Reactive Oxygen Species Emission Research Articles

Metal Toxicity: Effects on Energy Metabolism in Fish.

Metals are dispersed in natural environments, particularly in the aquatic environment, and accumulate, causing adverse effects on aquatic life. Moreover, chronic polymetallic water pollution is a common problem, and the biological effects of exposure to complex mixtures of metals are the most difficult to interpret. In this review, metal toxicity is examined with a focus on its impact on energy metabolism. Mechanisms regulating adenosine triphosphate (ATP) production and reactive oxygen species (ROS) emission are considered in their dual roles in the development of cytotoxicity and cytoprotection, and mitochondria may become target organelles of metal toxicity when the transmembrane potential is reduced below its phosphorylation level. One of the main consequences of metal toxicity is additional energy costs, and the metabolic load can lead to the disruption of oxidative metabolism and enhanced anaerobiosis.

Activation of the Nrf-2 pathway improves the mitochondrial phenotype in skeletal muscle cells

Aging muscle displays a reduction in mitochondrial content, along with elevated reactive oxygen species (ROS) emission and reduced levels of antioxidant enzymes. These characteristics enhance tissue susceptibility to oxidative damage, facilitating sub-optimal cellular metabolism and subsequent muscle atrophy. While these perturbations to metabolic homeostasis are a natural course of the aging process, there has been ongoing research investigating how to implement interventions to reduce this decline in muscle health. Regular exercise is one possible treatment modality, however this may be unsuitable for some populations who are unable to participate due to their health status. The objective of this study was to examine the activation of the Nrf-2 pathway using the nutraceutical Sulforaphane (SFN) and evaluate its role as a potential exercise mimetic. We hypothesized that SFN would act through similar pathways activated with chronic exercise to improve mitochondrial content, respiration, and ROS emission. To address our objective, C2C12 myotubes were treated with SFN for various timepoints basally, and in the presence of the Complex-I inhibitor rotenone to induce oxidative stress. Western blotting revealed an 8-fold increase in Nrf-2 nuclear localization following 24hrs of SFN treatment, which subsequently led to the upregulation of antioxidant enzymes NQO1, Heme Oxygenase-1, catalase, glutathione reductase and Glucose-6-Phosphate Dehydrogenase by 2-to 3-fold. These protein changes, were accompanied by dramatic reductions in both total cellular and mitochondrial ROS emission with SFN, detected with CellROX Green and MitoSOX Red respectively. Interestingly, this led to an increase in mitochondrial membrane potential, detected via MitoTracker Red (MTR), despite rotenone-induced oxidative stress. Western blotting revealed TFAM upregulation at 24hrs, suggesting an increase in mitochondrial DNA (mtDNA) replication and transcription. This was corroborated by an increase in COX subunit I protein, a mitochondrially-encoded subunit of cytochrome c oxidase, as well as MitoTracker Green and MTR staining by 48hrs. Mitochondrial respiration was evaluated using Seahorse analysis, demonstrating a marked increase in ATP production as well as basal and maximal respiration. Together these data suggest that activation of the Nrf-2 pathway via SFN may lead to adaptations similar to chronic exercise. Additionally, SFN may have applications in disorders characterized by mitochondrial loss and high ROS emission. To fully elucidate the precise mechanisms underlying this agent, SFN will be combined with chronic contractile activity to determine if these two interventions operate through independent pathways. This work was supported by funds from the Natural Science and Engineering Research Council (NSERC). Sabrina Champsi is the recipient of the NSERC Alexander Graham Bell Canada Graduate Scholarship-Master’s (CGS-M). David A. Hood is the holder of a Canadian Research Chair in Cell Physiology. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Oxidative stress in the remaining muscle after volumetric muscle loss contributes to impaired mitochondrial bioenergetics and is attenuated by SS-31

