H2O2 Emission Research Articles

The Switching of the Type of a ROS Signal from Mitochondria: The Role of Respiratory Substrates and Permeability Transition

Flashes of superoxide anion (O2−) in mitochondria are generated spontaneously or during the opening of the permeability transition pore (mPTP) and a sudden change in the metabolic state of a cell. Under certain conditions, O2− can leave the mitochondrial matrix and perform signaling functions beyond mitochondria. In this work, we studied the kinetics of the release of O2− and H2O2 from isolated mitochondria upon mPTP opening and the modulation of the metabolic state of mitochondria by the substrates of respiration and oxidative phosphorylation. It was found that mPTP opening leads to suppression of H2O2 emission and activation of the O2− burst. When the induction of mPTP was blocked by its antagonists (cyclosporine A, ruthenium red, EGTA), the level of substrates of respiration and oxidative phosphorylation and the selective inhibitors of complexes I and V determined the type of reactive oxygen species (ROS) emitted by mitochondria. It was concluded that upon complete and partial reduction and complete oxidation of redox centers of the respiratory chain, mitochondria emit H2O2, O2−, and nothing, respectively. The results indicate that the mPTP- and substrate-dependent switching of the type of ROS leaving mitochondria may be the basis for O2−- and H2O2-selective redox signaling in a cell.

Mitohormesis during advanced stages of Duchenne muscular dystrophy reveals a redox-sensitive creatine pathway that can be enhanced by the mitochondrial-targeting peptide SBT-20

Mitochondrial creatine kinase (mtCK) regulates the “fast” export of phosphocreatine to support cytoplasmic phosphorylation of ADP to ATP which is more rapid than direct ATP export. Such “creatine-dependent” phosphate shuttling is attenuated in several muscles, including the heart, of the D2.mdx mouse model of Duchenne muscular dystrophy at only 4 weeks of age. However, the degree to which creatine-dependent and -independent systems of phosphate shuttling progressively worsen or potentially adapt in a hormetic manner throughout disease progression remains unknown. Here, we performed a series of proof-of-principle investigations designed to determine how phosphate shuttling pathways worsen or adapt in later disease stages in D2.mdx (12 months of age). We also determined whether changes in creatine-dependent phosphate shuttling are linked to alterations in mtCK thiol redox state. In permeabilized muscle fibres prepared from cardiac left ventricles, we found that 12-month-old male D2.mdx mice have reduced creatine-dependent pyruvate oxidation and elevated complex I-supported H2O2 emission (mH2O2). Surprisingly, creatine-independent ADP-stimulated respiration was increased and mH2O2 was lowered suggesting that impairments in the faster mtCK-mediated phosphocreatine export system resulted in compensation of the alternative slower pathway of ATP export. The apparent impairments in mtCK-dependent bioenergetics occurred independent of mtCK protein content but were related to greater thiol oxidation of mtCK and a more oxidized cellular environment (lower GSH:GSSG). Next, we performed a proof-of-principle study to determine whether creatine-dependent bioenergetics could be enhanced through chronic administration of the mitochondrial-targeting, ROS-lowering tetrapeptide, SBT-20. We found that 12 weeks of daily treatment with SBT-20 (from day 4–∼12 weeks of age) increased respiration and lowered mH2O2 only in the presence of creatine in D2.mdx mice without affecting calcium-induced mitochondrial permeability transition activity. In summary, creatine-dependent mitochondrial bioenergetics are attenuated in older D2.mdx mice in relation to mtCK thiol oxidation that seem to be countered by increased creatine-independent phosphate shuttling as a unique form of mitohormesis. Separate results demonstrate that creatine-dependent bioenergetics can also be enhanced with a ROS-lowering mitochondrial-targeting peptide. These results demonstrate a specific relationship between redox stress and mitochondrial hormetic reprogramming during dystrophin deficiency with proof-of-principle evidence that creatine-dependent bioenergetics could be modified with mitochondrial-targeting small peptide therapeutics.

Mitochondrial bioenergetics are not associated with myofibrillar protein synthesis rates.

