in Malaria.

The Fcγ receptor IIIb (FcγRIIIb) is a low-affinity receptor of IgG and is essential in neutrophil-mediated effector functions. Different allelic forms of FcγRIIIb carrying human neutrophil antigen (HNA-1a, -1b, -1c, and -1d) have been identified. Here, we have generated stable transfected HEK293 cell lines expressing HNA-1aa, -1bb, and -1bc. Of these, cells expressing HNA-1bc interacted significantly stronger (binding affinities, 2.277 versus 0.743) with human IgG than cells expressing the HNA-1aa or -1bb alloforms. The higher affinity of IgG toward the HNA-1c alloform was confirmed using neutrophils derived from German blood donors. Neutrophils from HNA-1abc-phenotyped individuals bound IgG significantly stronger (1.825 versus 0.903) than did neutrophils from HNA-1ab-typed individuals. These findings were confirmed by surface plasmon resonance (SPR) analysis demonstrating that recombinant HNA-1bc had a higher affinity (dissociation constant [Kd ], 7.24 × 10-6 M) than recombinant HNA-1bb (Kd , 1.15 × 10-5 M) against normal IgG. Finally, we demonstrated that Plasmodium falciparum merozoites opsonized with human IgG affinity purified against P. falciparum glutamate-rich protein (GLURP) enhanced stronger reactive oxygen species (ROS) emission in neutrophils obtained from HNA-1abc donors than in neutrophils from HNA-1ab donors. Collectively, these results indicate that the amino acid substitution Ala78Asp resulting in the HNA-1c allotype leads to higher affinity toward human IgG, enhancement of neutrophil activation, and possibly effective clearance of malaria by intracellular ROS.

Effect of Tim23 knockdown in vivo on mitochondrial protein import and retrograde signaling to the UPRmt in muscle.

The mitochondrial unfolded protein response (UPRmt) is a protein quality control mechanism that strives to achieve proteostasis in the face of misfolded proteins. Because of the reliance of mitochondria on both the nuclear and mitochondrial genomes, a perturbation of the coordination of these genomes results in a mitonuclear imbalance in which holoenzymes are unable to assume mature stoichiometry and thereby activates the UPRmt. Thus, we sought to perturb this genomic coordination by using a systemic antisense oligonucleotide (in vivo morpholino) targeted to translocase of the inner membrane channel subunit 23 (Tim23), the major channel of the inner membrane. This resulted in a 40% reduction in Tim23 protein content, a 32% decrease in matrix-destined protein import, and a trend to elevate reactive oxygen species (ROS) emission under maximal respiration conditions. This import defect activated the C/EBP homologous protein (CHOP) branch of the UPRmt, as evident from increases in caseinolytic mitochondrial matrix peptidase proteolytic subunit (ClpP) and chaperonin 10 (cpn10) but not the activating transcription factor 5 (ATF5) arm. Thus, in the face of proteotoxic stress, CHOP and ATF5 could be activated independently to regain proteostasis. Our second aim was to investigate the role of proteolytically derived peptides in mediating retrograde signaling. Peptides released from the mitochondrion following basal proteolysis were isolated and incubated with import reactions. Dose- and time-dependent effect of peptides on protein import was observed. Our data suggest that mitochondrial proteolytic byproducts exert an inhibitory effect on protein import, possibly to reduce excessive protein import as a potential negative feedback mechanism. The inhibition of import into the organelle also serves a retrograde function, possibly via ROS emission, to modify nuclear gene expression and ultimately improve folding capacity.

Effect of Insulin Deprivation on Brain Mitochondrial ATP Production and Mitochondrial Proteome in Diabetic Mice