Volumetric muscle loss (VML) injury results in the non-recoverable loss of muscle mass and function. Our previous studies indicate mitochondrial dysfunction in the remaining muscle fibers after VML injury, marked by changes in mitochondrial respiration, membrane potential, and enzyme activities. In particular, mitochondrial membrane potential is hyperpolarized during the first 14-days post-injury, a condition that could lead to the production of reactive oxygen species (ROS). When ROS emissions exceed the antioxidant buffering capacity, cellular damage can ensue. The primary objective of this study was to determine ROS production, ROS emission, and antioxidant buffering capacity of remaining muscle fibers after VML injury in the first month after injury. The secondary objective of this study was to determine if a mitochondrial antioxidant (SS-31) could mitigate mitochondrial dysfunction after VML injury. We hypothesized that VML injury will alter the redox status of the remaining muscle. Study 1: Male C57BL/6J mice (n=40) were randomized to Uninjured or VML-injured cohorts. ROS (H2O2) emission/production and endogenous antioxidant buffering capacity were analyzed fluorometrically during live muscle fiber respiration (Oroboros O2k) at 3-, 7-, 14-, 21-, and 30-days post-injury. Study 2: Male C57BL/6J mice (n=32) were randomized into two groups: VML and VML+SS-31. SS-31 is a mitochondrial-targeted peptide with ROS-scavenging properties. VML+SS-31 received SS-31 (8mg/kg/day) for 14 days, and the VML received equal volume saline injections. At 14- and 28-days post-VML, mitochondrial respiration and electron conductance, ROS emission, and antioxidant buffering capacity were assessed in permeabilized gastrocnemius muscle fibers from the muscle remaining after the initial injury. Group differences were detected through ANOVA and unpaired t-test. ROS emission and antioxidant buffering capacity were different between VML-injured and Uninjured mice through 14-days post-injury, specifically at 3-, 7-, and 14-days, mitochondrial ROS emission was significantly higher (+28%, p<0.001), and antioxidant buffering capacity was less (-28%, p=0.002) in VML mice compared to Uninjured mice. Two weeks of SS-31 treatment lead to greater muscle fiber respiration and electron conductance (p≤0.008) at both 14- and 28-days post-VML. Importantly, at 14-days post-injury, mitochondrial ROS emission was 71% lower in VML+SS-31 than VML. At both 14- and 28-days post-injury, antioxidant buffering capacity was 12% greater in VML+SS-31 (p≤0.003) compared to VML. Oxidative stress is a feature of early VML pathophysiology marked by greater ROS emission and less antioxidant buffering capacity. Attenuating oxidative stress during the first two-weeks post-VML injury improves mitochondrial respiration. W81XWH-20-1-0885 and R01AR078903 to SG and JC. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

CuPc-Fe@BSA nanocomposite: Intracellular acid-sensitive aggregation for enhanced sonodynamic and chemo-therapy

Due to their rigid π-conjugated macrocyclic structure, organic sonosensitizers face significant aggregation in physiological conditions, hindering the production of reactive oxygen species (ROS). An acid-sensitive nanoassembly was developed to address this issue and enhance sonodynamic therapy (SDT) and emission. Initially, copper phthalocyanine (CuPc) was activated using a H2SO4-assisted hydrothermal method to introduce multiple functional groups (–COOH, –OH, and −SO3H), disrupting strong π–π stacking and promoting ROS generation and emission. Subsequently, negatively charged CuPc-SO4 was incorporated into bovine serum albumin (BSA) to form CuPc-Fe@BSA nanoparticles (10 nm) with Fe3+ ions serving as linkers. In acidic conditions, protonation of CuPc-SO4 and BSA weakened the interactions, leading to Fe3+ release and nanostructure dissociation. Protonated CuPc-SO4 tended to self-aggregate into nanorods. This acidity-sensitive aggregation is vital for achieving specific accumulation within the tumor microenvironment (TME), thereby enhancing retention and SDT efficacy. Prior to this, the nanocomposites demonstrated cycling stability under neutral conditions. Additionally, the released Fe ions exhibited mimicry of glutathione peroxidase and peroxidase activity for chemotherapy (CDT). The synergistic effect of SDT and CDT increased intracellular oxidative stress, causing mitochondrial injury and ferroptosis. Furthermore, the combined therapy induced immunogenic cell death (ICD), effectively activating anticancer immune responses and suppressing metastasis and recurrence.

Mitochondrial peptides derived from exercising skeletal muscle increase oxidative stress in microvascular endothelial cells

Introduction: Mitochondria-derived peptides are newly discovered small bioactive peptides thought to communicate metabolic status to other cells. Two such peptides, MOTS-c and Humanin, have been shown to have particularly strong local and systemic regulatory effects on metabolism and oxidative stress in tissues, but their effect specifically on vascular endothelial cells remains unclear. Furthermore, it has been proposed that skeletal muscle is a source of MOTS-c and humanin during exercise but this has not been directly shown. Methods: In seven healthy young males, muscle interstitial fluid was collected from microdialysis probes in the thigh muscle, blood was drawn from femoral arterial and venous catheters and thigh muscle biopsies were obtained before and after one acute bout of exercise. In addition, muscle biopsies were obtained before and after 14 days of leg exercise training. Samples were analysed for MOTS-c, Humanin protein and mRNA. To test the effect of MOTS-c on the microvasculature, human endothelial cells derived from the muscle biopsies were incubated for three days with MOTS-c and analysed for mitochondrial respiration and formation of reactive oxygen species (ROS). Furthermore, mice were injected with MOTS-c for 4-weeks to investigate the in-vivo effect of vessels and microvascular endothelial cells. Data was analyzed by linear mixed models performed with R. Results: Acute exercise resulted in an 88% and 55% ( p<0.001) increase in MOTS-c and humanin concentration, respectively, in muscle dialysate, with no effect on the femoral arterial-venous plasma concentration differences. Both MOTS-c and humanin mRNA levels increased (by 20 and 26%; p<0.05) after acute exercise and were 43% and 35% ( p<0.05) higher, respectively, after 14 days of training. A period of MOTS-c treatment of endothelial cells increased mitochondrial respiration and ROS formation by 3-fold and 5-fold, respectively ( p<0.01). Muscle derived endothelial cells from mice injected for 4-weeks presented a similar mitochondrial respiration but a 4-fold ( p<0.05) higher ROS emission. Conclusion: interstitium in response to exercise, muscle is not a likely source of these peptides in plasma. Our data from in vitro and in vivo experiments suggest that treatment with MOTS-c does not influence endothelial metabolism but leads to increased oxidative stress in microvascular endothelial cells. The funding by Novo Nordisk Foundation (application number: 0070369) is gratefully acknowledged. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Migration increases mitochondrial oxidative capacity without increasing reactive oxygen species emission in a songbird.