Mitochondria represent key organelles influencing cellular homeostasis and have been implicated in the signalling events regulating protein synthesis. We examined whether mitochondrial bioenergetics (oxidative phosphorylation and reactive oxygen species (H2O2) emission, ROS) measured in vitro in permeabilized muscle fibres represent regulatory factors for integrated daily muscle protein synthesis rates and skeletal muscle mass changes across the spectrum of physical activity, including free-living and bed-rest conditions: n=19 healthy, young men (26±4years, 23.4±3.3kg/m2) and following 12weeks of resistance-type exercise training: n=10 healthy older men (70±3years, 25.2±2.1kg/m2). Additionally, we evaluated the direct relationship between attenuated mitochondrial ROS emission and integrated daily myofibrillar and sarcoplasmic protein synthesis rates in genetically modified mice (mitochondrial-targeted catalase, MCAT). Neither oxidative phosphorylation nor H2O2 emission were associated with muscle protein synthesis rates in healthy young men under free-living conditions or following 1week of bed rest (both P>0.05). Greater increases in GSSG concentration were associated with greater skeletal muscle mass loss following bed rest (r=-0.49, P<0.05). In older men, only submaximal mitochondrial oxidative phosphorylation (corrected for mitochondrial content) was positively associated with myofibrillar protein synthesis rates during exercise training (r=0.72, P<0.05). However, changes in oxidative phosphorylation and H2O2 emission were not associated with changes in skeletal muscle mass following training (both P>0.05). Additionally, MCAT mice displayed no differences in myofibrillar (2.62±0.22 vs. 2.75±0.15%/day) and sarcoplasmic (3.68±0.35 vs. 3.54±0.35%/day) protein synthesis rates when compared with wild-type mice (both P>0.05). Mitochondrial oxidative phosphorylation and reactive oxygen emission do not seem to represent key factors regulating muscle protein synthesis or muscle mass regulation across the spectrum of physical activity.

Interactions of binary mixtures of metals on rainbow trout (Oncorhynchus mykiss) heart mitochondrial H2O2 homeodynamics

For continuous pumping of blood, the heart needs a constant supply of energy (ATP) that is primarily met via oxidative phosphorylation in the mitochondria of cardiomyocytes. However, sustained high rates of electron transport for energy conversion redox reactions predisposes the heart to the production of reactive oxygen species (ROS) and oxidative stress. Mitochondrial ROS are fundamental drivers of responses to environmental stressors including metals but knowledge of how combinations of metals alter mitochondrial ROS homeodynamics remains sparse. We explored the effects and interactions of binary mixtures of copper (Cu), cadmium (Cd), and zinc (Zn), metals that are common contaminants of aquatic systems, on ROS (hydrogen peroxide, H2O2) homeodynamics in rainbow trout (Oncorhynchus mykiss) heart mitochondria. Isolated mitochondria were energized with glutamate-malate or succinate and exposed to a range of concentrations of the metals singly and in equimolar binary concentrations. Speciation analysis revealed that Cu was highly complexed by glutamate or Tris resulting in Cu2+ concentrations in the picomolar to nanomolar range. The concentration of Cd2+ was 7.2–7.5 % of the total while Zn2+ was 15 % and 21 % of the total during glutamate-malate and succinate oxidation, respectively. The concentration-effect relationships for Cu and Cd on mitochondrial H2O2 emission depended on the substrate while those for Zn were similar during glutamate-malate and succinate oxidation. Cu + Zn and Cu + Cd mixtures exhibited antagonistic interactions wherein Cu reduced the effects of both Cd and Zn, suggesting that Cu can mitigate oxidative distress caused by Cd or Zn. Binary combinations of the metals acted additively to reduce the rate constant and increase the half-life of H2O2 consumption while concomitantly suppressing thioredoxin reductase and stimulating glutathione peroxidase activities. Collectively, our study indicates that binary mixtures of Cu, Zn, and Cd act additively or antagonistically to modulate H2O2 homeodynamics in heart mitochondria.

Altered Hepatic Mitochondrial Bioenergetics and Oxidant Emission with Ischemia and Reperfusion

