Mitochondrial Oxygen Utilization Research Articles

Fasting promotes acute hypoxic adaptation by suppressing mTOR-mediated pathways

Rapid adaptation to a hypoxic environment is an unanswered question that we are committed to exploring. At present, there is no suitable strategy to achieve rapid hypoxic adaptation. Here, we demonstrate that fasting preconditioning for 72 h reduces tissue injuries and maintains cardiac function, consequently significantly improving the survival rates of rats under extreme hypoxia, and this strategy can be used for rapid hypoxic adaptation. Mechanistically, fasting reduces blood glucose and further suppresses tissue mTOR activity. On the one hand, fasting-induced mTOR inhibition reduces unnecessary ATP consumption and increases ATP reserves under acute hypoxia as a result of decreased protein synthesis and lipogenesis; on the other hand, fasting-induced mTOR inhibition improves mitochondrial oxygen utilization efficiency to ensure ATP production under acute hypoxia, which is due to the significant decrease in ROS generation induced by enhanced mitophagy. Our findings highlight the important role of mTOR in acute hypoxic adaptation, and targeted regulation of mTOR could be a new strategy to improve acute hypoxic tolerance in the body.

Cardiac Rehabilitation in Heart Failure.

Heart failure (HF) is a complex clinical syndrome caused by a structural and/or functional cardiac abnormality, resulting in reduced organ perfusion. The goals of treatment in patients with HF are to improve functional capacity and quality of life, and to reduce mortality. Cardiac rehabilitation (CR) including exercise training is one of the treatment options, and current guidelines recommend CR as safe and effective for patients with HF. CR has been known to improve exercise capacity and quality of life, minimize HF progression, and lower mortality in patients with HF. Improvement of vascular endothelial function, activation of the neurohormonal system, increase of mitochondrial oxygen utilization in peripheral muscles, and increase of chronotropic responses are possible mechanisms of the beneficial effects of exercise-based CR in HF. Although CR has been shown to decrease morbidity and mortality, it is underutilized in clinical practice. Despite the existence of concrete evidence of clinical benefits, the CR participation rates of patients with HF range from only 14% to 43% worldwide, with high dropout rates after enrollment. These low participation rates have been attributed to several barriers, including patient factors, professional factors, and service factors. The motivation for participating in CR and for overcoming the patients' barriers for CR before discharge should be provided to each patient. Current guidelines strongly recommend applying a CR program to all eligible patients with HF.

Elevated ROS depress mitochondrial oxygen utilization efficiency in cardiomyocytes during acute hypoxia.

Mitochondria are important sites for the production of ATP and the generation of ROS in cells. However, whether acute hypoxia increases ROS generation in cells or affects ATP production remains unclear, and therefore, monitoring the changes in ATP and ROS in living cells in real time is important. In this study, cardiomyocytes were transfected with RoGFP for ROS detection and MitGO-Ateam2 for ATP detection, whereby ROS and ATP production in cardiomyocytes were respectively monitored in real time. Furthermore, the oxygen consumption rate (OCR) of cardiomyocytes was measured. Similar results were produced for adult and neonatal rat cardiomyocytes. Hypoxia (1% O2) reduced the basal OCR, ATP-linked OCR, and maximal OCR in cardiomyocytes compared with these OCR levels in the cardiomyocytes in the normoxic group (21% O2). However, ATP-linked OCR, normalized to maximal OCR, was increased during hypoxia, indicating that the electron leakage of complex III exacerbated the increase of ATP-linked oxygen consumption during hypoxia and vice versa. Combined with the result that cardiomyocytes expressing MitGO-Ateam2 showed a significant decrease in ATP production during hypoxia compared with that of normoxic group, acute hypoxia might depress the mitochondrial oxygen utilization efficiency of the cardiomyocytes. Moreover, cardiomyocytes expressing Cyto-RoGFP or IMS-RoGFP showed an increase in ROS generation in the cytosol and the mitochondrial intermembrane space (IMS) during hypoxia. All of these results indicate that acute hypoxia generated more ROS in complex III and increased mitochondrial oxygen consumption, leading to less ATP production. In conclusion, acute hypoxia depresses the mitochondrial oxygen utilization efficiency by decreasing ATP production and increasing oxygen consumption as a result of the enhanced ROS generation at mitochondrial complex III.