Birds remodel their flight muscle metabolism prior to migration to meet the physiological demands of migratory flight, including increases in both oxidative capacity and defence against reactive oxygen species. The degree of plasticity mediated by changes in these mitochondrial properties is poorly understood but may be explained by two non-mutually exclusive hypotheses: variation in mitochondrial quantity or in individual mitochondrial function. We tested these hypotheses using yellow-rumped warblers (Setophaga coronata), a Nearctic songbird which biannually migrates 2000-5000 km. We predicted higher flight muscle mitochondrial abundance and substrate oxidative capacity, and decreased reactive oxygen species emission in migratory warblers captured during autumn migration compared with a short-day photoperiod-induced non-migratory phenotype. We assessed mitochondrial abundance via citrate synthase activity and assessed isolated mitochondrial function using high-resolution fluororespirometry. We found 60% higher tissue citrate synthase activity in the migratory phenotype, indicating higher mitochondrial abundance. We also found 70% higher State 3 respiration (expressed per unit citrate synthase) in mitochondria from migratory warblers when oxidizing palmitoylcarnitine, but similar H2O2 emission rates between phenotypes. By contrast, non-phosphorylating respiration was higher and H2O2 emission rates were lower in the migratory phenotype. However, flux through electron transport system complexes I-IV, II-IV and IV was similar between phenotypes. In support of our hypotheses, these data suggest that flight muscle mitochondrial abundance and function are seasonally remodelled in migratory songbirds to increase tissue oxidative capacity without increasing reactive oxygen species formation.

Postnatal growth restriction alters myocardial mitochondrial energetics in mice.

Postnatal growth restriction (PGR) can increase the risk of cardiovascular disease (CVD) potentially due to impairments in oxidative phosphorylation (OxPhos) within cardiomyocyte mitochondria. The purpose of this investigation was to determine if PGR impairs cardiac metabolism, specifically OxPhos. FVB (Friend Virus B-type) mice were fed a normal-protein (NP: 20% protein), or low-protein (LP: 8% protein) isocaloric diet 2weeks before mating. LP dams produce ∼20% less milk, and pups nursed by LP dams experience reduced growth into adulthood as compared to pups nursed by NP dams. At birth (PN1), pups born to dams fed the NP diet were transferred to LP dams (PGR group) or a different NP dam (control group: CON). At weaning (PN21), all mice were fed the NP diet. At PN22 and PN80, mitochondria were isolated for respirometry (oxygen consumption rate, ) and fluorimetry (reactive oxygen species emission, ) analysis measured as baseline respiration (LEAK) and with saturating ADP (OxPhos). Western blotting at PN22 and PN80 determined protein abundance of uncoupling protein 3, peroxiredoxin-6, voltage-dependent anion channel and adenine nucleotide translocator 1 to provide further insight into mitochondrial function. ANOVAs with the main effects of diet, sex and age with α-level of 0.05 was set a priori. Overall, PGR (7.8±1.1) had significant (P=0.01) reductions in respiratory control in complex I when compared to CON (8.9±1.0). In general, our results show that PGR led to higher electron leakage in the form of free radical production and reactive oxygen species emission. No significant diet effects were found in protein abundance. The observed reduced respiratory control and increased ROS emission in PGR mice may increase risk for CVD in mice.

Effects of Aftermarket Electronic Cigarette Pods on Device Power Output and Nicotine, Carbonyl, and ROS Emissions.