Rationale: Ischemia-reperfusion injury (IRI) is inevitable in liver transplantation (LT) which can be mitigated by normothermic machine perfusion (NMP) before LT. The current NMP liver viability criteria for LT mainly rely on bile production and perfusate lactate levels, factors that can also be affected by external variables such as NMP conditions and perfusate additives. Incorporating mitochondrial bioenergetic data could improve the predictive value of current NMP viability criteria for LT. Thus, the goal of this study was to evaluate mitochondrial oxygen consumption rate (OCR), membrane potential (Δψ), and H2O2 oxidant emission in control livers and in livers with IRI and to explore underlying mechanisms responsible for any observed differences. Methods: Healthy control livers and livers exposed to 60 minutes warm ischemia time (WIT) through in situ clamping of the portal vein and hepatic artery followed by 60 minutes reperfusion (IRI) were harvested from adult male Sprague-Dawley rats. Mitochondria from control and IRI livers were isolated using established protocols and assessed for their bioenergetics responses. Three different substrate combinations, namely, pyruvate+malate (PM), glutamate+malate (GM), and succinate were used, followed by addition(s) of ADP and the uncoupler FCCP. Two different ADP addition protocols were designed to determine how different substrates influence mitochondrial OCR, Δψ, and H2O2 emission during oxidative phosphorylation (OxPhos) between control and IRI conditions. In one protocol, a single saturated dose of ADP addition, and in the other, sequentially increasing doses of ADP additions were made following substrate addition. The OCR and Δψ were measured simultaneously using a dual chamber Oroboros Oxygraph-2k Instrument coupled to a fluorometer using the TMRM dye. The H2O2 emission was measured spectrofluorometrically using the amplex red and horseradish peroxidase assay. Results: The measured data show that the kinetics and effciency of OxPhos defining mitochondrial OCR and Δψ responses in the liver are negatively affected by IRI, characterized by enhanced H2O2 emission, in a substrate-dependent manner. Interestingly, the respiratory rates are higher when GM and succinate are utilized as substrates, whereas the use of PM shows comparatively lower respiration in both conditions. Control mitochondria exhibited higher respiratory rates than IRI mitochondria irrespective of the substrate utilized. The ADP-induced state 3 OCR in IRI mitochondria was 50% lower than that in control mitochondria resulting in doubling the duration of state 3 OCR. Similar differences were observed in Δψ for both ADP addition protocols. Compromised mitochondrial bioenergetics during IRI was observed using both single and sequentially increasing doses of ADP and was concomitant with a higher rate of H2O2 emission. Conclusion: This study provided novel quantitative data demonstrating the substrate specific changes in mitochondrial bioenergetics in hepatic IRI, which can be used to improve the NMP viability criteria for LT. The obtained bioenergetics and H2O2 emission data indicate that, during IRI, mitochondrial function is highly affected which may critically impact energy dependent metabolism and lactate/pyruvate ratio. NIH R01-HL151587. 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.

Effects of aging and exercise on Mitochondria-ER contacts (MERCs) in skeletal muscle

Mitochondrial dysfunction is a prominent factor contributing to age-associated loss of muscle mass and force (a.k.a., sarcopenia). Yet, events preceding age-related mitochondrial dysfunction, which may allow the development of new therapies, remain poorly understood. Physical coupling of the outer mitochondrial membrane (OMM) to the endoplasmic or sarcoplasmic reticulum (ER), termed mitochondria ER contact sites (MERCs), modulate several mitochondrial processes influencing cellular function. In an energetically demanding and primarily post-mitotic tissue, we hypothesized that alterations in MERCs correlate with the development of sarcopenia. We also hypothesized that MERCs respond to exercise training and may contribute to beneficial muscular remodeling in older adults. To address these, we studied three age groups of male C57BL6N mice: young adults (Y, 5 mo. of age), old (O, 20 mo. of age), and very old (VO, 31 mo. of age). An additional group of old mice underwent 8-9 weeks of treadmill training (OET). Muscle mass and contractile function decreased with aging; VO animals displayed ~25% decline in maximum tetanic force vs. Y mice (p&lt;0.0001). Fatigability (assessed via repetitive sub-maximal contractions) increased by ~10% with aging but was attenuated in the OET group (p&lt;0.001). In saponin-permeabilized muscle fibers, aging did not affect mitochondrial oxidative capacity. However, H2O2 emission was nearly doubled in the O group (p&lt;0.01) and this trend was partially alleviated by exercise training. Assessed via TEM, aging decreased the size of MERCs (i.e., length of SR and OMM apposition) by around 20% (p&lt;0.01). Interestingly, OET animals displayed ~50% increase in MERC coverage (i.e., proportion of OMM covered by SR; p&lt;0.0001). Immunoblots of cellular MERC fractions revealed that MERC proteins associated with calcium exchange were altered with age and exercise. The OMM channel, VDAC1, was reduced by 20% in O muscles (p=0.02), while Grp75, a tether protein, was ~25% higher in OET (p=0.02). Quantitative proteomic analyses revealed various MERC proteins being modified by aging and exercise. Our findings indicate that changes in MERC structure and protein composition may underlie early events preceding the development of sarcopenia and that exercise-mediated adaptations at MERCs may contribute to the beneficial adaptations in aging skeletal muscle. Supported by the University of Iowa Fraternal Order of Eagles Diabetes Research Center (UIowa FOEDRC). 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.