Hyperpolarized [1,4-13C]fumarate imaging detects microvascular complications and hypoxia mediated cell death in diabetic nephropathy

Today, there is a general lack of prognostic biomarkers for development of renal disease and in particular diabetic nephropathy. Increased glycolytic activity, lactate accumulation and altered mitochondrial oxygen utilization are hallmarks of diabetic kidney disease. Fumarate hydratase activity has been linked to mitochondrial dysfunction as well as activation of the hypoxia inducible factor, induction of apoptosis and necrosis. Here, we investigate fumarate hydratase activity in biofluids in combination with the molecular imaging probe, hyperpolarized [1,4-13C2]fumarate, to identify the early changes associated with hemodynamics and cell death in a streptozotocin rat model of type 1 diabetes. We found a significantly altered hemodynamic signature of [1,4-13C2]fumarate in the diabetic kidneys as well as an systemic increased metabolic conversion of fumarate-to-malate, indicative of increased cell death associated with progression of diabetes, while little to no renal specific conversion was observed. This suggest apoptosis as the main cause of cell death in the diabetic kidney. This is likely resulting from an increased reactive oxygen species production following uncoupling of the electron transport chain at complex II. The mechanism coupling the enzyme leakage and apoptotic phenotype is hypoxia inducible factor independent and seemingly functions as a protective mechanism in the kidney cells.

Augmentation of Whole-Body Metabolic Status by Mind-Body Training: Synchronous Integration of Tissue- and Organ-Specific Mitochondrial Function.

The objective of our concise review is to elaborate an evidence-based integrative medicine model that incorporates functional linkages of key aspects of cortically-driven mind-body training procedures to biochemical and molecular processes driving enhanced cellular bioenergetics and whole-body metabolic advantage. This entails the adoption of a unified biological systems approach to selectively elucidate basic biochemical and molecular events responsible for achieving physiological relaxation of complex cellular structures. We provide accumulated evidence in support of the potential synergy of voluntary breathing exercises in combination with meditation and/or complementary cognitive tasks to promote medically beneficial enhancements in whole-body relaxation, anti-stress mechanisms, and restorative sleep. Accordingly, we propose that the widespread metabolic and physiological advantages emanating from a sustained series of complementary mind-body exercises will ultimately engender enhanced functional integration of cortical and limbic areas controlling voluntary respiratory processes with autonomic brainstem neural pattern generators. Finally, a unified mechanism is proposed that links behaviorally-mediated enhancements of whole-body metabolic advantage to optimization of synchronous regulation of mitochondrial oxygen utilization via recycling of nitrite and nitric oxide by iron-sulfur centers of coupled respiratory complexes and nitrite reductases.

Spermidine ameliorates the neuronal aging by improving the mitochondrial function in vitro.

Changes in mitochondrial structure and function are the initial factors of cell aging. Spermidine has an antiaging effect, but its effect on neuronal aging and mitochondrial mechanisms is unclear. In this study, mouse neuroblastoma (N2a) cells were treated with d‑galactose (d‑Gal) to establish cell aging to investigate the antiaging effect and mechanisms of spermidine. Changes in the cell cycle and β-galactosidase activity were analyzed to evaluate the extent of cell aging. Stabilities of mitochondrial mRNA and mitochondrial membrane potential (MMP) were evaluated in the process of cell aging under different treatments. The mitochondrial function was also evaluated using the Seahorse Metabolic Analysis System combined with ATP production. The unfolded protein response (UPR) of the N2a cells was analyzed under different treatments. Results showed that spermidine pretreatment could delay the cell aging and could maintain the mitochondrial stability during d‑Gal treatment. Spermidine increased the proportion of cells in the S phase and maintained the MMP. The oxygen utilization and ATP production in the N2a cells were reduced by d‑Gal treatment but were partially rescued by the spermidine pretreatment. Spermidine ameliorated the N2a cell aging by promoting the autophagy and inhibiting the apoptosis except the UPR. These results showed that spermidine could ameliorate the N2a cell aging by maintaining the mitochondrial mRNA transcription, MMP and oxygen utilization during the d‑Gal treatment.

Investigating mitochondrial dysfunction in human lung cells exposed to redox-active PM components