Aftermarket pods designed to operate with prevalent electronic nicotine delivery system (ENDS) products such as JUUL are marketed as low-cost alternatives that allow the use of banned flavored liquids. Subtle differences in the design or construction of aftermarket pods may intrinsically modify the performance of the ENDS device and the resulting nicotine and toxicant emissions relative to the original equipment manufacturer's product. In this study, we examined the electrical output of a JUUL battery and the aerosol emissions when four different brands of aftermarket pods filled with an analytical-grade mixture of propylene glycol, glycerol, and nicotine were attached to it and puffed by machine. The aerosol emissions examined included total particulate matter (TPM), nicotine, carbonyl compounds (CCs), and reactive oxygen species (ROS). We also compared the puff-resolved power and TPM outputs of JUUL and aftermarket pods. We found that all aftermarket pods drew significantly greater electrical power from the JUUL battery during puffing and had different electrical resistances and resistivity. In addition, unlike the case with the original pods, we found that with the aftermarket pods, the power provided by the battery did not vary greatly with flow rate or puff number, suggesting impairment of the temperature control circuitry of the JUUL device when used with the aftermarket pods. The greater power output with the aftermarket pods resulted in up to three times greater aerosol and nicotine output than the original product. ROS and CC emissions varied widely across brands. These results highlight that the use of aftermarket pods can greatly modify the performance and emissions of ENDS. Consumers and public health authorities should be made aware of the potential increase in the level of toxicant exposure when aftermarket pods are employed.

Abstract 13835: Glutaminolysis Boosts Cardiac Performance Under High Workload via Enhancement of Mitochondrial GSH

Introduction: Glutamine, the most abundant non-essential amino acids in circulation, serves as an important energy source in proliferating cells. Previous evidence has indicated that glutamine is not a primary carbon source for the tricarboxylic acid cycle in cardiomyocytes under physiological workload. However, current clinical studies reveal that impaired glutaminolysis is associated with heart failure and an increased risk of cardiovascular events. Hypothesis: We hypothesized that impaired glutaminolysis would compromise cardiac function, especially under conditions of increased workload. Methods: We analyzed cardiac structure and function in a mouse model with cardiac-specific knockdown of glutaminase (GLS), a key enzyme of glutaminolysis. Cardiac reserve was assessed by isoproterenol stress echocardiography. We performed transcriptomic, 13 C isotopic labeling, and reactive oxygen species emission analyses to investigate the underlying mechanism. Results: GLS deficiency impaired cardiac reserve and exercise capacity in mice, but had no discernible effects on cardiac structure or function under baseline condition. Inhibition of glutaminolysis in pressure-overloaded hearts further exacerbated cardiac dysfunction, indicating the critical role of glutaminolysis in cardiomyocytes under high workload. Mechanistically, glutaminolysis maintained cardiomyocyte function by balancing intracellular redox status through the production of glutathione (GSH), particularly in mitochondria. Inhibition of glutaminolysis decreased both cytosolic and mitochondrial GSH levels, resulting in compromised cell contraction in isolated cardiomyocytes under high workload. Additionally, heterologous expression of an engineered mitochondrial GSH biosynthetic enzyme increased mitochondrial GSH levels and enhanced cardiac reserve in GLS knockout mice. Conclusions: Our findings establish the previously unrecognized role of glutaminolysis in cardiomyocytes, maintaining cardiac reserve via upregulating mitochondrial GSH levels. This suggests that targeting glutaminolysis is a potential strategy for improving cardiac reserve and managing cardiac diseases.

Effects of ROS pathway inhibitors and NADH and FADH2 linked substrates on mitochondrial bioenergetics and ROS emission in the heart and kidney cortex and outer medulla

Mitochondria are major sources of reactive oxygen species (ROS), which play important roles in both physiological and pathological processes. However, the specific contributions of different ROS production and scavenging components in the mitochondria of metabolically active tissues such as heart and kidney cortex and outer medulla (OM) are not well understood. Therefore, the goal of this study was to determine contributions of different ROS production and scavenging components and provide detailed comparisons of mitochondrial respiration, bioenergetics, ROS emission between the heart and kidney cortex and OM using tissues obtained from the same Sprague-Dawley rat under identical conditions and perturbations. Specifically, data were obtained using both NADH-linked substrate pyruvate + malate and FADH2-linked substrate succinate followed by additions of inhibitors of different components of the electron transport chain (ETC) and oxidative phosphorylation (OxPhos) and other ROS production and scavenging systems. Currently, there is limited data available for the mitochondria of kidney cortex and OM, the two major energy-consuming tissues in the body only next to the heart, and scarce quantitative information on the interplay between mitochondrial ROS production and scavenging systems in the three tissues. The findings from this study demonstrate significant differences in mitochondrial respiratory and bioenergetic functions and ROS emission among the three tissues. The results quantify the rates of ROS production from different complexes of the ETC, identify the complexes responsible for variations in mitochondrial membrane depolarization and regulations of ROS production, and quantify the contributions of ROS scavenging enzymes towards overall mitochondrial ROS emission. These findings advance our fundamental knowledge of tissue-specific and substrate-dependent mitochondrial respiratory and bioenergetic functions and ROS emission. This is important given the critical role that excess ROS production, oxidative stress, and mitochondrial dysfunction in the heart and kidney cortex and OM play in the pathogenesis of cardiovascular and renal diseases, including salt-sensitive hypertension.