Accumulating evidence indicates a strong association between diabetic status and degenerative brain disease, including cognitive impairment. Here we hypothesized that although insulin is not necessary for glucose metabolism in brain, like in skeletal muscle insulin is critical for mitochondrial function in brain. We measured oxygen consumption rate (OCR), maximal ATP production rate (MAPR), and reactive oxygen species (ROS) production in isolated brain mitochondria of streptozotocin (STZ) diabetic mice. STZ mice deprived of insulin treatment for 96 hours (D-I), in comparison to nondiabetic mice (ND) showed a significant reduction in State 3 MAPR [1.77±0.14 vs. 2.35±0.27 (pmol/s/ug)], phosphorylation efficiency [0.42±0.04 vs. 0.54±0.07 (mol ATP produced/mol of oxygen consumed)] and respiratory control ratio [2.95±0.16 vs. 3.12±0.18] indicating reduced mitochondrial coupling efficiency during insulin deprivation. Citrate synthase and cytochrome c oxidase activities were significantly decreased in the hypothalamus of D-I compared ND. Surprisingly, no significant differences in ROS emission were detected between ND and D-I suggesting additional factors, potentially elevated ketone bodies, may protect against increased oxidative stress in the D-I brain. Proteomics analysis of the cerebrum found post-translational modification involving oxidative damage of several mitochondrial proteins that potentially cause tau phosphorylation and neurofibrillary degeneration in D-I vs. ND. In STZ mice with continued insulin treatment (D+I), state 3 OCR, MAPR, and phosphorylation efficiency were comparable to ND, demonstrating the restorative effects of insulin and euglycemia on brain mitochondrial function. We conclude that insulin deprivation and hyperglycemia significantly alter brain mitochondrial function. Insulin treatment and control of glycemia could have salutary effects on these parameters. Disclosure G. Ruegsegger: None. M. Shankarappa: None. P. Summer: None. S. Gopala: None. P. Zabielski: None. K. Klaus: None. S. Dasari: None. I.R. Lanza: None. K. Nair: None.

Particle-bound reactive oxygen species (PB-ROS) emissions and formation pathways in residential wood smoke under different combustion and aging conditions

Abstract. Wood combustion emissions can induce oxidative stress in the human respiratory tract by reactive oxygen species (ROS) in the aerosol particles, which are emitted either directly or formed through oxidation in the atmosphere. To improve our understanding of the particle-bound ROS (PB-ROS) generation potential of wood combustion emissions, a suite of smog chamber (SC) and potential aerosol mass (PAM) chamber experiments were conducted under well-determined conditions for different combustion devices and technologies, different fuel types, operation methods, combustion regimes, combustion phases, and aging conditions. The PB-ROS content and the chemical properties of the aerosols were quantified by a novel ROS analyzer using the DCFH (2′,7′-dichlorofluorescin) assay and a high-resolution time-of-flight aerosol mass spectrometer (HR-ToF-AMS). For all eight combustion devices tested, primary PB-ROS concentrations substantially increased upon aging. The level of primary and aged PB-ROS emission factors (EFROS) were dominated by the combustion device (within different combustion technologies) and to a greater extent by the combustion regimes: the variability within one device was much higher than the variability of EFROS from different devices. Aged EFROS under bad combustion conditions were ∼ 2–80 times higher than under optimum combustion conditions. EFROS from automatically operated combustion devices were on average 1 order of magnitude lower than those from manually operated devices, which indicates that automatic combustion devices operated at optimum conditions to achieve near-complete combustion should be employed to minimize PB-ROS emissions. The use of an electrostatic precipitator decreased the primary and aged ROS emissions by a factor of ∼ 1.5 which is however still within the burn-to-burn variability. The parameters controlling the PB-ROS formation in secondary organic aerosol were investigated by employing a regression model, including the fractions of the mass-to-charge ratios m∕z 44 and 43 in secondary organic aerosol (SOA; f44−SOA and f43−SOA), the OH exposure, and the total organic aerosol mass. The regression model results of the SC and PAM chamber aging experiments indicate that the PB-ROS content in SOA seems to increase with the SOA oxidation state, which initially increases with OH exposure and decreases with the additional partitioning of semi-volatile components with lower PB-ROS content at higher OA concentrations, while further aging seems to result in a decay of PB-ROS. The results and the special data analysis methods deployed in this study could provide a model for PB-ROS analysis of further wood or other combustion studies investigating different combustion conditions and aging methods.

Calcium Signaling and Reactive Oxygen Species in Mitochondria.