Impact of exercise training on endothelial metabolism and skeletal muscle angiogenic potential in type 2 diabetes

Type 2 diabetes (T2D) is associated with vascular endothelial dysfunction and microvascular rarefaction in skeletal muscle tissue (1). Although endothelial dysfunction in metabolic disease is proposed to be associated with altered endothelial cell metabolism, there is to date a paucity of human data to support this notion. The aim of this study was to investigate the metabolism in microvascular endothelial cells derived from skeletal muscle biopsies. Moreover, as exercise training is known to reverse endothelial dysfunction and promote angiogenesis, the effect of a period of exercise training was assessed. Microvascular endothelial cells were isolated from m. vastus lateralis biopsies taken from obese individuals with (n = 7) and without (n = 8) T2D before and after 12 weeks of aerobic exercise training. Mitochondrial respiration, hydrogen peroxide (H2O2) emission, and nitric oxide (NO) formation were determined in the cells by the Oroboros Oxygraph-2K. In addition, angiogenic-, metabolic- and antioxidant enzymes and proteins, cell proliferation, and skeletal muscle capillarization were determined. At baseline, there was no difference between endothelial cells from obese control participants and T2D obese participants for any of the parameters measured: glycolytic capacity, as evidenced by a similar lactate production and PFK-1 protein content, respiratory capacity, H2O2 emission, NO emission, endothelial NO synthase protein (eNOS) content. The training period led to an overall increase in the respiratory capacity in endothelial cells from both groups (main effect, P = 0.021) whereas mitochondrial H2O2 emission remained unaltered. There was no change in glycolytic capacity, superoxide dismutase 2 or eNOS protein contents, or NO emission. Training did not influence the capillary-to-muscle fiber ratio in either group. The results of this study show that aerobic exercise training of obese control and obese diabetic subjects leads to an increase in the respiratory capacity of muscle microvascular endothelial cells. The data also indicate that there are no differences in endothelial metabolism and NO production between obese control and obese diabetic individuals. However, as the study is ongoing and only half of the subjects have been included at this time, power is limited and results may differ upon completion. Data from all subjects will be presented at the conference. Funding: The study is funded by Novo Nordisk A/S. 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.

MFN2 overexpression in skeletal muscles of young and old mice causes a mild hypertrophy without altering mitochondrial respiration and H2O2 emission.

Sarcopenia, the aging-related loss of muscle mass and function, is a debilitating process negatively impacting the quality of life of affected individuals. Although the mechanisms underlying sarcopenia are incompletely understood, impairments in mitochondrial dynamics, including mitochondrial fusion, have been proposed as a contributing factor. However, the potential of upregulating mitochondrial fusion proteins to alleviate the effects of aging on skeletal muscles remains unexplored. We therefore hypothesized that overexpressing Mitofusin 2 (MFN2) in skeletal muscle invivo would mitigate the effects of aging on muscle mass and improve mitochondrial function. MFN2 was overexpressed in young (7 mo) and old (24 mo) male mice for 4 months through intramuscular injections of an adeno-associated viruses. The impacts of MFN2 overexpression on muscle mass and fiber size (histology), mitochondrial respiration, and H2O2 emission (Oroboros fluororespirometry), and various signaling pathways (qPCR and western blotting) were investigated. MFN2 overexpression increased muscle mass and fiber size in both young and old mice. No sign of fibrosis, necrosis, or inflammation was found upon MFN2 overexpression, indicating that the hypertrophy triggered by MFN2 overexpression was not pathological. MFN2 overexpression even reduced the proportion of fibers with central nuclei in old muscles. Importantly, MFN2 overexpression had no impact on muscle mitochondrial respiration and H2O2 emission in both young and old mice. MFN2 overexpression attenuated the increase in markers of impaired autophagy in old muscles. MFN2 overexpression may be a viable approach to mitigate aging-related muscle atrophy and may have applications for other muscle disorders.

Chronic kidney disease and left ventricular diastolic dysfunction (CKD-LVDD) alter cardiac expression of mitochondria-related genes in swine

Cardiovascular disease and heart failure doubles in patients with chronic kidney disease (CKD), but the underlying mechanisms remain obscure. Mitochondria are central to maintaining cellular respiration and modulating cardiomyocyte function. We took advantage of our novel swine model of CKD and left ventricular diastolic dysfunction (CKD-LVDD) to investigate the expression of mitochondria-related genes and potential mechanisms regulating their expression. CKD-LVDD and normal control pigs (n=6/group, 3 males/3 females) were studied for 14 weeks. Renal and cardiac hemodynamics were quantified by multidetector-CT, echocardiography, and pressure-volume loop studies, respectively. Mitochondrial morphology (electron microscopy) and function (Oroboros) were assessed ex vivo. In randomly selected pigs (n=3/group), cardiac mRNA-, MeDIP-, and miRNA-sequencing (seq) were performed to identify mitochondria-related genes and study their pre- and post -transcriptional regulation. CKD-LVDD exhibited cardiac mitochondrial structural abnormalities and elevated mitochondrial H2O2 emission but preserved mitochondrial function. Cardiac mRNA-seq identified 862 mitochondria-related genes, of which 69 were upregulated and 33 downregulated (fold-change ≥2, false discovery rate≤0.05). Functional analysis showed that upregulated genes were primarily implicated in processes associated with oxidative stress, whereas those downregulated mainly participated in respiration and ATP synthesis. Integrated mRNA/miRNA/MeDIP-seq analysis showed that upregulated genes were modulated predominantly by miRNAs, whereas those downregulated were by miRNA and epigenetic mechanisms. CKD-LVDD alters cardiac expression of mitochondria-related genes, associated with mitochondrial structural damage but preserved respiratory function, possibly reflecting intrinsic compensatory mechanisms. Our findings may guide the development of early interventions at stages of cardiac dysfunction in which mitochondrial injury could be prevented, and the development of LVDD ameliorated.