Dietary nitrate preserves mitochondrial bioenergetics and mitochondrial protein synthesis rates during short-term immobilization in mice.

Skeletal muscle disuse reduces muscle protein synthesis rates and induces atrophy, events associated with decreased mitochondrial respiration and increased reactive oxygen species. Given that dietary nitrate can improve mitochondrial bioenergetics, we examined whether nitrate supplementation attenuates disuse-induced impairments in mitochondrial function and muscle protein synthesis rates. Female C57Bl/6N mice were subjected to single-limb casting (3 or 7days) and consumed drinking water with or without 1mM sodium nitrate. Compared with the contralateral control limb, 3days of immobilization lowered myofibrillar fractional synthesis rates (FSR, P< 0.0001), resulting in muscle atrophy. Although FSR and mitophagy-related proteins were higher in subsarcolemmal (SS) compared with intermyofibrillar (IMF) mitochondria, immobilization for 3days decreased FSR in both SS (P=0.009) and IMF (P=0.031) mitochondria. Additionally, 3days of immobilization reduced maximal mitochondrial respiration, decreased mitochondrial protein content, and increased maximal mitochondrial reactive oxygen species emission, without altering mitophagy-related proteins in muscle homogenate or isolated mitochondria (SS and IMF). Although nitrate consumption did not attenuate the decline in muscle mass or myofibrillar FSR, intriguingly, nitrate completely prevented immobilization-induced reductions in SS and IMF mitochondrial FSR. In addition, nitrate prevented alterations in mitochondrial content and bioenergetics after both 3 and 7days of immobilization. However, in contrast to 3days of immobilization, nitrate did not prevent the decline in SS and IMF mitochondrial FSR after 7days of immobilization. Therefore, although nitrate supplementation was not sufficient to prevent muscle atrophy, nitrate may represent a promising therapeutic strategy to maintain mitochondrial bioenergetics and transiently preserve mitochondrial protein synthesis rates during short-term muscle disuse. KEY POINTS: Alterations in mitochondrial bioenergetics (decreased respiration and increased reactive oxygen species) are thought to contribute to muscle atrophy and reduced protein synthesis rates during muscle disuse. Given that dietary nitrate can improve mitochondrial bioenergetics, we examined whether nitrate supplementation could attenuate immobilization-induced skeletal muscle impairments in female mice. Dietary nitrate prevented short-term (3day) immobilization-induced declines in mitochondrial protein synthesis rates, reductions in markers of mitochondrial content, and alterations in mitochondrial bioenergetics. Despite these benefits and the preservation of mitochondrial content and bioenergetics during more prolonged (7 day) immobilization, nitrate consumption did not preserve skeletal muscle mass or myofibrillar protein synthesis rates. Overall, although dietary nitrate did not prevent atrophy, nitrate supplementation represents a promising nutritional approach to preserve mitochondrial function during muscle disuse.

Effect of methamphetamine on ultraweak photon emission and level of reactive oxygen species in male rat brain

All living cells, including neurons, generate ultra-weak photon emission (UPE) during biological activity, and in particular, in the brain, it has been shown that UPE is correlated with neuronal activity and associated metabolic processes. Various intracellular factors, as well as external factors, can reduce or increase the intensity of UPE. In this study, we have used Methamphetamine (METH) as one potentially effective external factor, which is a substance that has the property of stimulating the central nervous system. METH can impair mitochondrial function by causing toxicity via various pathways, including an increase in the number of mitochondria, hyperthermia, the increased metabolic activity of the brain, and the production of glutamate and excess calcium. In addition to mitochondrial dysfunction, METH alters cellular homeostasis, leading to cell damage and the production of excess ROS. The aim of this study is to measure and compare the UPE intensity and reactive oxygen species (ROS) levels of the prefrontal, motor, and visual cortex before and after METH administration. Twenty male rats were randomly assigned to two groups, the control, and METH groups. In the control group, 2 h after injection of normal saline and without any intervention, and in the experimental group 2 h after IP injection of 20 mg/kg METH, sections were prepared from three areas: prefrontal, motor, and V1-V2 cortex, which were used to evaluate the emission of UPE using a photomultiplier tube (PMT) device and to evaluate the amount of ROS. The results showed that the amount of ROS and UPE in the experimental group in all three areas significantly increased compared to the control group. So, METH increases UPE and ROS in the prefrontal, motor, and visual regions, and there is a direct relationship between UPE intensity and ROS production. Therefore, UPE may be used as a dynamic reading tool to monitor oxidative metabolism in physiological processes related to ROS and METH research. Also, the results of this experiment may create a new avenue to test the hypothesis that the excess in UPE generation may lead to the phenomenon of phosphene and visual hallucinations.