In heart failure, alterations of Na+ and Ca2+ handling, energetic deficit, and oxidative stress in cardiac myocytes are important pathophysiological hallmarks. Mitochondria are central to these processes because they are the main source for ATP, but also reactive oxygen species (ROS), and their function is critically controlled by Ca2+ During physiological variations of workload, mitochondrial Ca2+ uptake is required to match energy supply to demand but also to keep the antioxidative capacity in a reduced state to prevent excessive emission of ROS. Mitochondria take up Ca2+ via the mitochondrial Ca2+ uniporter, which exists in a multiprotein complex whose molecular components were identified only recently. In heart failure, deterioration of cytosolic Ca2+ and Na+ handling hampers mitochondrial Ca2+ uptake and the ensuing Krebs cycle-induced regeneration of the reduced forms of NADH (nicotinamide adenine dinucleotide) and NADPH (nicotinamide adenine dinucleotide phosphate), giving rise to energetic deficit and oxidative stress. ROS emission from mitochondria can trigger further ROS release from neighboring mitochondria termed ROS-induced ROS release, and cross talk between different ROS sources provides a spatially confined cellular network of redox signaling. Although low levels of ROS may serve physiological roles, higher levels interfere with excitation-contraction coupling, induce maladaptive cardiac remodeling through redox-sensitive kinases, and cell death through mitochondrial permeability transition. Targeting the dysregulated interplay between excitation-contraction coupling and mitochondrial energetics may ameliorate the progression of heart failure.

Effects of Buffer Composition and Substrates on Calcium‐stimulated Mitochondrial Respiration and Free Radical Emission

Mitochondrial ATP synthesis is tightly coupled to oxygen consumption in a highly regulated process; however, unavoidable byproducts in the form of reactive oxygen species (ROS) are formed. An important regulator of this process is calcium, but the precise relationship between calcium and free radical production is obscure. Therefore, we measured ROS production under a variety of experimental conditions that mimic the pathological environmental setting. The Oroborous oxygraph system was used to simultaneously measure ROS emission and the oxygen consumption rate from isolated cardiac guinea pig mitochondria energized with different substrates and challenged with different calcium concentrations. The Amplex Red assay was used to quantify ROS emission rates. The substrates used utilized a mixture of the carbohydrate and fatty acid dependent metabolic pathways. Two respiration buffers, one lactobionate‐based and the other potassium chloride‐based, were used to quantify the differences in respiration and ROS emission rates. Relative to the potassium chloride‐based respiration buffer, the lactobionate‐based buffer resulted in higher ADP‐stimulated respiration rates and higher ROS emission rates for each substrate combination. On average, the presence of calcium depressed ROS emission rates during the leak respiration, sodium/calcium cycling, and ADP‐stimulated states. The lower respiration rates in the potassium chloride‐based buffer is likely due to the presence of inhibitory levels of chloride. The high chloride levels also appear to depress ROS emission. Overall, these data refute the hypothesis that calcium stimulates ROS emission from isolated mitochondria. Studies are ongoing to further characterize ROS emission from cardiac guinea pig mitochondria and elucidate the relationship between calcium and ROS emission.Support or Funding InformationR00‐HL121160This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Autophagy promotes cancer chemotherapy‐induced oxidative stress and skeletal muscle dysfunction

Chemotherapy‐induced cachexia is a debilitating condition characterized by fatigue and muscle wasting. The onset of muscle weakness in patients often limits treatment, resulting in reduced quality of life and increased morbidity. Our previous findings show that the chemotherapeutic agent doxorubicin (DOX) causes myotoxicity as a result of reactive oxygen species (ROS) generation during intracellular metabolism of DOX by the mitochondria, and subsequent activation of the autophagy/lysosomal system. We hypothesize that DOX‐induced accelerated autophagy results in the elimination of vital cellular organelles and proteins, resulting in functional deficits to the muscle tissue. To prevent elevated activity of the autophagy/lysosomal system, we administered a dominant negative mutation of the autophagy protein ATG5 (rAAV‐dnATG5) via intramuscular injection of the rat soleus muscle four weeks prior to treatment. Animals received either DOX (20 mg/kg ip) or saline. Two days after chemotherapy, soleus muscle specific force production and fiber cross‐sectional area were significantly reduced in control animals. These changes corresponded with deficits to mitochondrial respiration, increased mitochondrial ROS emission and the increased appearance of autophagic vacuoles within the muscle. In contrast, rAAV‐dnATG5 administration in DOX treated animals prevented the conjugation of ATG12 and ATG5 thereby reducing autophagosome formation within the soleus muscle. Interestingly, the protein expression of catalase and superoxide dismutase 2 (SOD2) was elevated in these animals, which may account for improved mitochondrial respiration and reduced mitochondrial free radical production. Finally, DOX‐induced muscle fiber atrophy and contractile dysfunction were prevented in rAAV‐dnATG5 animals. These findings indicate that accelerated autophagy is an underlying cause of chemotherapy‐induced oxidative damage and skeletal muscle weakness.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Exercise-induced reductions in mitochondrial ADP sensitivity contribute to the induction of gene expression and mitochondrial biogenesis through enhanced mitochondrial H2O2 emission

