Mitochondrial H2O2 Research Articles

Novel Role of CETP in Macrophages: Reduction of Mitochondrial Oxidants Production and Modulation of Cell Immune-Metabolic Profile.

Plasma cholesteryl ester transfer protein (CETP) activity diminishes HDL-cholesterol levels and thus may increase atherosclerosis risk. Experimental evidence suggests CETP may also exhibit anti-inflammatory properties, but local tissue-specific functions of CETP have not yet been clarified. Since oxidative stress and inflammation are major features of atherogenesis, we investigated whether CETP modulates macrophage oxidant production, inflammatory and metabolic profiles. Comparing macrophages from CETP-expressing transgenic mice and non-expressing littermates, we observed that CETP expression reduced mitochondrial superoxide anion production and H2O2 release, increased maximal mitochondrial respiration rates, and induced elongation of the mitochondrial network and expression of fusion-related genes (mitofusin-2 and OPA1). The expression of pro-inflammatory genes and phagocytic activity were diminished in CETP-expressing macrophages. In addition, CETP-expressing macrophages had less unesterified cholesterol under basal conditions and after exposure to oxidized LDL, as well as increased HDL-mediated cholesterol efflux. CETP knockdown in human THP1 cells increased unesterified cholesterol and abolished the effects on mitofusin-2 and TNFα. In summary, the expression of CETP in macrophages modulates mitochondrial structure and function to promote an intracellular antioxidant state and oxidative metabolism, attenuation of pro-inflammatory gene expression, reduced cholesterol accumulation, and phagocytosis. These localized functions of CETP may be relevant for the prevention of atherosclerosis and other inflammatory diseases.

P53 drives necroptosis via downregulation of sulfiredoxin and peroxiredoxin 3

Mitochondrial dysfunction is a key contributor to necroptosis. We have investigated the contribution of p53, sulfiredoxin, and mitochondrial peroxiredoxin 3 to necroptosis in acute pancreatitis. Late during the course of pancreatitis, p53 was localized in mitochondria of pancreatic cells undergoing necroptosis. In mice lacking p53, necroptosis was absent, and levels of PGC-1α, peroxiredoxin 3 and sulfiredoxin were upregulated. During the early stage of pancreatitis, prior to necroptosis, sulfiredoxin was upregulated and localized into mitochondria. In mice lacking sulfiredoxin with pancreatitis, peroxiredoxin 3 was hyperoxidized, p53 localized in mitochondria, and necroptosis occurred faster; which was prevented by Mito-TEMPO. In obese mice, necroptosis occurred in pancreas and adipose tissue. The lack of p53 up-regulated sulfiredoxin and abrogated necroptosis in pancreas and adipose tissue from obese mice. We describe here a positive feedback between mitochondrial H2O2 and p53 that downregulates sulfiredoxin and peroxiredoxin 3 leading to necroptosis in inflammation and obesity.

Chronic developmental hypoxia alters mitochondrial oxidative capacity and reactive oxygen species production in the fetal rat heart in a sex-dependent manner.

Insufficient oxygen supply (hypoxia) during fetal development leads to cardiac remodeling and a predisposition to cardiovascular disease in later life. Previous work has shown hypoxia causes oxidative stress in the fetal heart and alters the activity and expression of mitochondrial proteins in a sex‐dependent manner. However, the functional effects of these modifications on mitochondrial respiration remain unknown. Furthermore, while maternal antioxidant treatments are emerging as a promising new strategy to protect the hypoxic fetus, whether these treatments convey similar protection to cardiac mitochondria in the male or female fetus has not been investigated. Therefore, using an established rat model, we measured the sex‐dependent effects of gestational hypoxia and maternal melatonin treatment on fetal cardiac mitochondrial respiration, reactive oxygen species (ROS) production, and lipid peroxidation. Pregnant Wistar rats were subjected to normoxia or hypoxia (13% oxygen) during gestational days (GDs) 6–20 (term ~22 days) with or without melatonin treatment (5 µg/ml in maternal drinking water). On GD 20, mitochondrial aerobic respiration and H2O2 production were measured in fetal heart tissue, together with lipid peroxidation and citrate synthase (CS) activity. Gestational hypoxia reduced maternal body weight gain (p < .01) and increased placental weight (p < .05) but had no effect on fetal weight or litter size. Cardiac mitochondria from male but not female fetuses of hypoxic pregnancy had reduced respiratory capacity at Complex II (CII) (p < .05), and an increase in H2O2 production/O2 consumption (p < .05) without any changes in lipid peroxidation. CS activity was also unchanged in both sexes. Despite maternal melatonin treatment increasing maternal and fetal plasma melatonin concentration (p < .001), melatonin treatment had no effect on any of the mitochondrial parameters investigated. To conclude, we show that gestational hypoxia leads to ROS generation from the mitochondrial electron transport chain and affects fetal cardiac mitochondrial respiration in a sex‐dependent manner. We also show that maternal melatonin treatment had no effect on these relationships, which has implications for the development of future therapies for hypoxic pregnancies.