Effect of skeletal muscle mitochondrial phenotype on H2O2 emission

Reactive oxygen species (ROS) are a key output of the skeletal muscle mitochondrial information processing system both at rest and during exercise. In skeletal muscle, mitochondrial ROS release depends on multiple factors; however, fiber-type specific differences remain ambiguous in part owing to the use of mitochondria from mammalian muscle that consist of mixed fibers. To elucidate fiber-type specific differences, we used mitochondria isolated from rainbow trout (Oncorhynchus mykiss) red and white skeletal muscles that consist of spatially distinct essentially pure red and white fibers. We first characterized the assay conditions for measuring ROS production (as H2O2) in isolated fish red and white skeletal muscle mitochondria (RMM and WMM) and thereafter compared the rates of emission during oxidation of different substrates and the responses to mitochondrial electron transport system (ETS) pharmacological modulators. Our results showed that H2O2 emission rates by RMM and WMM can be quantified using the same protein concentration and composition of the Amplex UltraRed-horseradish peroxidase (AUR-HRP) detection system. For both RMM and WMM, protein normalized H2O2 emission rates were highest at the lowest protein concentration tested and decreased exponentially thereafter. However, the absolute values of H2O2 emission rates depended on the calibration curves used to convert fluorescent signals to H2O2 while the trends depended on the normalization strategy. We found substantial qualitative and quantitative differences between RMM and WMM in the H2O2 emission rates depending on the substrates being oxidized and their concentrations. Similarly, pharmacological modulators of the ETS altered the magnitudes and trends of the H2O2 emission differently in RMM and WMM. While comparable concentrations of substrates elicited maximal albeit quantitively different emission rates in RMM and WMM, different concentrations of pharmacological ETS modulators may be required for maximal H2O2 emission rates depending on muscle fiber-type. Taken together, our study suggests that biochemical differences exist in RMM compared with WMM that alter substrate oxidation and responses to ETS modulators resulting in fiber-type specific mitochondrial H2O2 emission rates.

The slow-release adiponectin analog ALY688-SR modifies early-stage disease development in the D2.mdx mouse model of Duchenne muscular dystrophy.

Fibrosis is associated with respiratory and limb muscle atrophy in Duchenne muscular dystrophy (DMD). Current standard of care partially delays the progression of this myopathy but there remains an unmet need to develop additional therapies. Adiponectin receptor agonism has emerged as a possible therapeutic target to lower inflammation and improve metabolism in mdx mouse models of DMD but the degree to which fibrosis and atrophy are prevented remain unknown. Here, we demonstrate that the recently developed slow-release peptidomimetic adiponectin analog, ALY688-SR, remodels the diaphragm of murine model of DMD on DBA background (D2.mdx) mice treated from days 7-28 of age during early stages of disease. ALY688-SR also lowered interleukin-6 (IL-6) mRNA but increased IL-6 and transforming growth factor-β1 (TGF-β1) protein contents in diaphragm, suggesting dynamic inflammatory remodeling. ALY688-SR alleviated mitochondrial redox stress by decreasing complex I-stimulated H2O2 emission. Treatment also attenuated fibrosis, fiber type-specific atrophy, and in vitro diaphragm force production in diaphragm suggesting a complex relationship between adiponectin receptor activity, muscle remodeling, and force-generating properties during the very early stages of disease progression in murine model of DMD on DBA background (D2.mdx) mice. In tibialis anterior, the modest fibrosis at this young age was not altered by treatment, and atrophy was not apparent at this young age. These results demonstrate that short-term treatment of ALY688-SR in young D2.mdx mice partially prevents fibrosis and fiber type-specific atrophy and lowers force production in the more disease-apparent diaphragm in relation to lower mitochondrial redox stress and heterogeneous responses in certain inflammatory markers. These diverse muscle responses to adiponectin receptor agonism in early stages of DMD serve as a foundation for further mechanistic investigations.NEW & NOTEWORTHY There are limited therapies for the treatment of Duchenne muscular dystrophy. As fibrosis involves an accumulation of collagen that replaces muscle fibers, antifibrotics may help preserve muscle function. We report that the novel adiponectin receptor agonist ALY688-SR prevents fibrosis in the diaphragm of D2.mdx mice with short-term treatment early in disease progression. These responses were related to altered inflammation and mitochondrial functions and serve as a foundation for the development of this class of therapy.