Acute exercise rapidly induces mitochondrial gene expression, however, the intracellular events regulating this process remain incompletely understood. The purpose of this study was to determine whether reductions in mitochondrial ADP sensitivity during exercise have a biological role in regulating mitochondrial-derived reactive oxygen species (ROS) production and the induction of mitochondrial biogenesis. Mitochondrial creatine kinase wildtype (WT) and knockout (KO) mice have divergent responses in ADP sensitivity during exercise, and we therefore used these mice to determine the relationship between mitochondrial ADP sensitivity, ROS production, and mitochondrial adaptations to exercise. In WT mice, acute exercise reduced mitochondrial ADP respiratory sensitivity and the ability of ADP to suppress ROS production, while increasing mitochondrial gene transcription (PGC-1α, PGC-1β and PDK4). In stark contrast, in KO mice, exercise increased ADP sensitivity, reduced mitochondrial ROS emission, and did not induce gene transcription. Despite the divergence in mRNA responses, exercise similarly induced calcium/calmodulin-dependent protein kinase II (CaMKII) and AMP-activated protein kinase (AMPK) phosphorylation in WT and KO mice, however only WT mice were associated with redox stress (4HNE). These data implicate acute changes in ADP sensitivity in mitochondrial adaptations to exercise. To further examine this we chronically exercise trained mice. While training increased mitochondrial content and reduced ADP sensitivity in WT mice, KO mice did not exhibit adaptations to exercise. Combined, these data suggest that exercise-induced attenuations in mitochondrial ADP sensitivity mediate redox signals that contribute to the induction of acute and chronic mitochondrial adaptations.

Metabolic Costs of Exposure to Wastewater Effluent Lead to Compensatory Adjustments in Respiratory Physiology in Bluegill Sunfish.

Municipal wastewater effluent is a major source of aquatic pollution and has potential to impact cellular energy metabolism. However, it is poorly understood whether wastewater exposure impacts whole-animal metabolism and whether this can be accommodated with adjustments in respiratory physiology. We caged bluegill sunfish (Lepomis macrochirus) for 21 days at two sites downstream (either 50 or 830 m) from a wastewater treatment plant (WWTP). Survival was reduced in fish caged at both downstream sites compared to an uncontaminated reference site. Standard rates of O2 consumption increased in fish at contaminated sites, reflecting a metabolic cost of wastewater exposure. Several physiological adjustments accompanied this metabolic cost, including an expansion of the gill surface area available for gas exchange (reduced interlamellar cell mass), a decreased blood-O2 affinity (which likely facilitates O2 unloading at respiring tissues), increased respiratory capacities for oxidative phosphorylation in isolated liver mitochondria (supported by increased succinate dehydrogenase, but not citrate synthase, activity), and decreased mitochondrial emission of reactive oxygen species (ROS). We conclude that exposure to wastewater effluent invokes a metabolic cost that leads to compensatory respiratory improvements in O2 uptake, delivery, and utilization.

Interspecific variation in brain mitochondrial complex I and II capacity and ROS emission in marine sculpins.