Mitochondrial and redox modifications in early stages of Huntington's disease

Deficits in mitochondrial function and redox deregulation have been attributed to Huntington's disease (HD), a genetic neurodegenerative disorder largely affecting the striatum. However, whether these changes occur in early stages of the disease and can be detected in vivo is still unclear. In the present study, we analysed changes in mitochondrial function and production of reactive oxygen species (ROS) at early stages and with disease progression. Studies were performed in vivo in human brain by PET using [64Cu]-ATSM and ex vivo in human skin fibroblasts of premanifest and prodromal (Pre-M) and manifest HD carriers. In vivo brain [64Cu]-ATSM PET in YAC128 transgenic mouse and striatal and cortical isolated mitochondria were assessed at presymptomatic (3 month-old, mo) and symptomatic (6–12 mo) stages. Pre-M HD carriers exhibited enhanced whole-brain (with exception of caudate) [64Cu]-ATSM labelling, correlating with CAG repeat number. Fibroblasts from Pre-M showed enhanced basal and maximal respiration, proton leak and increased hydrogen peroxide (H2O2) levels, later progressing in manifest HD. Mitochondria from fibroblasts of Pre-M HD carriers also showed reduced circularity, while higher number of mitochondrial DNA copies correlated with maximal respiratory capacity. In vivo animal PET analysis showed increased accumulation of [64Cu]-ATSM in YAC128 mouse striatum. YAC128 mouse (at 3 months) striatal isolated mitochondria exhibited a rise in basal and maximal mitochondrial respiration and in ATP production, and increased complex II and III activities. YAC128 mouse striatal mitochondria also showed enhanced mitochondrial H2O2 levels and circularity, revealed by brain ultrastructure analysis, and defects in Ca2+ handling, supporting increased striatal susceptibility. Data demonstrate both human and mouse mitochondrial overactivity and altered morphology at early HD stages, facilitating redox unbalance, the latter progressing with manifest disease.

A hemicyanine-based near-infrared fluorescent probe for visualizing biothiols fluctuations induced by Ag+ in mitochondria

A novel near-infrared fluorescent probe CySNC based on hemicyanine was developed to monitor fluctuations of biothiols in response to oxidative stress caused by heavy metal ion poisoning. In the presence of biothiols, CySNC showed “turn-on” fluorescence at 772 nm with low limits of detection (0.12 μM for Cys, 0.15 μM for Hcy and 0.14 μM for GSH). The probe CySNC had excellent fluorescence response to Cys/Hcy/GSH under physiological conditions and the response time for Cys/Hcy/GSH was 15 min, 35 min and 28 min, respectively. In addition, CySNC was applied for detecting endogenous and exogenous biothiols in mitochondria with low cytotoxicity. More importantly, CySNC successfully visualized biothiols fluctuations induced by Ag+ in mitochondria and confirmed the relationship between oxidative stress and biothiols levels by stimulation with Ag+ and H2O2 in mitochondria, which provided a new train of thought to unravel the mechanisms of heavy ion poisoning.

Absence of Gem1 (mammalian Miro/Rhot) mitigates alpha-synuclein toxicity in a yeast model of Parkinson's disease

Alpha-synuclein aggregation is a hallmark of Parkinson's disease (PD). Mutants A30P and A53T alpha-synuclein are known to exacerbate the toxicity of alpha-synuclein, which includes oxidative stress, mitochondrial and endoplasmic reticulum (ER) dysfunction. Saccharomyces cerevisiae (budding yeast) is a cellular model widely used to investigate mechanisms underlying neurodegenerative disorders, such as PD. In yeast, Gem1 (Miro/Rhot mammalian orthologue) coordinates mitochondrial dynamics and ER homeostasis, which is impaired in the presence of mutant alpha-synuclein and can lead to cell death. In this study, A30P or A53T alpha-synuclein were expressed in wild type or ΔGem (deletion of Gem1 gene) yeast strains. ΔGem cells presented decreased viability and increased mitochondrial H2O2 production and ER stress compared to wild type cells. However, in the presence of mutant alpha-synuclein, ΔGem cells showed increased growth compared to cells that do not express mutant alpha-synuclein. ΔGem cells expressing A53T alpha-synuclein also presented reduced ER stress and increased ability to deal with oxidative stress. Together, our results suggest that deletion of Gem1 activates pathways that strengthen cells against other stressful agents such as the presence of mutant alpha-synuclein.

Comprehensive investigations of key mitochondrial metabolic changes in senescent human fibroblasts.