High Fat Diet-Induced Obesity Dysregulates Splenic B Cell Mitochondrial Activity.

Diet-induced obesity impairs mitochondrial respiratory responses in tissues that are highly metabolically active, such as the heart. However, less is known about the impact of obesity on the respiratory activity of specific cell types, such as splenic B cells. B cells are of relevance, as they play functional roles in obesity-induced insulin resistance, inflammation, and responses to infection. Here, we tested the hypothesis that high-fat-diet (HFD)-induced obesity could impair the mitochondrial respiration of intact and permeabilized splenic CD19+ B cells isolated from C57BL/6J mice and activated ex vivo with lipopolysaccharide (LPS). High-resolution respirometry was used with intact and permeabilized cells. To reveal potential mechanistic targets by which HFD-induced obesity dysregulates B cell mitochondria, we conducted proteomic analyses and 3D serial block face scanning electron microscopy (SBFEM). High-resolution respirometry revealed that intact LPS-stimulated B cells of obese mice, relative to controls, displayed lower ATP-linked, as well as maximal uncoupled, respiration. To directly investigate mitochondrial function, we used permeabilized LPS-stimulated B cells, which displayed increased H2O2 emission and production with obesity. We also examined oxidative phosphorylation efficiency simultaneously, which revealed that oxygen consumption and ATP production were decreased in LPS-stimulated B cells with obesity relative to controls. Despite minimal changes in total respiratory complex abundance, in LPS-stimulated B cells of obese mice, three of the top ten most downregulated proteins were all accessory subunits of respiratory complex I. SBFEM showed that B cells of obese mice, compared to controls, underwent no change in mitochondrial cristae integrity but displayed increased mitochondrial volume that was linked to bioenergetic function. Collectively, these results establish a proof of concept that HFD-induced obesity dysregulates the mitochondrial bioenergetic metabolism of activated splenic B cells.

Exhaustive exercise alters native and site-specific H2O2 emission in red and white skeletal muscle mitochondria

Mitochondrial reactive oxygen species (ROS) homeostasis is intricately linked to energy conversion reactions and entails regulation of the mechanisms of ROS production and removal. However, there is limited understanding of how energy demand modulates ROS balance. Skeletal muscle experiences a wide range of energy requirements depending on the intensity and duration of exercise and therefore is an excellent model to probe the effect of altered energy demand on mitochondrial ROS production. Because in most fish skeletal muscle exists essentially as pure spatially distinct slow-twitch red oxidative and fast-twitch white glycolytic fibers, it provides a natural system for investigating how functional specialization affects ROS homeostasis. We tested the hypothesis that acute increase in energy demand imposed by exhaustive exercise will increase mitochondrial H2O2 emission to a greater extent in red muscle mitochondria (RMM) compared with white muscle mitochondria (WMM). We found that native H2O2 emission rates varied by up to 6-fold depending on the substrate being oxidized and muscle fiber type, with RMM emitting at higher rates with glutamate-malate and palmitoylcarnitine while WMM emitted at higher rates with succinate and glyceral-3-phosphate. Exhaustive exercise increased the native and site-specific H2O2 emission rates; however, the maximal emission rates depended on the substrate, fiber type and redox site. The H2O2 consumption capacity and activities of individual antioxidant enzymes including the glutathione- and thioredoxin-dependent peroxidases as well as catalase were higher in RMM compared with WMM indicating that the activity of antioxidant defense system does not explain the differences in H2O2 emission rates in RMM and WMM. Overall, our study suggests that substrate selection and oxidation may be the key factors determining the rates of ROS production in RMM and WMM following exhaustive exercise.