Environmental hypoxia presents a metabolic challenge for animals because it inhibits mitochondrial respiration and can lead to the generation of reactive oxygen species (ROS). We investigated the interplay between O2 use for aerobic respiration and ROS generation among sculpin fishes (Cottidae, Actinopterygii) that are known to vary in whole-animal hypoxia tolerance. We hypothesized that mitochondria from hypoxia-tolerant sculpins would show more efficient O2 use with a higher phosphorylation efficiency and lower ROS emission. We showed that brain mitochondria from more hypoxia-tolerant sculpins had lower complex I and higher complex II flux capacities compared with less hypoxia-tolerant sculpins, but these differences were not related to variation in phosphorylation efficiency (ADP/O) or mitochondrial coupling (respiratory control ratio). The hypoxia-tolerant sculpins had higher mitochondrial H2O2 emission per O2 consumed (H2O2/O2) under oligomycin-induced state 4 conditions compared with less hypoxia-tolerant sculpins. An in vitro redox challenge experiment revealed species differences in how well mitochondria defend their glutathione redox status when challenged with high levels of reduced glutathione, but the redox challenge elicited the same H2O2/O2 in all species. Furthermore, in vitro anoxia recovery lowered absolute H2O2 emission (H2O2 per mg mitochondrial protein) in all species and negatively impacted state 3 respiration rates in some species, but the responses were not related to hypoxia tolerance. Overall, we clearly demonstrate a relationship between hypoxia tolerance and complex I and II flux capacities in sculpins, but the differences in complex flux capacity do not appear to be directly related to variation in ROS metabolism.

The guardian of the genome p53 regulates exercise‐induced mitochondrial plasticity beyond organelle biogenesis

The Guardian of the Genome p53 has been established as a potent tumour suppressor. However, culminating from seminal findings in rodents more than a decade ago, several studies have demonstrated that p53 is required to maintain basal mitochondrial function [ie, respiration and reactive oxygen species (ROS) homeostasis]. Specifically, via its role(s) as a tumour suppressor, p53 intimately surveys cellular DNA damage, in particular mitochondrial DNA (mtDNA), to ensure that the mitochondrial network is carefully monitored and cell viability is upheld, because aberrant mtDNA damage leads to apoptosis and widespread cellular perturbations. Indeed, data from rodents and humans have demonstrated that p53 forms an integral component of the exercise-induced signal transduction network regulating skeletal muscle mitochondrial remodelling. In response to exercise-induced disruptions to cellular homeostasis that have the potential to harm mtDNA (eg, contraction-stimulated ROS emissions), appropriate p53-regulated, mitochondrial turnover responses prevail to protect the genome and ultimately facilitate a shift from aerobic glycolysis to oxidative phosphorylation, adaptations critical for endurance-based exercise that are commensurate with p53's role as a tumour suppressor. Despite these observations, several discrepancies exist between rodent and human studies pinpointing p53 subcellular trafficking from nuclear-to-mitochondrial compartments following acute exercise. Such interspecies differences in p53 activity and the plausible p53-mediated adaptations to chronic exercise training will be discussed herein.

Crosstalk between autophagy and oxidative stress regulates proteolysis in the diaphragm during mechanical ventilation.

Mechanical ventilation (MV) results in the rapid development of ventilator-induced diaphragm dysfunction (VIDD). While the mechanisms responsible for VIDD are not fully understood, recent data reveal that prolonged MV activates autophagy in the diaphragm, which may occur as a result of increased cellular reactive oxygen species (ROS) production. Therefore, we tested the hypothesis that (1) accelerated autophagy is a key contributor to VIDD; and that (2) oxidative stress is required to increase the expression of autophagy genes in the diaphragm. Our findings reveal that targeted inhibition of autophagy in the rat diaphragm prevented MV-induced muscle atrophy and contractile dysfunction. Attenuation of VIDD in these animals occurred as a result of increased diaphragm concentration of the antioxidant catalase and reduced mitochondrial ROS emission, which corresponded to reductions in the activity of calpain and caspase-3. To determine if increased ROS production is required for the upregulation of autophagy biomarkers in the diaphragm, rats that were administered the mitochondrial-targeted peptide SS-31 during MV. Results from this study demonstrated that mitochondrial ROS production in the diaphragm during MV is required for the increased expression of key autophagy genes (i.e. LC3, Atg7, Atg12, Beclin1 and p62), as well as for increased activity of cathepsin L. Together, these data reveal that autophagy is required for VIDD, and that autophagy inhibition reduces MV-induced diaphragm ROS production and prevents a positive feedback loop whereby increased autophagy is stimulated by oxidative stress, resulting in further increases in ROS and autophagy.