There is a paucity of detailed data related to the effect of senescence on the mitochondrial antioxidant capacity and redox state of senescent human cells. Activities of TCA cycle enzymes, respiratory chain complexes, hydrogen peroxide (H2O2), superoxide anions (SA), lipid peroxides (LPO), protein carbonyl content (PCC), thioredoxin reductase 2 (TrxR2), superoxide dismutase 2 (SOD2), glutathione peroxidase 1 (GPx1), glutathione reductase (GR), reduced glutathione (GSH), and oxidized glutathione (GSSG), along with levels of nicotinamide cofactors and ATP content were measured in young and senescent human foreskin fibroblasts. Primary and senescent cultures were biochemically identified by monitoring the augmented cellular activities of key glycolytic enzymes including phosphofructokinase, lactate dehydrogenase, and glycogen phosphorylase, and accumulation of H2O2, SA, LPO, PCC, and GSSG. Citrate synthase, aconitase, α-ketoglutarate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, isocitrate dehydrogenase, and complex I-III, II-III, and IV activities were significantly diminished in P25 and P35 cells compared to P5 cells. This was accompanied by significant accumulation of mitochondrial H2O2, SA, LPO, and PCC, along with increased transcriptional and enzymatic activities of TrxR2, SOD2, GPx1, and GR. Notably, the GSH/GSSG ratio was significantly reduced whereas NAD+/NADH and NADP+/NADPH ratios were significantly elevated. Metabolic exhaustion was also evident in senescent cells underscored by the severely diminished ATP/ADP ratio. Profound oxidative stress may contribute, at least in part, to senescence pointing at a potential protective role of antioxidants in aging-associated disease.

Abstract 128: SIRT3 supports anchorage-independent survival of ovarian cancer cells

Abstract Metabolic changes during tumor progression have numerous consequences on cancer cells, including an increased risk of cell death due to production of reactive oxygen species (ROS). Antioxidant defense adaptations thus accompany metabolic alterations as essential survival mechanisms during metastasis. We observe that oxidative phosphorylation is increased when ovarian cancer (OC) cells transition to anchorage independence (a-i), an important step during metastasis in the peritoneal cavity. To combat the rise in oxidative stress resulting from this metabolic shift, a rapid increase in the sirtuin 3 (SIRT3)/superoxide dismutase 2 (SOD2) axis occurs to reduce the mitochondrial superoxide anion surge and to maintain cellular viability. We demonstrate that this upregulation occurs in transcription, translation, and SIRT3-mediated regulation of SOD2 activity. However, the fate of the hydrogen peroxide (H2O2) resulting from superoxide dismutation through SIRT3/SOD2 remains unknown. RNAseq analysis of attached versus a-i conditions revealed a significant downregulation of peroxiredoxin 3, a major scavenger of H2O2 in the mitochondria, suggesting that OCs have a net increase in mitochondrial H2O2 in a-i. At sub-lethal levels, H2O2 serves as a second messenger of redox signaling, primarily via protein cysteine oxidation, which has been implicated with metastasis. We thus hypothesize that increases in SIRT3/SOD2/H2O2 play a role as mediators of redox signaling during OC anchorage-independent survival, spheroid formation and metastasis. Using the HyPer7 redox probe and manipulation of SIRT3 and SOD2 expression, H2O2 generation and localization in a-i are being assessed using 3D fluorescence microscopy. Moreover, siRNA mediated knock-down of SIRT3 in a-i leads to the downregulation of genes involved in specific signaling pathways, including NF-kB signaling, the inflammatory response, and metabolism. Mass-spec studies are underway to identify specific SIRT3 target proteins upstream of these transcriptional changes, by assessing global cysteine oxidation and the cellular acetylome to determine the role of H2O2-dependent redox signaling and SIRT3-dependent protein de-acetylation, respectively. Given the increase in SIRT3-dependent inflammatory signaling in a-i, we are exploring how the SIRT3/SOD2/H2O2 axis influences OC interaction with tumor associated macrophages and mesenchymal stem cells, commonly found in the ascites tumor environment. This work demonstrates a multi-faceted role for the SIRT3/SOD2/H2O2 axis in a-i survival, a key step during OC metastasis. Citation Format: Priscilla Tang, Yeon Soo Kim, Piyushi Gupta-Vallur, Amal T. Elhaw, Zaineb Javed, Weihua Pan, Nadine Hempel. SIRT3 supports anchorage-independent survival of ovarian cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 128.

Short-term exposure to a clinical dose of metformin increases skeletal muscle mitochondrial H2O2 emission and production in healthy, older adults: A randomized controlled trial.