255-LB: Liver Pyruvate Carboxylase Deletion in Mice Prevents Fibrosis from a NASH Diet

Obesity and T2D are closely associated with Nonalcoholic Fatty Liver Disease, which can advance to Nonalcoholic Steatohepatitis (NASH) with fibrosis. We previously found that liver pyruvate carboxylase (PC) is required for hepatic gluconeogenesis and the concentration of metabolites essential to mediating redox state and antioxidant function. Liver PC knockout (LPCKO) mice are protected from HFD-induced hyperinsulinemia and glucose intolerance despite retaining excess liver fat and exhibiting reduced antioxidant capacity. Here, we report that despite impaired antioxidant markers and increased inflammatory markers on a HFD, PC loss protected the liver from fibrosis and other indices of liver damage induced by a long-term NASH diet. LPCKO and control mice were fed a high fat, high cholesterol Western Diet (WD) for 13-14 months. After 10 mos. of WD, markers of insulin resistance were assessed by lactate/pyruvate, glucose, and insulin tolerance tests. After sacrifice, liver damage was assessed by serum markers and histology. Oxidative stress and inflammation were evaluated by gene expression, MDA concentration, GSH/GSSG and NADPH/NADP+ redox states, and H2O2 emission potential in isolated mitochondria. LPCKO-WD mice were more sensitive to an insulin tolerance test and exhibited lower fasting blood glucose and better glycemic control during a lactate/pyruvate or glucose tolerance test than controls. Liver malate/pyruvate indicated substantially lower NADPH/NADP+ in LPCKO-WD mice, but there were no differences in MDA levels, GSH/GSSG, and H2O2 emission potential. Despite similar hepatic steatosis, inflammation, and ballooning between LPCKO-WD and controls, fibrosis and serum markers of liver injury were markedly lower in LPCKO-WD mice. The data suggest that liver PC deletion improves glycemia and insulin resistance during chronic Western Diet exposure by reducing hepatic gluconeogenesis and prevents fibrosis independent of steatosis, inflammation, ballooning, and oxidative stress. Disclosure M. R. Iñigo-Vollmer: None. B. Kucejova: None. M. N. Mizerska: None. S. Khan: None. P. A. Szweda: None. H. Zaki: None. L. I. Szweda: None. X. Fu: None. S. C. Burgess: None.

Pre- and Post-Sexual Maturity Liver-specific ERα Knockout Does Not Impact Hepatic Mitochondrial Function.

Compared with males, premenopausal women and female rodents are protected against hepatic steatosis and present with higher functioning mitochondria (greater hepatic mitochondrial respiration and reduced H2O2 emission). Despite evidence that estrogen action mediates female protection against steatosis, mechanisms remain unknown. Here we validated a mouse model with inducible reduction of liver estrogen receptor alpha (ERα) (LERKO) via adeno-associated virus (AAV) Cre. We phenotyped the liver health and mitochondrial function of LERKO mice (n = 10-12 per group) on a short-term high-fat diet (HFD), and then tested whether timing of LERKO induction at 2 timepoints (sexually immature: 4 weeks old [n = 11 per group] vs sexually mature: 8-10 weeks old [n = 8 per group]) would impact HFD-induced outcomes. We opted for an inducible LERKO model due to known estrogen-mediated developmental programming, and we reported both receptor and tissue specificity with our model. Control mice were ERαfl/fl receiving AAV with green fluorescent protein (GFP) only. Results show that there were no differences in body weight/composition or hepatic steatosis in LERKO mice with either short-term (4-week) or chronic (8-week) high-fat feeding. Similarly, LERKO genotype nor timing of LERKO induction (pre vs post sexual maturity) did not alter hepatic mitochondrial O2 and H2O2 flux, coupling, or OXPHOS protein. Transcriptomic analysis showed that hepatic gene expression in LERKO was significantly influenced by developmental stage. Together, these studies suggest that hepatic ERα is not required in female protection against HFD-induced hepatic steatosis nor does it mediate sexual dimorphism in liver mitochondria function.

Enhanced Levels of Peroxisome-Derived H2O2 Do Not Induce Pexophagy but Impair Autophagic Flux in HEK-293 and HeLa Cells

Peroxisomes are functionally specialized organelles that harbor multiple hydrogen peroxide (H2O2)-producing and -degrading enzymes. Given that this oxidant functions as a major redox signaling agent, peroxisomes have the intrinsic ability to mediate and modulate H2O2-driven processes, including autophagy. However, it remains unclear whether changes in peroxisomal H2O2 (po-H2O2) emission impact the autophagic process and to which extent peroxisomes with a disturbed H2O2 metabolism are selectively eliminated through a process called "pexophagy". To address these issues, we generated and validated HEK-293 and HeLa pexophagy reporter cell lines in which the production of po-H2O2 can be modulated. We demonstrate that (i) po-H2O2 can oxidatively modify multiple selective autophagy receptors and core autophagy proteins, (ii) neither modest nor robust levels of po-H2O2 emission act as a prime determinant of pexophagy, and (iii) high levels of po-H2O2 impair autophagic flux by oxidative inhibition of enzymes involved in LC3II formation. Unexpectedly, our analyses also revealed that the autophagy receptor optineurin can be recruited to peroxisomes, thereby triggering pexophagy. In summary, these findings lend support to the idea that, during cellular and organismal aging, peroxisomes with enhanced H2O2 release can escape pexophagy and downregulate autophagic activity, thereby perpetuating the accumulation of damaged and toxic cellular debris.