Mitochondrial ROS as a regulator of synaptic function.

Exacerbated emission of reactive oxygen species (ROS) from presynaptic mitochondria is a well-studied hallmark of several neurodegenerative diseases, including amyotrophic lateral sclerosis. Outside of the context of disease, the potential role of mitochondrial ROS in presynaptic function and plasticity remains largely understudied. Here, we investigated this potential role by combining electrophysiological recordings, confocal imaging, immunohistochemical staining, and optogenetics using a well-established protocol for induction and measurement of synaptic potentiation in drosophila neuromuscular preparations. We have previously found that optogenetic induction of ROS emission from presynaptic mitochondria expressing mito-killer red was accompanied by an increase in spontaneous mini junction potentials. The increase in electrical activity did not appear to be associated with major synaptic structure alterations such as the formation of presynaptic filopodia or the growth of ghost boutons. However, in existing boutons, we observed an increase in active zone markers such as nc82/Brp. We have not detected Wnt1/Wg release from synaptic boutons suggesting the involvement of other signaling pathways that underlie observed changes in electrophysiological activity mediated by ROS emission. Future studies will further inquire the role of mitochondrial ROS in synaptic potentiation, as well as the potential signaling targets of mitochondrial ROS in the presynaptic structure.

Ryanodine receptor hyperactivity promotes mitochondrial dysfunction via calpain-dependent OPA1 proteolysis.

Hereditary RyR2 gain-of function mutations are linked to the highly malignant arrhythmia syndrome, catecholaminergic polymorphic tachycardia (CPVT). A mutation-mediated increase in RyR2 activity not only results in unstable sarcoplasmic reticulum Ca2+ release, but also alters mitochondrial structure and function, leading to excessive emission of damaging reactive oxygen species (ROS) by the organelle. However, the exact mechanisms underlying RyR2 hyperactivity-dependent mitochondrial damage remain elusive. We hypothesized that RyR2 hyperactivity activates the intermembrane space (IMS)-residing mitochondrial Ca2+-dependent protease calpain, driving proteolysis of OPA1, a protein responsible for tight cristae arrangement. Concomitant changes in cristae architecture promotes mito-ROS production, thereby aggravating the CPVT phenotype. To test this, we generated a unique CPVT RyR2-S2222L(+/-) rat model. Electron microscopy demonstrated significantly increased mitochondrial cristae diameter in tissue slices from CPVT rat hearts. Biochemical analysis showed reduced OPA1 expression in isolated CPVT myocytes, suggesting that mitochondrial ultrastructural changes in CPVT could be driven by Ca2+-dependent proteolysis of OPA1 by calpain. Importantly, specific inhibition of calpain via adeno-associated virus mediated expression of IMS-targeted calpastatin reduced the incidence of ventricular tachycardia in CPVT rats. At the cellular level, adenoviral expression of IMS-CAST restored OPA1 expression levels and significantly improved intracellular Ca2+ homeostasis in CPVT, indicative of normalized RyR2 function. Furthermore, IMS-calpain inhibition attenuated the reduction in mitochondrial matrix Ca2+ and increase in mito-ROS emission observed in CPVT myocytes using genetic probes mtRCamp1h and MLS-HyPer7. All protective effects were lost in myocytes with additional shRNA-mediated knockdown of OPA1. We conclude that RyR2 hyperactivity promotes mitochondrial structural damage via IMS-calpain-mediated degradation of OPA1 and contributes to proarrhythmic remodeling in CPVT.

Restoration of mitochondrial Ca2+ and redox homeostasis by enhancement of SK channels rescues Ca2+cycling in HFpEF.

Heart failure is the leading cause of death in the postindustrial world. While many effective therapies are available to treat heart failure with reduced ejection fraction (HFrEF), these approaches fail to improve outcomes in heart failure with preserved ejection fraction (HFpEF). Small conductance Ca2+-activated K+ (SK) channels have recently emerged as an attractive target to improve defective mitochondrial function and reduce emission of damaging reactive oxygen species (ROS) by the organelle. The goal of the present study was to test enhancement of SK as a new strategy to restore abnormal intracellular Ca2+ cycling and mitochondrial redox and Ca2+ homeostasis in ventricular myocytes from HFpEF rat hearts. To achieve this goal as a model of HFpEF we employed ZSF1 obese rats that demonstrate diastolic dysfunction, unchanged fractional shortening and ejection fraction. The confocal Ca2+ and ROS imaging experiments were carried out in ventricular myocytes isolated from lean and obese ZSF1 rats. Mitochondrial matrix [Ca2+] and ROS were assessed in ventricular myocytes expressing matrix-targeted biosensors mtRCamp1h and MLS-HyPer-7, respectively. To increase SK channel activity, we employed adenovirus-mediated overexpression of rat SK channel type 2 and pharmacological SK channel enhancer NS309 . Periodically paced ventricular myocytes isolated from obese ZSF1 rat hearts exhibited enhanced propensity to spontaneous sarcoplasmic reticulum (SR) Ca2+ release, increased mito-ROS production and a dramatic increase in mitochondrial [Ca2+] when exposed to beta-adrenergic agonist isoproterenol. Enhancement of SK channels in ZSF1 obese myocytes prevented mitochondrial Ca2+ overload, reduced mito-ROS emission to the control levels, and improved cytosolic Ca2+ cycling, reducing diastolic SR Ca2+ release. In summary, enhancement of SK channels shows high therapeutic potential in HFpEF by limiting mitochondrial Ca2+ uptake, thereby reducing oxidative stress restraining excessive SR Ca2+ release during diastole.