Impact of prolonged overfeeding on skeletal muscle mitochondria in healthy individuals.

Reduced mitochondrial capacity in skeletal muscle has been observed in obesity and type 2 diabetes. In humans, the aetiology of this abnormality is not well understood but the possibility that it is secondary to the stress of nutrient overload has been suggested. To test this hypothesis, we examined whether sustained overfeeding decreases skeletal muscle mitochondrial content or impairs function. Twenty-six healthy volunteers (21 men, 5 women, age 25.3±4.5years, BMI 25.5±2.4kg/m2) underwent a supervised protocol consisting of 8weeks of high-fat overfeeding (40% over baseline energy requirements). Before and after overfeeding, we measured systemic fuel oxidation by indirect calorimetry and performed skeletal muscle biopsies to measure mitochondrial gene expression, content and function in vitro. Mitochondrial function in vivo was measured by 31P NMR spectroscopy. With overfeeding, volunteers gained 7.7±1.8kg (% change 9.8±2.3). Overfeeding increased fasting NEFA, LDL-cholesterol and insulin concentrations. Indirect calorimetry showed a shift towards greater reliance on lipid oxidation. In skeletal muscle tissue, overfeeding increased ceramide content, lipid droplet content and perilipin-2 mRNA expression. Phosphorylation of AMP-activated protein kinase was decreased. Overfeeding increased mRNA expression of certain genes coding for mitochondrial proteins (CS, OGDH, CPT1B, UCP3, ANT1). Despite the stress of nutrient overload, mitochondrial content and mitochondrial respiration in muscle did not change after overfeeding. Similarly, overfeeding had no effect on either the emission of reactive oxygen species or on mitochondrial function in vivo. Skeletal muscle mitochondria are significantly resilient to nutrient overload. The lower skeletal muscle mitochondrial oxidative capacity in human obesity is likely to be caused by reasons other than nutrient overload per se. ClinicalTrials.gov NCT01672632.

Abstract 24032: Exercise Instigates Apoptosis-inducing Factor Nuclear Translocation and Myocyte Death in Arrhythmogenic Cardiomyopathy

Exercise increases disease penetrance in arrhythmogenic cardiomyopathy (ACM). Yet, how exercise contributes to disease pathogenesis is unclear. Mitochondria potentiate reactive oxygen species (ROS) generation during exercise that are scavenged by antioxidants, such as thioredoxin-2 (Trx2). Here we tested if deficits in Trx2-based ROS buffering act as substrates for exercise-induced cardiac apoptosis in ACM. Homozygote Desmoglein-2 mutant mice ( Dsg2 mut/mut ) model ACM features, thus WT and Dsg2 mut/mut mice were enrolled in a 10 week swimming protocol to evaluate survival and post-effort cardiac function, ROS production by EPR, and mitochondrial ROS-gating protein levels. Survival rate after swimming was 91% (20/22) in WT, but only 60% (15/25) in Dsg2 mut/mut mice (p=0.008). Of the survivors, Dsg2 mut/mut mice displayed cardiac dysfunction (Ejection Fraction 57±4 vs 84±0.4% in WT; n≥14/cohort, P<0.001) and increased bouts of non-sustained VT. Swimming augmented ROS emission in Dsg2 mut/mut right ventricles (RV) than in WT (25±3 vs 15±1 Gauss/total protein [G/tp]; P<0.05) and amplified ROS release from Dsg2 mut/mut left ventricles (LV) (39±5 G/tp vs mutant RV; P<0.05). Isolated mutant mitochondria showed reduced NADH generation and peroxiredoxin-3, Trx2 and Trx2 reductase (Trx2R) protein levels, with impaired Trx2R activity (all P<0.05 vs WT in RV&LV). When mitochondria bound, Apoptosis-Inducing Factor (AIF) acts as a NADH oxidoreductase, yet upon oxidation, AIF translocates to the nucleus and initiates apoptosis. After exercise, Dsg2 mut/mut hearts displayed marked AIF nuclear and chromatin-bound protein levels and increased AIF immunostained nuclei vs WT. Additionally, direct H 2 O 2 exposure or Trx2 inhibition (by auranofin) in Dsg2 mut/mut embryonic stem-cell derived myocytes elevated AIF-nuclear translocation and apoptosis (via AnnexinV/PI FaCS analysis). Our study reveals a novel causal link between exercise-evoked cardiac redox imbalance and aberrant AIF-Trx2 signaling in ACM, which was associated with increased apoptosis, propensity of arrhythmias and sudden cardiac death. These findings offer a new targetable mechanism for preventing one of the most cited, yet poorly understood, pathological phenotypes (apoptosis) in ACM.