Metformin is the most commonly prescribed medication to treat diabetes. Emerging evidence suggests that metformin could have off target effects that might help promote healthy muscle aging, but these effects have not been thoroughly studied in glucose tolerant older individuals. The purpose of this study was to investigate the short-term effects of metformin consumption on skeletal muscle mitochondrial bioenergetics in healthy older adults. We obtained muscle biopsy samples from 16 healthy older adults previously naïve to metformin and treated with metformin (METF; 3F, 5M), or placebo (CON; 3F, 5M), for two weeks using a randomized and blinded study design. Samples were analyzed using high-resolution respirometry, immunofluorescence, and immunoblotting to assess muscle mitochondrial bioenergetics, satellite cell (SC) content, and associated protein markers. We found that metformin treatment did not alter maximal mitochondrial respiration rates in muscle compared to CON. In contrast, mitochondrial H2O2 emission and production were elevated in muscle samples from METF versus CON (METF emission: 2.59±0.72 SE Fold, P=0.04; METF production: 2.29±0.53 SE Fold, P=0.02). Furthermore, the change in H2O2 emission was positively correlated with the change in type 1 myofiber SC content and this was biased in METF participants (Pooled: R2=0.5816, P=0.0006; METF: R2=0.674, P=0.0125). These findings suggest that acute exposure to metformin does not impact mitochondrial respiration in aged, glucose-tolerant muscle, but rather, influences mitochondrial-free radical and SC dynamics. NCT03107884, clinicaltrials.gov.

213-LB: Combined Oral Contraceptives Drive Inactivity and Hepatic Mitochondrial Oxidative Stress in Female Mice

Background: It is well established that compared to age-matched men, pre-menopausal women are protected against metabolic disease (obesity, diabetes, hepatic steatosis) . However, both epidemiological data on menopause and pre-clinical ovariectomy models show that disruption of estrogen signaling increases disease susceptibility. Combined oral hormonal contraceptives (OC) induce loss of normal estrogen cycling, and despite widespread use, the impact of OCs on metabolic health and disease remains poorly understood. Methods: Female C57BL/6J mice (7-8 wks old) were treated with high-fat diet (HFD; CON) or HFD + OCs (2mg estradiol/kg HFD, 200mg progestin/kg HFD; OC) for 12 wks. In each treatment group, mice either remained sedentary or had access to voluntary running wheels (VWR; n=12 per group) . Liver and skeletal muscle mitochondrial function (respiratory capacity, H2O2, coupling) were measured along with body composition, glucose tolerance, energy expenditure, and intrahepatic triglyceride. Results: OCs decreased VWR, spontaneous physical activity, and total energy expenditure. OC treated mice had lower adiposity and hepatic triglyceride compared to CON mice, with no difference in glucose tolerance. Despite similar maximal respiratory capacity, OCs induced robust increases in hepatic mitochondrial H2O2 across all respiratory states and with both fat and carbohydrate-based substrates. There were no differences in muscle mitochondrial outcomes. Conclusions: When combined with HFD, OC use lowers physical activity and energy expenditure while inducing tissue-specific effects on mitochondrial function in mice. These data provide pause for use of OCs in the context of elevated metabolic disease risk. Future work should explore whether OC-induced changes in physical activity blunts the ability of exercise to prevent or treat insulin resistance and steatosis and whether prolonged OC exposure leads to liver injury via elevated oxidative stress. Disclosure K. Fuller: None. C. S. Mccoin: None. H. Stierwalt: None. J. P. Thyfault: None. Funding AHA (20PRE35120098) , VA Merit Review Grant (1I01BX002567-01)

Evaluating the Protective Effect of Ginsenoside Re on Hippocampal Mitochondria in a Rat Model of Vascular Dementia

Vascular dementia (VD) is a common disease among elderly individuals that results in cognitive dysfunction. Although pharmaceutical treatments are used to temporarily improve the symptoms, these have side effects, including mental disorders and epilepsy. Therefore, identifying therapeutic strategies for VD which limit side effects would be advantageous. Previous studies have demonstrated the involvement of oxidative stress in the pathogenesis of VD. As the major saponin type of Panax ginseng, Ginsenoside Re, exerts anti-oxidation and anti-inflammation effects, Re may have a therapeutic role in the management of VD. Our aim was to evaluate this potential therapeutic role of Re in a rat model of VD. We compared mitochondrial function in hippocampal neurons in groups with or without Re administration. Our findings show that Re administration can reverse the effects of VD on mitochondrial structure and function in hippocampal neurons, including protecting against a reduction in the number of mitochondria and preventing mitochondrial vacuolization. The relative expression levels of COX IV and PDH-A1 also increased after Re treatment, with a reduction in mitochondrial H2O2 production, in a time-and dose-dependent manner. These improvements in mitochondrial function protected against VD-associated cognitive dysfunction, measured by performance in a Morris water maze. Based on these findings, we concluded that Ginsenoside Re may ameliorate cognitive impairment in VD by inhibiting mitochondrial oxidative stress in the brain. Collectively, we provide valuable experimental data which could inform the development of safe and effective drugs for the management of VD from ischemia.