Independent, but not co-supplementation, with nitrate and resveratrol improves glucose tolerance and reduces markers of cellular stress in high-fat-fed male mice.

Independent supplementation with nitrate (NIT) and resveratrol (RSV) enriches various aspects of mitochondrial biology in key metabolic tissues. Although RSV is known to activate Sirt1 and initiate mitochondrial biogenesis, the metabolic benefits elicited by dietary nitrate appear to be dependent on 5'-adenosine monophosphate-activated protein kinase (AMPK)-mediated signaling events, a process also linked to the activation of Sirt1. Although the benefits of individual supplementation with these compounds have been characterized, it is unknown if co-supplementation may produce superior metabolic adaptations. Thus, we aimed to determine if treatment with combined +NIT and +RSV (+RN) could additively alter metabolic adaptations in the presence of a high-fat diet (HFD). Both +RSV and +NIT improved glucose tolerance compared with HFD (P < 0.05); however, this response was attenuated following combined +RN supplementation. Within skeletal muscle, all supplements increased mitochondrial ADP sensitivity compared with HFD (P < 0.05), without altering mitochondrial content. Although +RSV and +NIT decreased hepatic lipid deposition compared with HFD (P < 0.05), this effect was abolished with +RN, which aligned with significant reductions in Sirt1 protein content (P < 0.05) after combined treatment, in the absence of changes to mitochondrial content or function. Within epididymal white adipose tissue (eWAT), all supplements reduced crown-like structure accumulation compared with HFD (P < 0.0001) and mitochondrial reactive oxygen species (ROS) emission (P < 0.05), alongside reduced adipocyte cross-sectional area (CSA) (P < 0.05), with the greatest effect observed after +RN treatment (P = 0.0001). Although the present data suggest additive changes in adipose tissue metabolism after +RN treatment, concomitant impairments in hepatic lipid homeostasis appear to prevent improvements in whole body glucose homeostasis observed with independent treatment, which may be Sirt1 dependent.

Reactive Oxygen Species Promote Nitrous Oxide (N2O) Emissions from Soil/Sediment during the Anoxic-Oxic Transition.

Reactive oxygen species (ROS)-induced element/pollutant geochemical processes in fluctuating anoxic-oxic areas have received increasing attention in recent years. Nitrous oxide (N2O) is a strong greenhouse gas; however, the relationship between ROS and N2O emissions in these areas has not been established. This work revealed the essential role of ROS in promoting N2O emissions in soil/sediment during the anoxic-oxic transition. ROS decreased the rate of nitrate reduction by 26-31% and increased N2O emissions by 8.8-31.3% (at 48 h). ROS-induced N2O emission was via inhibiting the step of N2O reduction. During the anoxic-oxic transition, the contribution of ROS to inhibit the step of N2O reduction was higher than 52.6%, demonstrating the important role of ROS. The downregulated relative transcription of the NosZ gene demonstrated inhibition at the gene level. Hydrogen peroxide was the dominant ROS species inhibiting N2O reduction, while the role of hydroxyl radicals was negligible, suggesting a different behavior of N2O emission with common pollutant conversion induced by ROS during the anoxic-oxic transition. This study demonstrated an overlooked factor in promoting N2O emission in the soil/sediment and appealed to a re-examination of the mechanism of N2O emissions in fluctuating anoxic-oxic areas.