Mitochondrial Creatine Kinase Counters ROS Emission in Failing Hearts

The failing heart is energy-starved and redox imbalanced. Understanding how, and where cardiac energetic and redox alterations intersect in the failing heart are paramount to understanding and treating heart failure (HF). Creatine kinase (CK) is the major myocardial energy reserve reaction and ATP buffer with isozymes in the cytosol (CK-M) and mitochondria (Mt-CK). CK activity is reduced in human HF and following transverse aortic constriction (TAC). Mt-CK is an octamer particularly prone to oxidative modifications. It is not known whether HF changes in Mt-CK contribute to alter myocardial redox state in HF. We found that, in failing TAC WT hearts, a marked rise in reactive oxygen species (ROS) emission occurs (+93%, n=13, p

A mitohormetic response to pro-oxidant exposure in the house mouse.

Mitochondria are hypothesized to display a biphasic response to reactive oxygen species (ROS) exposure. In this study, we evaluated the time course changes in mitochondrial performance and oxidative stress in house mice following X-irradiation. Forty-eight mice were equally divided among six groups, including a nonirradiated control and five experimental groups that varied in time between X-ray exposure and euthanasia (1 h and 1, 4, 7, and 10 days after X-irradiation). We measured parameters associated with mitochondrial respiratory function and ROS emission from isolated liver and skeletal muscle mitochondria and levels of oxidative damage and antioxidants in liver, skeletal muscle, and heart tissues. Mitochondrial function dropped initially after X-irradiation but recovered quickly and was elevated 10 days after the exposure. Hydrogen peroxide production, lipid peroxidation, and protein carbonylation showed inverse U-shaped curves, with levels returning to control or lower than control, 10 days after X-irradiation. Enzymatic antioxidants and markers for mitochondrial biogenesis exhibited a tissue-specific response after irradiation. These data provide the first chronological description of the mitohormetic response after a mild dose of irradiation and highlight the protective response that cells display to ROS exposure. This study also provides valuable information and application for future mitochondrial and oxidative stress studies in numerous physiological settings.

Mitochondrial respiration and ROS emission during β-oxidation in the heart: An experimental-computational study.

Lipids are main fuels for cellular energy and mitochondria their major oxidation site. Yet unknown is to what extent the fuel role of lipids is influenced by their uncoupling effects, and how this affects mitochondrial energetics, redox balance and the emission of reactive oxygen species (ROS). Employing a combined experimental-computational approach, we comparatively analyze β-oxidation of palmitoyl CoA (PCoA) in isolated heart mitochondria from Sham and streptozotocin (STZ)-induced type 1 diabetic (T1DM) guinea pigs (GPs). Parallel high throughput measurements of the rates of oxygen consumption (VO2) and hydrogen peroxide (H2O2) emission as a function of PCoA concentration, in the presence of L-carnitine and malate, were performed. We found that PCoA concentration < 200 nmol/mg mito protein resulted in low H2O2 emission flux, increasing thereafter in Sham and T1DM GPs under both states 4 and 3 respiration with diabetic mitochondria releasing higher amounts of ROS. Respiratory uncoupling and ROS excess occurred at PCoA > 600 nmol/mg mito prot, in both control and diabetic animals. Also, for the first time, we show that an integrated two compartment mitochondrial model of β-oxidation of long-chain fatty acids and main energy-redox processes is able to simulate the relationship between VO2 and H2O2 emission as a function of lipid concentration. Model and experimental results indicate that PCoA oxidation and its concentration-dependent uncoupling effect, together with a partial lipid-dependent decrease in the rate of superoxide generation, modulate H2O2 emission as a function of VO2. Results indicate that keeping low levels of intracellular lipid is crucial for mitochondria and cells to maintain ROS within physiological levels compatible with signaling and reliable energy supply.