The impact of high-fat feeding and parkin overexpression on skeletal muscle mass, mitochondrial respiration, and H2O2 emission.

Obesity is a major risk factor for developing various health problems, including insulin resistance and type 2 diabetes. Although controversial, accumulation of mitochondrial dysfunction, and notably an increase in mitochondrial reactive oxygen species (ROS) production, was proposed as a key contributor leading to obesity-induced insulin resistance. Here, our goal was to investigate whether Parkin overexpression, a key regulator of the removal of dysfunctional mitochondria through mitophagy, could confer protection against obesity-induced mitochondrial dysfunction. To this end, intramuscular injections of adeno-associated viruses (AAVs) were performed to overexpress Parkin in limb muscle of 6-mo-old mice fed a control diet (CD) or a high-fat diet (HFD) for 12 wk. An AAV-expressing the green fluorescent protein (GFP) was used as control. HFD increased fat mass, altered glycemia, and resulted in insulin resistance. Parkin overexpression resulted in an increase in muscle mass in both CD and HFD mice. In CD mice, Parkin overexpression increased maximal mitochondrial respiration and lowered H2O2 emission. HFD increased mitochondrial respiration and, surprisingly, also lowered H2O2 emission. Parkin overexpression did not significantly impact mitochondrial function in HFD mice. Taken altogether, our results indicate that Parkin overexpression positively impacts muscle and mitochondrial health under basal conditions and challenges the notion that intrinsic mitochondrial dysfunction is involved in the development of insulin resistance caused by high-fat feeding.

Muscle weakness precedes atrophy during cancer cachexia and is linked to muscle-specific mitochondrial stress.

Muscle weakness and wasting are defining features of cancer-induced cachexia. Mitochondrial stress occurs before atrophy in certain muscles, but the possibility of heterogeneous responses between muscles and across time remains unclear. Using mice inoculated with Colon-26 cancer, we demonstrate that specific force production was reduced in quadriceps and diaphragm at 2 weeks in the absence of atrophy. At this time, pyruvate-supported mitochondrial respiration was lower in quadriceps while mitochondrial H2O2 emission was elevated in diaphragm. By 4 weeks, atrophy occurred in both muscles, but specific force production increased to control levels in quadriceps such that reductions in absolute force were due entirely to atrophy. Specific force production remained reduced in diaphragm. Mitochondrial respiration increased and H2O2 emission was unchanged in both muscles versus control while mitochondrial creatine sensitivity was reduced in quadriceps. These findings indicate muscle weakness precedes atrophy and is linked to heterogeneous mitochondrial alterations that could involve adaptive responses to metabolic stress. Eventual muscle-specific restorations in specific force and bioenergetics highlight how the effects of cancer on one muscle do not predict the response in another muscle. Exploring heterogeneous responses of muscle to cancer may reveal new mechanisms underlying distinct sensitivities, or resistance, to cancer cachexia.

Impact of Aging and Lifelong Exercise Training on Mitochondrial Function and Network Connectivity in Human Skeletal Muscle.

Aging is associated with metabolic decline in skeletal muscle, which can be delayed by physical activity. Moreover, both lifelong and short-term exercise training have been shown to prevent age-associated fragmentation of the mitochondrial network in mouse skeletal muscle. However, whether lifelong endurance exercise training exerts the same effects in human skeletal muscle is still not clear. Therefore, the aim of the present study was to examine the effect of volume-dependent lifelong endurance exercise training on mitochondrial function and network connectivity in older human skeletal muscle. Skeletal muscle complex I+II-linked mitochondrial respiration per tissue mass was higher, but intrinsic complex I+II-linked mitochondrial respiration was lower in highly trained older subjects than in young untrained, older untrained, and older moderately trained subjects. Mitochondrial volume and connectivity were higher in highly trained older subjects than in untrained and moderately trained older subjects. Furthermore, the protein content of the ADP/ATP exchangers ANT1 + 2 and VDAC was higher and of the mitophagic marker parkin lower in skeletal muscle from the highly trained older subjects than from untrained and moderately trained older subjects. In contrast, H2O2 emission in skeletal muscle was not affected by either age or exercise training, but SOD2 protein content was higher in highly trained older subjects than in untrained and moderately trained older subjects. This suggests that healthy aging does not induce oxidative stress or mitochondrial network fragmentation in human skeletal muscle, but high-volume exercise training increases mitochondrial volume and network connectivity, thereby increasing oxidative capacity in older human skeletal muscle.