Exposure to ambient particulate matter (PM) causes cardiopulmonary morbidity and mortality through mechanisms that involve oxidative stress. 1,2-naphthoquinone (1,2-NQ) is a ubiquitous component of PM and a potent redox-active electrophile. We previously reported that 1,2-NQ increases mitochondrial H2O2 production through an unidentified mechanism. We sought to characterize the effects of 1,2-NQ exposure on mitochondrial respiration as a source of H2O2 in human airway epithelial cells. We measured the effects of acute exposure to 1,2-NQ on oxygen consumption rate (OCR) in the human bronchial epithelial cell line BEAS-2B and mitochondrial preparations using extracellular flux analysis. Complex-specific assays and NADPH depletion by glucose deprivation distinguished between mitochondrial and non-mitochondrial oxygen utilization. 1,2-NQ exposure of BEAS cells caused a rapid, marked dose-dependent increase in OCR that was independent of mitochondrial respiration, exceeded the OCR observed after mitochondrial uncoupling, and remained sensitive to NADPH depletion, implicating extra-mitochondrial redox cycling processes. Similar effects were observed with the environmentally relevant redox-cycling quinones 1,4-naphthoquinone and 9,10-phenanthrenequinone, but not with quinones that do not redox cycle, such as 1,4-benzoquinone. In mitochondrial preparations, 1,2-NQ caused a decrease in Complex I-linked substrate oxidation, suggesting impairment of pyruvate utilization or transport, a novel mechanism of mitochondrial inhibition by an environmental exposure. This study also highlights the methodological utility and challenges in the use of extracellular flux analysis to elucidate the mechanisms of action of redox-active electrophiles present in ambient air.

Reducing Fibrinogen through Platelet Additive Solutions Improves Mitochondrial Function and Reduces Reactive Oxygen Species in Stored Platelets

BackgroundThe platelet storage lesion is mitigated by refrigeration in platelet additive solutions (PAS), but questions remain regarding effects of cold storage (4°C) on mitochondrial function. Understanding underlying mechanisms responsible for enhanced hemostatic function of 4°C-stored PAS platelets will be critical for such improvements in platelet transfusion. We hypothesized that storage of platelet concentrates at 4°C leads to GPIIb-IIIa activation and thus aggregate formation through fibrinogen binding, and that this could be prevented by storing platelets in platelet additive solution (PAS) without compromising platelet function.MethodsApheresis platelets in plasma (AP) or apheresis platelets in PAS were stored at 22°C or 4°C for up to 15 days. Measurements include platelet counts, blood gases, flow cytometry analysis, intracellular reactive oxygen species (ROS) levels, and high resolution respirometry. To explore the effect of fibrinogen on storage, platelets were treated with abciximab to inhibit GPIIb-IIIa binding and aggregation. Donor fibrinogen levels were measured before being collected in PAS; samples were separated into minibags for comparison of control 4°C PAS-stored platelets to 4°C PAS-stored platelets with fibrinogen added back to original donor levels (PAS-FBN).ResultsMitochondrial respiration was lower in platelets stored at 4°C compared to 22°C in both AP and PAS; however, PAS-stored platelets retained lower mitochondrial respiration over the time course. Additionally, maximal mitochondrial oxygen utilization (oxidative burst capacity) was better preserved in 4°C-stored platelets; PAS demonstrated less overall mitochondrial exhaustion at 4°C compared to 4°C-AP samples (Figure 1). PAS-FBN resulted in reduced oxidative burst capacity over 15 days of storage which was consistent with loss of platelet function observed through flow cytometry analysis including increased phosphatidylserine expression, microparticles, and GPIb shedding. Mitochondrial intracellular ROS levels were lower in all 4°C-stored samples compared to their respective RT-stored groups. PAS-FBN resulted in significantly lower platelet counts (Day 5: BL= 1.1x106±.04plt/µL, PAS = 1.1 x106±.06plt/µL, PAS-FBN = .61 x106±.06plt/µL; P<0.001) compared to 4°C PAS-stored platelets and displayed marked increases in visible aggregates in the storage bag. PAS-stored platelets resulted in significantly lower levels of intracellular ROS compared to AP at both RT and 4°C conditions and PAS-FBN (P<0.05). 4°C-AP-stored samples treated with abciximab (AP-Ab) rescued the loss in platelet count commonly encountered in 4°C-AP storage (Day 5: BL = 1112±18, AP-4°C = 720±77, AP-Ab = 1085±28;P<0.001) and resulted in less intracellular ROS compared to untreated 4°C-AP samples at Day 5 (Figure 2).DiscussionMitochondrial damage and ROS production may contribute to loss of platelet viability during storage, whereas cold storage is known to preserve platelet function. Our previous work has shown that use of PAS in stored platelets prevents aggregate formation, enhances platelet function, results in less oxidant stress, and preserves both mitochondrial function and potential compared to AP-stored platelets. Here we demonstrate that reduction in fibrinogen levels of 4°C PAS -stored platelets plays a key role in the underlying mechanism for PAS-stored platelet enhancement. Taken together, these data indicate that cold storage combined with PAS preserves platelet viability both by maintaining energy potential and preventing degradation due to enhanced ROS. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Successful Treatment of Aluminum Phosphide Poisoning with Continuous Veno-venous Hemofiltration: A Case Report