Oxidative stress monitoring in iPSC-derived motor neurons using genetically encoded biosensors of H2O2

Oxidative stress plays an important role in the development of neurodegenerative diseases, being either the initiator or part of a pathological cascade that leads to the neuron’s death. Genetically encoded biosensors of oxidative stress demonstrated their general functionality and overall safety in various systems. However, there is still insufficient data regarding their use in the research of disease-related phenotypes in relevant model systems, such as human cells. Here, we establish an approach for monitoring the redox state of live motor neurons with SOD1 mutations associated with amyotrophic lateral sclerosis. Using CRISPR/Cas9, we insert genetically encoded biosensors of cytoplasmic and mitochondrial H2O2 in the genome of induced pluripotent stem cell (iPSC) lines. We demonstrate that the biosensors remain functional in motor neurons derived from these iPSCs and reflect the differences in the stationary redox state of the neurons with different genotypes. Moreover, we show that the biosensors respond to alterations in motor neuron oxidation caused by either environmental changes or cellular stress. Thus, the obtained platform is suitable for cell-based research of neurodegenerative mechanisms.

Regulation of kidney mitochondrial function by caloric restriction.

Caloric restriction (CR) prevents obesity and increases resilience against pathological stimuli in laboratory rodents. At the mitochondrial level, protection promoted by CR in the brain and liver is related to higher Ca2+ uptake rates and capacities, avoiding Ca2+-induced mitochondrial permeability transition. Dietary restriction has also been shown to increase kidney resistance against damaging stimuli; if these effects are related to similar mitochondrial adaptations has not been uncovered. Here, we characterized changes in mitochondrial function in response to 6 mo of CR in rats and measured bioenergetic parameters, redox balance, and Ca2+ homeostasis. CR promoted an increase in succinate-supported mitochondrial oxygen consumption rates. Although CR prevents mitochondrial reactive oxygen species production in many tissues, in kidney, we found that mitochondrial H2O2 release was enhanced in a succinate-dependent manner. Surprisingly, and opposite to the effects observed in the brain and liver, mitochondria from CR animals were more prone to Ca2+-induced mitochondrial permeability transition, in a manner reversed by the antioxidant dithiothreitol. CR mitochondria also displayed higher Ca2+ uptake rates, which were not accompanied by changes in Ca2+ efflux rates or related to altered inner mitochondrial membrane potentials or amounts of the mitochondrial Ca2+ uniporter. Instead, increased mitochondrial Ca2+ uptake rates in CR kidneys correlated with loss of mitochondrial Ca2+ uptake protein 2 (MICU2), a mitochondrial Ca2+ uniporter modulator. Interestingly, MICU2 is also modulated by CR in the liver, suggesting that it has a broader diet-sensitive regulatory role controlling mitochondrial Ca2+ homeostasis. Together, our results highlight the organ-specific bioenergetic, redox, and ionic transport results of CR, with some unexpected deleterious effects in the kidney.NEW & NOTEWORTHY Prevention of obesity through caloric restriction (CR) is well known to protect many tissues but has been poorly studied in kidneys. Here, we determined the effects of long-term CR in rat kidney mitochondria, which are central players in energy metabolism and aging. Surprisingly, we found that the diet increased mitochondrial reactive oxygen production and permeability transition. This suggests that the kidneys respond differently to restricted diets and may be more susceptible under CR.

Substrate‐Dependent Differential Regulation of Mitochondrial Bioenergetics and ROS emission in the Heart and Kidney Cortex and Outer Medulla