Dopamine/BDNF loss underscores narcosis cognitive impairment in divers: a proof of concept in a dry condition

PurposeDivers can experience cognitive impairment due to inert gas narcosis (IGN) at depth. Brain-derived neurotrophic factor (BDNF) rules neuronal connectivity/metabolism to maintain cognitive function and protect tissues against oxidative stress (OxS). Dopamine and glutamate enhance BDNF bioavailability. Thus, we hypothesized that lower circulating BDNF levels (via lessened dopamine and/or glutamate release) underpin IGN in divers, while testing if BDNF loss is associated with increased OxS.MethodsTo mimic IGN, we administered a deep narcosis test via a dry dive test (DDT) at 48 msw in a multiplace hyperbaric chamber to six well-trained divers. We collected: (1) saliva samples before DDT (T0), 25 msw (descending, T1), 48 msw (depth, T2), 25 msw (ascending, T3), 10 min after decompression (T4) to dopamine and/or reactive oxygen species (ROS) levels; (2) blood and urine samples at T0 and T4 for OxS too. We administered cognitive tests at T0, T2, and re-evaluated the divers at T4.ResultsAt 48 msw, all subjects experienced IGN, as revealed by the cognitive test failure. Dopamine and total antioxidant capacity (TAC) reached a nadir at T2 when ROS emission was maximal. At decompression (T4), a marked drop of BDNF/glutamate content was evidenced, coinciding with a persisting decline in dopamine and cognitive capacity.ConclusionsDivers encounter IGN at – 48 msw, exhibiting a marked loss in circulating dopamine levels, likely accounting for BDNF-dependent impairment of mental capacity and heightened OxS. The decline in dopamine and BDNF appears to persist at decompression; thus, boosting dopamine/BDNF signaling via pharmacological or other intervention types might attenuate IGN in deep dives.

Enduring Reactive Oxygen Species Emission Causes Aberrant Protein S-Glutathionylation Transitioning Human Aortic Valve Cells from a Sclerotic to a Stenotic Phenotype.

Aims: During calcific aortic valve stenosis (CAVS) progression, oxidative stress and endothelial dysfunction mark the initial pathogenic steps with a parallel dysregulation of the antioxidant systems. Here, we tested whether oxidation-induced protein S-glutathionylation (P-SSG) accounts for a phenotypic switch in human aortic valvular tissue, eventually leading to calcium deposition. Next, we tested whether countering this reactive oxygen species (ROS) surge would prevent these perturbations. Results: We employed state-of-the-art technologies, such as electron paramagnetic resonance (EPR), liquid chromatography-tandem mass spectrometry, imaging flow-cytometry, and live-cell imaging on human excised aortic valves and primary valve endothelial cells (VECs). We observed that a net rise in EPR-detected ROS emission marked the transition from fibrotic to calcific in human CAVS specimens, coupled to a progressive increment in P-SSG deposition. In human VECs (hVECs), treatment with 2-acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanylthiocarbonylamino)phenylthiocarbamoylsulfanyl]propionic acid triggered highly oxidizing conditions prompting P-SSG accumulation, damaging mitochondria, and inducing endothelial nitric oxide synthase uncoupling. All the events conjured up in morphing these cells from their native endothelial phenotype into a damaged calcification-inducing one. As proof of principle, the use of the antioxidant N-acetyl-L-cysteine prevented these alterations. Innovation: Borne as a compensatory system to face excessive oxidative burden, with time, P-SSG contributes to the morphing of hVECs from their innate phenotype into a damaged one, paving the way to calcium deposition. Conclusion: Our data suggest that, in the human aortic valve, unremitted ROS emission along with a P-SSG build-up occurs and accounts, at least in part, for the morphological/functional changes leading to CAVS. Antioxid. Redox Signal. 37, 1051-1071.

Adaptive increases in respiratory capacity and O2 affinity of subsarcolemmal mitochondria from skeletal muscle of high-altitude deer mice.

Aerobic energy demands have led to the evolution of complex mitochondrial reticula in highly oxidative muscles, but the extent to which metabolic challenges can be met with adaptive changes in physiology of specific mitochondrial fractions remains unresolved. We examined mitochondrial mechanisms supporting adaptive increases in aerobic performance in deer mice (Peromyscus maniculatus) adapted to the hypoxic environment at high altitude. High-altitude and low-altitude mice were born and raised in captivity, and exposed as adults to normoxia or hypobaric hypoxia (12kPa O2 for 6-8weeks). Subsarcolemmal and intermyofibrillar mitochondria were isolated from the gastrocnemius, and a comprehensive substrate titration protocol was used to examine mitochondrial physiology and O2 kinetics by high-resolution respirometry and fluorometry. High-altitude mice had greater yield, respiratory capacity for oxidative phosphorylation, and O2 affinity (lower P50 ) of subsarcolemmal mitochondria compared to low-altitude mice across environments, but there were no species difference in these traits in intermyofibrillar mitochondria. High-altitude mice also had greater capacities of complex II relative to complexes I + II and higher succinate dehydrogenase activities in both mitochondrial fractions. Exposure to chronic hypoxia reduced reactive oxygen species (ROS) emission in high-altitude mice but not in low-altitude mice. Our findings suggest that functional changes in subsarcolemmal mitochondria contribute to improving aerobic performance in hypoxia in high-altitude deer mice. Therefore, physiological variation in specific mitochondrial fractions can help overcome the metabolic challenges of life at high altitude.