Address for Correspondence: Ayca Acikalin, Department of Emergency Medicine, Cukurova University School of Medicine, Adana, Turkey E-mail: aycaacikalin@yahoo.com ©Copyright 2016 by Emergency Physicians Association of Turkey Available online at www.jemcr.org Introduction Aluminum phosphide (AP) is a pesticide that has been used since the 1940s (1). It is a metal phosphide compound (Na3P, AlP) and is used as a rodenticide and insecticide. AP in an evaporable tablet form is used in wheat storage to reduce the number of mice attacks. This form causes fatalities, in suicidal exposure in particular. In countries such as Turkey, Iran, and India, public awareness of deadly exposure results in oral intake during suicide attempts (1). Gastric hydrochloric acid (HCl) causes the release of phosphine in the stomach after swallowing metal phosphide compounds. Phosphine induces cellular lipid peroxidation by means of the inhibition of mitochondrial cytochrome oxidase and oxygen utilization. This influence usually presents with hypotension, acidosis, and multiple organ failure (MOF) resistant to treatment. Mortality is extremely high and there is no identified antidote. Gastric irrigation with potassium and the replacement of intravenous magnesium have been suggested for treatment (1).

Gene Expression Profiling Reveals Key Mitochondrial Gene Changes in Stored Platelets

IntroductionAs of June 2015 the FDA has approved an alternative procedure under 21 CFR 640.120 that allows for storage of apheresis platelets at refrigerator temperature (1-6 C; 4°C) without agitation for up to 3 days for use in the resuscitation of actively bleeding patients. Understanding underlying mechanisms responsible for enhanced hemostatic function at 4°C will be critical for such improvements in platelet transfusion. We hypothesized that 4°C platelets display better mitochondrial respiratory function for up to 7 days compared to standard 5-day RT platelets and that mitochondrial gene expression differences between RT and 4°C -stored platelets will correlate with mitochondrial function.MethodsPlatelets were collected from healthy donors by apheresis according to an IRB-approved protocol. Apheresis platelets (AP) were rested for 1 h before allocation into platelet minibags (Blood Cell Storage, Seattle, WA) and stored for 4 storage durations (Baseline (BL), Day 3, 5, and 7). Mitochondrial respiration, maximal oxygen utilization, and individual mitochondrial complex-dependent respiration were assessed with high-resolution respirometry (O2k, Oroboros). Mitochondrial ROS generation in response to storage condition or stimulation (to assess oxidative burst capacity as a measure of function) was visualized with fluorescent imaging and assayed with flow cytometry using a superoxide stain (Life Technologies). Total RNA was extracted both immediately following apheresis (BL) and on Day 5 from RT and 4°C-stored platelets using Trizol (Molecular Research Center, Cincinnati, OH) after centrifuging the platelets at 900 x g for 10 min. Platelet RNA was quantified using the NanoDrop 2000. RNA quality was examined using gel electrophoresis with the Reliant Gel System (Cambrex, Rockland, ME). Platelet mitochondrial gene expression analysis was evaluated using the 96-well RT2 Profiler PCR Array (Qiagen, Valencia, CA) which profiled 84 mitochondria-focused targets and 12 control genes per sample. Gene expression data analysis was based on the ΔΔCt method with normalization of the raw data to housekeeping genes located on each 96-well plate.ResultsMitochondrial respiration was lower in platelets stored at 4°C compared to RT on Days 3, 5, and 7 (Day 5= -57%±0.3; P < 0.05), demonstrating that refrigeration slows metabolism. Additionally, maximal mitochondrial oxygen utilization (electron transport system capacity) was better preserved in platelets stored at 4°C (Figure 1). Fluorescent imaging and flow cytometry demonstrated that mROS generation was higher in RT-stored platelets compared to 4°C, reflecting mitochondrial damage. Mitochondrial burst during de novo mROS generation due to stimulation was also preserved at 4°C. Mitochondrial gene expression studies revealed distinct differences in expression profiles for 4°C versus RT-stored platelets after 5 days of storage when normalized to BL measures. Storage at 4°C resulted in significantly greater preservation of 15 gene products at Day 5 (P<0.05). In contrast, Day 5 RT samples resulted in a marked decrease or loss of gene products when compared to BL levels of gene expression (P<0.05).DiscussionPlatelet mitochondrial respiratory function (mitochondrial respiration and maximal oxygen utilization) decreased in RT-stored platelets over 7 days, but the impairment was attenuated by 4°C storage. We previously noted that intracellular ROS flux was higher at room temperature, and here the gene expression analysis in combination with oximetry data showed that mitochondrial damage is likely responsible. Furthermore, gene expression profiling of mitochondrial-related genes revealed that distinct differences exist in key mitochondrial genes between the storage conditions. This work illustrates that 4°C storage of platelets preserved and enhanced critical mitochondrial genes compared to RT; this finding combined with improved mitochondrial respiratory measures and reduced ROS demonstrates a significant improvement in current efforts to mitigate platelet dysfunction. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract P188: Mitochondrial Redox Phenotype Contributes to Angiotensin Induced Hypertension