Hybridization increases mitochondrial production of reactive oxygen species in sunfish.

Mitochondrial dysfunction and oxidative stress have been suggested to be possible mechanisms underlying hybrid breakdown, as a result of mito-nuclear incompatibilities in respiratory complexes of the electron transport system. However, it remains unclear whether hybridization increases the production of reactive oxygen species (ROS) by mitochondria. We used high-resolution respirometry and fluorometry on isolatedliver mitochondria to examine mitochondrial physiology and ROSemission in naturally occurring hybrids of pumpkinseed (Lepomis gibbosus) and bluegill (L. macrochirus). ROS emission was greater in hybridsthan in both parent species when respiration was supported bycomplex I (but not complex II)substrates, and was associated with increases in lipid peroxidation.However, respiratory capacitiesfor oxidative phosphorylation, phosphorylation efficiency, and O2 kinetics inhybrids were intermediate between those in parental species. Flux control ratios of capacities for electron transport (measured in uncoupled mitochondria) relative to oxidative phosphorylation suggested that the limiting influence of the phosphorylation system is reduced in hybrids. This likely helped offset impairments in electron transport capacity and complex III activity, but contributed to augmenting ROS production.Therefore, hybridization can increase mitochondrial ROS production, in support of previous suggestions that mitochondrial dysfunction can induce oxidative stress and thus contribute to hybridbreakdown.

Pre-ischaemic mitochondrial substrate constraint by inhibition of malate-aspartate shuttle preserves mitochondrial function after ischaemia-reperfusion.

Pre-ischaemic administration of aminooxiacetate (AOA), an inhibitor of the malate-aspartate shuttle (MAS), provides cardioprotection against ischaemia-reperfusion injury. The underlying mechanism remains unknown. We examined whether transient inhibition of the MAS during ischaemia and early reperfusion by AOA treatment could prevent mitochondrial damage at later reperfusion. The AOA treatment preserved mitochondrial respiratory capacity with reduced mitochondrial oxidative stress during late reperfusion to the same extent as ischaemic preconditioning (IPC). However, AOA treatment, but not IPC, reduced the myocardial interstitial concentration of tricarboxylic acid cycle intermediates at the onset of reperfusion. The results obtained in the present study demonstrate that metabolic regulation by inhibition of the MAS at the onset of reperfusion may be beneficial for the preservation of mitochondrial function during late reperfusion in an IR-injured heart. Mitochondrial dysfunction plays a central role in ischaemia-reperfusion (IR) injury. Pre-ischaemic administration of aminooxyacetate (AOA), an inhibitor of the malate-aspartate shuttle (MAS), provides cardioprotection against IR injury, although the underlying mechanism remains unknown. We hypothesized that a transient inhibition of the MAS during ischaemia and early reperfusion could preserve mitochondrial function at later phase of reperfusion in the IR-injured heart to the same extent as ischaemic preconditioning (IPC), which is a well-validated cardioprotective strategy against IR injury. In the present study, we show that pre-ischaemic administration of AOA preserved mitochondrial complex I-linked state 3 respiration and fatty acid oxidation during late reperfusion in IR-injured isolated rat hearts. AOA treatment also attenuated the excessive emission of mitochondrial reactive oxygen species during state 3 with complex I-linked substrates during late reperfusion, which was consistent with reduced oxidative damage in the IR-injured heart. As a result, AOA treatment reduced infarct size after reperfusion. These protective effects of MAS inhibition on the mitochondria were similar to those of IPC. Intriguingly, the protection of mitochondrial function by AOA treatment appears to be different from that of IPC because AOA treatment, but not IPC, downregulated myocardial tricarboxilic acid (TCA)-cycle intermediates at the onset of reperfusion. MAS inhibition thus preserved mitochondrial respiratory capacity and decreased mitochondrial oxidative stress during late reperfusion in the IR-injured heart, at least in part, via metabolic regulation of TCA cycle intermediates in the mitochondria at the onset of reperfusion.