RationaleThe kinetics and efficiency of mitochondrial oxidative phosphorylation (OxPhos) is determined by the availability and utilization of particular respiratory substrates. Although the heart and kidney represent the two major energy consuming organs in the body, the substrate dependency of their mitochondrial OxPhos has not been characterized for these two organs within the same species and same individual animal.MethodThe effects of different substrate combinations on the kinetics and efficiency of OxPhos were characterized in mitochondria isolated from the heart, renal cortex, and renal outer medulla (OM) of adult Sprague‐Dawley rats. The substrate combinations included pyruvate+malate (PM), glutamate+malate (GM), palmitoyl‐carnitine+malate (PCM), alpha‐ketoglutarate+malate (AM), and succinate±rotenone (Suc±Rot). Mitochondrial bioenergetics (O2 consumption and membrane potential) and H2O2 emission were studied using two protocols: addition of a fixed [ADP] and sequential additions of incremental [ADP] in the presence of different substrates to determine how these substrates influence mitochondrial functional markers in each of these tissues.ResultsResults show that the kinetics and efficiency of mitochondrial OxPhos are highly dependent on the substrates used and this dependency is tissue‐specific. Heart mitochondria showed higher respiratory rates and OxPhos efficiencies for all substrates used in comparison to kidney mitochondria. Renal cortex mitochondrial respiratory rates were higher than OM mitochondria, but OM mitochondria OxPhos efficiencies were higher than cortex mitochondria. It was also found that heart mitochondria do not synthesize ATP efficiently from ADP with Suc in the absence of Rot (blocker of complex I). This may be due to (i) accumulation of oxaloacetate (OAA) and/or (ii) occurrence of reverse electron transfer (RET). In contrast, cortex and OM mitochondria achieved the same respiratory rates with Suc±Rot suggesting that efficient conversion of ADP to ATP by kidney mitochondria can occur in the presence of Suc. Similar differences were observed in mitochondrial membrane potential. However, differences in H2O2 emission in the presence of Suc±Rot were observed in heart mitochondria and to a lesser extent in OM mitochondria, but not in cortex mitochondria. The bioenergetics and H2O2 emission data observed with Suc±Rot indicate that OAA accumulation and RET play a more prominent regulatory role in heart mitochondria than in kidney mitochondria.ConclusionThis study provided novel quantitative data demonstrating that the choice of respiratory substrates affects mitochondrial bioenergetics and H2O2 emission in a tissue‐specific manner.

Role of NOX2 in Ceramide‐Induced Human Microvascular Endothelial Dysfunction

Endothelial dysfunction, or the loss of nitric oxide (NO)‐mediated dilation to shear stress (flow‐induced dilation; FID), is observed in human adipose arterioles from patients with coronary artery disease (CAD). FID is maintained during CAD by release of the mitochondrial‐derived, pro‐inflammatory dilator, H2O2. Ceramide, a sphingolipid that when elevated in plasma is an independent risk factor for future cardiac events, also induces this transition in human arterioles (10µM, 16‐20hrs). The initial events leading to ceramide‐induced endothelial dysfunction are unknown. Here we hypothesize that activation of NADPH oxidase 2 (NOX2), an endothelial membrane enzyme activated by acute stress, occurs prior to the formation of mitochondrial H2O2 in arterioles exposed to chronic ceramide. Human arterioles (100‐250µm in diameter) were dissected from discarded surgical adipose tissues and prepared for videomicroscopy. Following preconstriction with endothelin‐1, internal diameters were measured in response to increased flow. To determine the minimum exposure time necessary to convert to H2O2‐dependent FID in arterioles exposed to ceramide, microvessels from healthy nonCAD adults were incubated with C2 ceramide for 30 min, 2 hrs, and 4 hrs. FID remained mediated by NO at 30min and 2hrs, however, following a 4hr incubation, FID was significantly impaired in the presence of PEG‐catalase (44.6% of maximal dilator capacity ±13.8 (SEM), n=8, p&lt;0.05, one‐way ANOVA*) compared to vehicle control (83.5%±5.0, n=8). The source of H2O2 generated during FID in arterioles treated for 4hr with ceramide was not mitochondrial as increases in fluorescent intensity of mito peroxy yellow 1 (mitoPY1) were not observed during maximal flow. To test whether NOX2 is the initial source of H2O2 during FID in arterioles exposed to 4hr ceramide, nonCAD arterioles were first treated with the NOX2 inhibitor GSK2795039 (NOX2i, 10‐6 M) or underwent intraluminal administration of siRNA to decrease NOX2 expression prior to treatment with ceramide. FID was reduced in vessels treated with both ceramide and the NOX2 inhibitor in the presence of cPTIO (NO scavenger) (50.2%±30.1, n=4* vs. NOX2 inhibitor and ceramide alone 80.8%±6.4, n=7). L‐NAME also impaired FID in vessels with reduced expression of NOX2 and subsequently treated with ceramide (10.8%±38.9, n=3* compared to siNOX2 and ceramide alone 85.5%±5.8, n=3). This suggests that activation of NOX2 may be the initial event in the conversion of NO‐ to H2O2‐dependent FID due to ceramide and may serve as a potential therapeutic target in those at risk for developing cardiovascular disease due to increased plasma ceramide.