Oxidant stress contributes to the initiation and progression of hypertension by contributing to endothelial dysfunction and/or causing perturbations in nitric oxide homeostasis. Differences in mitochondrial function may contribute to this process and provide insight into why age of onset and clinical outcomes differ amongst individuals form distinct ethnic groups. We have previously demonstrated that variations in normal mitochondrial function and oxidant production exist in endothelial cells from individuals of Caucasian and African American ethnicity and that this variation contributes to endothelial dysfunction. To model these distinct mitochondrial redox phenotypes we used the C57Bl/6J (6J) and C57Bl/6NJ (NJ) that also display unique mitochondrial redox phenotypes due to the differential expression nicotinamide nucleotide transhydrogenase. Interestingly, we found that the 6J animals had a significantly higher systolic blood pressure compared to the NJ animals (116 ± 0.96 vs 102 ± 2.39 mmHg) that was exacerbated by angiotensin II induced hypertension (159 ± 8.25 vs 136 ± 4.33 mmHg). Vascular oxidative stress was assessed in these animals by evaluating mitochondrial oxygen utilization and oxidant production in primary aortic endothelial cells (MAEC) from these animals. Bioenergetic analysis indicates that compared to NJ, 6J MAEC utilized significantly less oxygen for ATP production, possess a lower maximal respiratory capacity (17.31 ± 1.84 vs 41.87 ± 2.95 pmol O 2 /min/ug protein), and have reduced electron leak. 6J MAEC also produce more superoxide compared to NJ in response to angiotensin II stimulation (0.08 ± 0.004 vs 0.11 ± 0.006 nmol/mg protein). Additionally, isolated renal mitochondria and vessel myography indicated differences in respiratory coupling ratios and endothelial dependent vasodilation amongst the groups. Taken together, these data indicate that differences in “normal” mitochondrial function amongst ethnic groups could contribute to a unique mitochondrial redox phenotype that influences individual susceptibility by contributing to endothelial dysfunction, providing important insights into the mechanisms that contribute to the development of hypertension.

Nitric Oxide Regulation of Mitochondrial Processes: Commonality in Medical Disorders

The vital status of diverse classes of eukaryotic mitochondria is reflected by the high degree of evolutionary modification functionally linked to ongoing multifaceted organelle development. From this teleological perspective, a logistical enhancement of eukaryotic cellular energy requirements indicates a convergence of metabolic processes within the mitochondrial matrix for optimal synthesis of ATP from ADP and inorganic phosphate and necessitates an evolutionarily driven retrofit of the primordial endosymbiont bacterial plasma membrane into the inner mitochondrial membrane. The biochemical complexity of eukaryotic inner membrane electron transport complexes linked to temporally-defined, state-dependent, fluctuations in mitochondrial oxygen utilization is capable of generating deleterious reactive oxygen species. Within this functional context, an extensive neurochemical literature supports the role of the free radical gas nitric oxide (NO) as a key signaling molecule involved in the regulation of multiple aspects of mitochondrial respiration/oxidative phosphorylation. Importantly, the unique chemical properties of NO underlie its rapid metabolism in vivo within a mechanistic spectrum of small oxidative molecules, free and protein-bound thiol adducts, and reversible binding to ferrous heme iron centers. Recent compelling work has identified a medically relevant dual regulation pathway for mitochondrial NO expression mediated by traditionally characterized NO synthases (NOS) and by enzymatic reduction of available cellular nitrite pools by a diverse class of cytosolic and mitochondrial nitrite reductases. Accordingly, our short review presents selected medically-based discussion topics relating to multi-faceted NO regulation of mitochondrial functions in human health and disease states.

MNRR1 (formerly CHCHD2) is a bi-organellar regulator of mitochondrial metabolism

Our understanding of stress-associated regulatory mechanisms for mitochondria remains incomplete. We now report a new regulator of mitochondrial metabolism, the coiled-coil–helix–coiled-coil–helix domain-containing protein 2 (CHCHD2) which, based on the functionality described here, is renamed MNRR1 (Mitochondria Nuclear Retrograde Regulator 1). It functions in a novel way by acting in two cellular compartments, mitochondria and nucleus. In normally growing cells most MNRR1 is located in mitochondria; during stress most MNRR1 is now located in the nucleus. MNRR1 is imported to the mitochondrial intermembrane space by a Mia40-mediated pathway, where it binds to cytochrome c oxidase (COX). This association is required for full COX activity. Decreased MNRR1 levels produce widespread dysfunction including reduced COX activity, membrane potential, and growth rate, and increased reactive oxygen species and mitochondrial fragmentation. In the nucleus, MNRR1 acts as a transcription factor, one of whose targets is the COX subunit 4 isoform, COX4I2, which is transcriptionally stimulated by hypoxia. This MNRR1-mediated stress response may provide an important survival mechanism for cells under conditions of oxidative or hypoxic stress, both in the acute phase by altering mitochondrial oxygen utilization and in the chronic phase by promoting COX remodeling.

A mitochondrial-targeted coenzyme q analog prevents weight gain and ameliorates hepatic dysfunction in high-fat-fed mice.

We hypothesized that the mitochondrial-targeted antioxidant, mitoquinone (mitoQ), known to have mitochondrial uncoupling properties, might prevent the development of obesity and mitigate liver dysfunction by increasing energy expenditure, as opposed to reducing energy intake. We administered mitoQ or vehicle (ethanol) to obesity-prone C57BL/6 mice fed high-fat (HF) or normal-fat (NF) diets. MitoQ (500 µM) or vehicle (ethanol) was added to the drinking water for 28 weeks. MitoQ significantly reduced total body mass and fat mass in the HF-fed mice but had no effect on these parameters in NF mice. Food intake was reduced by mitoQ in the HF-fed but not in the NF-fed mice. Average daily water intake was reduced by mitoQ in both the NF- and HF-fed mice. Hypothalamic expression of neuropeptide Y, agouti-related peptide, and the long form of the leptin receptor were reduced in the HF but not in the NF mice. Hepatic total fat and triglyceride content did not differ between the mitoQ-treated and control HF-fed mice. However, mitoQ markedly reduced hepatic lipid hydroperoxides and reduced circulating alanine aminotransferase, a marker of liver function. MitoQ did not alter whole-body oxygen consumption or liver mitochondrial oxygen utilization, membrane potential, ATP production, or production of reactive oxygen species. In summary, mitoQ added to drinking water mitigated the development of obesity. Contrary to our hypothesis, the mechanism involved decreased energy intake likely mediated at the hypothalamic level. MitoQ also ameliorated HF-induced liver dysfunction by virtue of its antioxidant properties without altering liver fat or mitochondrial bioenergetics.

Abstract 327: Mitochondrial Functional Variation Contributes to Endothelial Cell Activation

Vascular oxidant stress contributes to endothelial dysfunction and plays a critical role in early stage cardiovascular disease (CVD) development. Changes in endothelial function due to oxidant stress may contribute to CVD initiation and progression through the development of a pro-inflammatory environment. Differences in mitochondrial function may contribute to this process and provide insight into why age of onset and clinical outcomes differ amongst individuals form distinct ethnic groups; but no reports demonstrate distinct mitochondrial functional parameters between normal cells. Consequently, we hypothesized that significant variations in normal mitochondrial function and oxidant production exist between endothelial cells from donors representing different ethnic groups. Aspects of mitochondrial oxygen utilization and oxidant production were assessed under basal and inflammatory conditions in human aortic endothelial cells (HAECs) isolated from African Americans (AA) and Caucasians (CA). Bioenergetic analysis indicates that compared to CA, AA HAEC utilized significantly less oxygen for ATP production, possess a lower maximal respiratory capacity, and have reduced electron leak. Significant differences in mitochondrial membrane potential, decreased expression of endothelial nitric oxide synthase, and increased levels of superoxide were also observed and AA HAEC supporting a pro-inflammatory phenotype. As a marker of endothelial cell activation, AA HAEC expressed increased levels of intercellular cell adhesion molecule-1 under both basal and inflammatory conditions that could be partially mitigated but treatment with the mitochondrially targeted antioxidant MitoTEMPO. These data demonstrate that fundamental differences exist in mitochondrial oxygen utilization and oxidant production between CA and AA HAEC and that these changes may affect endothelial cell activation. These findings are consistent with the hypothesis that differences in “normal” mitochondrial function amongst ethnic groups could influence individual susceptibility by contributing to vascular inflammation, providing important insights into the mechanisms that contribute human CVD development.