Regulation of Mitochondrial Calcium Transport by Caloric Restriction in Rat Kidney

Caloric restriction (CR) in laboratory rodents prevents obesity, promotes healthy aging, and increases resilience against several pathological stimuli. At the mitochondrial level, protection promoted by CR in the brain and liver is related to higher calcium uptake rates and retention capacity, preventing Ca2+‐induced mitochondrial permeability transition. Dietary restriction has been demonstrated to increase kidney resistance against damaging stimuli such as ischemia/reperfusion, but if these effects are related to similar mitochondrial adaptations had not yet been uncovered. Here, we characterized changes in mitochondrial function in response to six months of CR in rats, measuring oxidative phosphorylation, redox balance and calcium homeostasis. CR promoted an increase in mitochondrial oxygen consumption rates under non‐phosphorylating (state 4) and uncoupled (state 3U) conditions. While CR prevents mitochondrial reactive oxygen species production in many tissues, in kidney we found that mitochondrial H2O2 release was enhanced in CR rats, although levels of carbonylated proteins and methionine sulfoxide were unchanged. Surprisingly, and opposite to the effects observed in brain and liver, mitochondria from CR animals are more prone to Ca2+‐induced mitochondrial permeability transition. CR mitochondria also displayed higher calcium uptake rates, which were not accompanied by changes in calcium efflux rates, nor related to altered inner mitochondrial membrane potentials, or the amounts of the Mitochondrial Calcium Uniporter (MCU). Instead, increased mitochondrial calcium uptake rates correlate with a loss of Mitochondrial Calcium Uptake 2 (MICU2), an MCU modulator, in CR kidneys. Interestingly, MICU2 is also modulated by CR in liver, suggesting it has a broader diet‐sensitive regulatory role determining mitochondrial calcium homeostasis. Together, our results remark the highly organ‐specific bioenergetic, redox, and ionic transport effects of CR. Specifically, we describe the regulation of the expression of MICU2, and its effects on mitochondrial calcium transport, as a novel and interesting aspect of the metabolic responses to dietary interventions.

Circulating Factors Provoke Endothelial Dysfunction in the Human Microcirculation Following Doxorubicin Chemotherapy

RationaleAnthracyclines (e.g., doxorubicin) are among the most effective anti‐cancer therapies (CTx), yet their use is limited by cardiotoxic side effects. We sought to characterize the impact of anthracycline‐based CTx on microvascular endothelial function in breast cancer patients and determine if circulating factors are sufficient to induce endothelial dysfunction.MethodsWe assessed microvascular endothelium‐dependent vasodilation and its underlying mechanisms in two groups of breast cancer patients: 1) CTx‐naïve controls and 2) CTx‐treated patients (one month post‐CTx). Adipose samples were obtained at the time of surgical procedures. Freshly‐dissected arterioles were cannulated and pressurized, then flow‐induced production of nitric oxide (NO) and mitochondria‐derived hydrogen peroxide (H2O2) were visualized with fluorescent indicator probes. Next, we assessed whether plasma obtained from breast cancer patients one month following completion of CTx would evoke endothelial dysfunction. Adipose arterioles from healthy donors were exposed to diluted plasma from either CTx patients or healthy controls. Vessels were cannulated and filled with 10% plasma for overnight incubation prior to assessing flow‐mediated dilation (FMD; videomicroscopy). Data are expressed as area under the curve (AUC) for percentage change in diameter across five flow gradients and are shown as mean ± SE for n = 3–9 per condition.ResultsIn the cross‐sectional study, FMD was ~65% lower in vessels from breast cancer patients who had undergone CTx compared to those who were CTx‐naïve (AUC: 23 ± 10 vs. 61 ± 6, P = 0.004). Preliminary results suggest flow‐induced NO production is abolished in vessels from patients one month after CTx (Panel A, fold change in NO: −0.2 ± 0.2 vs. CTx‐naïve group: 11.3 ± 5.6; P = 0.08), whereas mitochondrial H2O2 production is elevated (Panel B, fold change in mtH2O2: 0.52 ± 0.25 vs. −0.51 ± 0.23; P = 0.03). FMD was also ~40% lower in vessels exposed to CTx plasma vs. healthy plasma (Panels C &amp; D, AUC: 45 ± 7 vs. 75 ± 7, P = 0.049). Degradation of hydrogen peroxide (H2O2) with PEG‐catalase further reduced FMD in vessels exposed to CTx plasma (AUC: 22 ± 12), whereas PEG‐catalase did not affect dilation in vessels exposed to healthy plasma (AUC: 62 ± 17). Inhibition of NO synthase (NOS) with L‐NAME restored FMD in vessels exposed to CTx plasma (AUC: 77 ± 6), while L‐NAME had minimal impact on dilation in vessels exposed to healthy plasma (AUC: 60 ± 9). Future studies are needed to determine whether this reflects NOS uncoupling.Summary &amp; ConclusionsMicrovascular endothelial function is impaired in breast cancer patients following doxorubicin chemotherapy. This is accompanied by a loss of NO and elevation of H2O2. Plasma obtained from breast cancer patients one month after cessation of CTx recapitulates these effects in otherwise healthy vessels, which suggest that circulating factors may contribute to endothelial dysfunction following treatment. Our preliminary findings reveal long‐term defects in the microcirculation that may contribute to adverse cardiovascular outcomes associated with doxorubicin chemotherapy.