Coenzyme Q supplementation in pulmonary arterial hypertension

Mitochondrial dysfunction is a fundamental abnormality in the vascular endothelium and smooth muscle of patients with pulmonary arterial hypertension (PAH). Because coenzyme Q (CoQ) is essential for mitochondrial function and efficient oxygen utilization as the electron carrier in the inner mitochondrial membrane, we hypothesized that CoQ would improve mitochondrial function and benefit PAH patients. To test this, oxidized and reduced levels of CoQ, cardiac function by echocardiogram, mitochondrial functions of heme synthesis and cellular metabolism were evaluated in PAH patients (N=8) in comparison to healthy controls (N=7), at baseline and after 12 weeks oral CoQ supplementation. CoQ levels were similar among PAH and control individuals, and increased in all subjects with CoQ supplementation. PAH patients had higher CoQ levels than controls with supplementation, and a tendency to a higher reduced-to-oxidized CoQ ratio. Cardiac parameters improved with CoQ supplementation, although 6-minute walk distances and BNP levels did not significantly change. Consistent with improved mitochondrial synthetic function, hemoglobin increased and red cell distribution width (RDW) decreased in PAH patients with CoQ, while hemoglobin declined slightly and RDW did not change in healthy controls. In contrast, metabolic and redox parameters, including lactate, pyruvate and reduced or oxidized gluthathione, did not change in PAH patients with CoQ. In summary, CoQ improved hemoglobin and red cell maturation in PAH, but longer studies and/or higher doses with a randomized placebo-controlled controlled design are necessary to evaluate the clinical benefit of this simple nutritional supplement.

Mitochondrial quality, dynamics and functional capacity in Parkinson’s disease cybrid cell lines selected for Lewy body expression

BackgroundLewy bodies (LB) are a neuropathological hallmark of Parkinson’s disease (PD) and other synucleinopathies. The role their formation plays in disease pathogenesis is not well understood, in part because studies of LB have been limited to examination of post-mortem tissue. LB formation may be detrimental to neuronal survival or merely an adaptive response to other ongoing pathological processes. In a human cytoplasmic hybrid (cybrid) neural cell model that expresses mitochondrial DNA from PD patients, we observed spontaneous formation of intracellular protein aggregates (“cybrid LB” or CLB) that replicate morphological and biochemical properties of native, cortical LB. We studied mitochondrial morphology, bioenergetics and biogenesis signaling by creating stable sub-clones of three PD cybrid cell lines derived from cells expressing CLB.ResultsCloning based on CLB expression had a differential effect on mitochondrial morphology, movement and oxygen utilization in each of three sub-cloned lines, but no long-term change in CLB expression. In one line (PD63CLB), mitochondrial function declined compared to the original PD cybrid line (PD63Orig) due to low levels of mtDNA in nucleoids. In another cell line (PD61Orig), the reverse was true, and cellular and mitochondrial function improved after sub-cloning for CLB expression (PD61CLB). In the third cell line (PD67Orig), there was no change in function after selection for CLB expression (PD67CLB).ConclusionsExpression of mitochondrial DNA derived from PD patients in cybrid cell lines induced the spontaneous formation of CLB. The creation of three sub-cloned cybrid lines from cells expressing CLB resulted in differential phenotypic changes in mitochondrial and cellular function. These changes were driven by the expression of patient derived mitochondrial DNA in nucleoids, rather than by the presence of CLB. Our studies suggest that mitochondrial DNA plays an important role in cellular and mitochondrial dysfunction in PD. Additional studies will be needed to assess the direct effect of CLB expression on cellular and mitochondrial function.

Mitochondrial proteomic analysis reveals deficiencies in oxygen utilization in medullary thick ascending limb of Henle in the Dahl salt-sensitive rat

The renal medullary thick ascending limb (mTAL) of the Dahl salt-sensitive (SS) rat is the site of enhanced NaCl reabsorption and excess superoxide production. In the present studies we isolated mitochondria from mTAL of SS and salt-resistant control strain SS.13(BN) rats on 0.4 and 8% salt diet for 7 days and performed a proteomic analysis. Purity of mTAL and mitochondria isolations exceeded 93.6 and 55%, respectively. Using LC/MS spectral analysis techniques we identified 96 mitochondrial proteins in four biological mTAL mitochondria samples, run in duplicate, as defined by proteins with a false discovery rate <5% and scan count ≥2. Seven of these 96 proteins, including IDH2, ACADM, SCOT, Hsp60, ATPA, EFTu, and VDAC2 were differentially expressed between the two rat strains. Oxygen consumption and high-resolution respirometry analyses showed that mTAL cells and the mitochondria in the outer medulla of SS rats fed high-salt diet exhibited lower rates of oxygen utilization compared with those from SS.13(BN) rats. These studies advance the conventional proteomic paradigm of focusing exclusively upon whole tissue homogenates to a focus upon a single cell type and specific subcellular organelle. The results reveal the importance of a largely unexplored role for deficiencies of mTAL mitochondrial metabolism and oxygen utilization in salt-induced hypertension and renal medullary oxidative stress.

Placental metabolic reprogramming: do changes in the mix of energy-generating substrates modulate fetal growth?

Insufficient oxygen leads to the cessation of growth in favor of cellular survival. Our unique model of high-altitude human pregnancy indicates that hypoxia-induced reductions in fetal growth occur at higher levels of oxygen than previously described. Fetal PO(2) is surprisingly high and fetal oxygen consumption unaffected by high altitude, whereas fetal glucose delivery and consumption decrease. Placental delivery of energy-generating substrates to the fetus is thus altered by mild hypoxia, resulting in maintained fetal oxygenation but a relative fetal hypoglycemia. Our data point to this altered mix of substrates as a potential initiating factor in reduced fetal growth, since oxygen delivery is adequate. These data support the existence, in the placenta, of metabolic reprogramming mechanisms, previously documented in tumor cells, whereby HIF-1 stimulates reductions in mitochondrial oxygen consumption at the cost of increased glucose consumption. Decreased oxygen consumption is not due to substrate (oxygen) limitation but rather results from active inhibition of mitochondrial oxygen utilization. We suggest that under hypoxic conditions, metabolic reprogramming in the placenta decreases mitochondrial oxygen consumption and increases anerobic glucose consumption, altering the mix of energy-generating substrates available for transfer to the fetus. Increased oxygen is available to support the fetus, but at the cost of less glucose availability, leading to a hypoglycemia-mediated decrease in fetal growth. Our data suggest that metabolic reprogramming may be an initiating step in the progression to more severe forms of fetal growth restriction and points to the placenta as the pivotal source of fetal programming in response to an adverse intrauterine environment.

Prevention of endotoxin-induced sarcoplasmic reticulum calcium leak improves mitochondrial and myocardial dysfunction*

Growing evidence suggests that mitochondria function is impaired in sepsis. Here, we tested the hypothesis that lipopolysaccharide would induce mitochondrial Ca2+ overload and oxygen utilization abnormalities as consequences of sarcoplasmic reticulum Ca2+ handling derangements that are typically observed in sepsis. As lipopolysaccharide-induced sarcoplasmic reticulum dysfunction was mainly characterized by reduced sarcoplasmic reticulum Ca2+ uptake and Ca2+ leak, we tested whether dantrolene, a sarco(endo)plasmic reticulum calcium ATPase leak inhibitor, would prevent mitochondrial and cardiac contractile dysfunction. Randomized controlled trial. Experimental laboratory. Male Sprague Dawley rats. Sepsis was induced by injection of endotoxin lipopolysaccharide (10 mg/kg/intravenously). Assessment of contractile function and Ca2+ handling was performed 4 hr after lipopolysaccharide. The relative contribution of the different Ca2+ transporters to relaxation in intact cardiomyocytes was studied during successive electrically evoked twitches and caffeine stimulation. Sarcoplasmic reticulum vesicles and mitochondria from ventricles of rats treated or not with lipopolysaccharide were prepared to evaluate Ca2+ uptake-release and oxygen fluxes, respectively. Effects of dantrolene (10 mg/kg) treatment in rats were evaluated in sarcoplasmic reticulum vesicles, mitochondria, and isolated hearts. Lipopolysaccharide challenge elicited cardiac contractile dysfunction that was accompanied by severe derangements in sarcoplasmic reticulum function, i.e., reduced Ca2+ uptake and increased sarcoplasmic reticulum Ca2+ leak. Functional sarcoplasmic reticulum changes were associated with modification in the status of phospholamban phosphorylation whereas SERCA was unchanged. Rises in mitochondrial Ca2+ content observed in lipopolysaccharide-treated rats coincided with derangements in mitochondrial oxygen efficacy, i.e., reduced respiratory control ratio. Administration of dantrolene in lipopolysaccharide-treated rats prevented mitochondrial Ca2+ overload and mitochondrial oxygen utilization abnormalities. Moreover, dantrolene treatment in lipopolysaccharide rats improved heart mitochondrial redox state and myocardial dysfunction. These experiments suggest that sarcoplasmic reticulum Ca2+ handling dysfunction is an early event during endotoxemia that could be responsible for, or contribute to, mitochondrial Ca2+ overload, metabolic failure, and cardiac dysfunction.