Factors affecting liver mitochondrial hydrogen peroxide emission.

Mitochondria are key cellular sources of reactive oxygen species (ROS) and contain at least 12 known sites on multiple enzymes that convert molecular oxygen to superoxide and hydrogen peroxide (H2O2). Quantitation of site-specific ROS emission is critical to understand the relative contribution of different sites and the pathophysiologic importance of mitochondrial ROS. However, factors that affect mitochondrial ROS emission are not well understood. We characterized and optimized conditions for maximal total and site-specific H2O2 emission during oxidation of standard substrates and probed the source of the high H2O2 emission in unenergized rainbow trout liver mitochondria. We found that mitochondrial H2O2 emission capacity depended on the substrate being oxidized, mitochondrial protein concentration, and composition of the ROS detection system. Contrary to our expectation, addition of exogenous superoxide dismutase reduced H2O2 emission. Titration of conventional mitochondrial electron transfer system (ETS) inhibitors over a range of conditions revealed that one size does not fit all; inhibitor concentrations evoking maximal responses varied with substrate and were moderated by the presence of other inhibitors. Moreover, the efficacy of suppressors of electron leak (S1QEL1.1 and S3QEL2) was low and depended on the substrate being oxidized. We found that H2O2 emission in unenergized rainbow trout liver mitochondria was suppressed by GKT136901 suggesting that it is associated with NADPH oxidase activity. We conclude that optimization of assay conditions is critical for quantitation of maximal H2O2 emission and would facilitate more valid comparisons of mitochondrial total and site-specific H2O2 emission capacities between studies, tissues, and species.

Nicotinamide riboside promotes Mfn2-mediated mitochondrial fusion in diabetic hearts through the SIRT1-PGC1α-PPARα pathway

Myocardial dysfunction is associated with an imbalance in mitochondrial fusion/fission dynamics in patients with diabetes. However, effective strategies to regulate mitochondrial dynamics in the diabetic heart are still lacking. Nicotinamide riboside (NR) supplementation ameliorated mitochondrial dysfunction and oxidative stress in both cardiovascular and aging-related diseases. This study investigated whether NR protects against diabetes-induced cardiac dysfunction by regulating mitochondrial fusion/fission and further explored the underlying mechanisms. Here, we showed an evident decrease in NAD+ (nicotinamide adenine dinucleotide) levels and mitochondrial fragmentation in the hearts of leptin receptor-deficient diabetic (db/db) mouse models. NR supplementation significantly increased NAD+ content in the diabetic hearts and promoted mitochondrial fusion by elevating Mfn2 level. Furthermore, NR-induced mitochondrial fusion suppressed mitochondrial H2O2 and O2•− production and reduced cardiomyocyte apoptosis in both db/db mice hearts and neonatal primary cardiomyocytes. Mechanistically, chromatin immunoprecipitation (ChIP) and luciferase reporter assay analyses revealed that PGC1α and PPARα interdependently regulated Mfn2 transcription by binding to its promoter region. NR treatment elevated NAD+ levels and activated SIRT1, resulting in the deacetylation of PGC1α and promoting the transcription of Mfn2. These findings suggested the promotion of mitochondrial fusion via oral supplementation of NR as a potential strategy for delaying cardiac complications in patients with diabetes.

Ratiometric Imaging of Mitochondrial Hydrogen Peroxide in Aβ42-Mediated Neurotoxicity.

Oxidative stress is important for the etiology and pathogenesis of Alzheimer's disease (AD). Research tools that can conveniently evaluate oxidative stress in AD models are expected to catalyze and accelerate research on AD. This study explored the use of genetically encoded fluorescent indicators (GEFIs) to detect mitochondrial oxidative stress in organotypic brain slices and AD mouse models. To enable ratiometric normalization and avoid tissue autofluorescence, we genetically fused a green fluorescent hydrogen peroxide (H2O2) indicator, HyPer7, with each of two selected, bright red fluorescent proteins (RFPs), mScarlet-I and tdTomato. The resultant indicators, namely, HyPerGRS and HyPerGRT, were tagged with mitochondrial targeting sequences and examined for localization and function in cultured HeLa cells and primary mouse neurons. We further utilized HyPerGRT, which is a genetic fusion of HyPer7 with tdTomato, to monitor mitochondrial H2O2 in response to the human β-amyloid 1-42 isoform (Aβ42) in cultured brain slices and an AD mouse model. Owing to the high sensitivity and low autofluorescence interference resulting from HyPerGRT, we successfully detected Aβ42-mediated mitochondrial H2O2 in these AD models. The results suggest that HyPerGRT is a valuable tool for studying mitochondrial oxidative stress in tissues and animals.