Mitochondrial Electron Transport Complex Research Articles

Regulation of gene expression by miR-144/451 during mouse erythropoiesis

AbstractThe microRNA (miRNA) locus miR-144/451 is abundantly expressed in erythrocyte precursors, facilitating their terminal maturation and protecting against oxidant stress. However, the full repertoire of erythroid miR-144/451 target messenger RNAs (mRNAs) and associated cellular pathways is unknown. In general, the numbers of mRNAs predicted to be targeted by an miRNA vary greatly from hundreds to thousands, and are dependent on experimental approaches. To comprehensively and accurately identify erythroid miR-144/451 target mRNAs, we compared gene knockout and wild-type fetal liver erythroblasts by RNA sequencing, quantitative proteomics, and RNA immunoprecipitation of Argonaute (Ago), a component of the RNA-induced silencing complex that binds miRNAs complexed to their target mRNAs. Argonaute bound ∼1400 erythroblast mRNAs in a miR-144/451–dependent manner, accounting for one-third of all Ago-bound mRNAs. However, only ∼100 mRNAs were stabilized after miR-144/451 loss. Thus, miR-144 and miR-451 deregulate <10% of mRNAs that they bind, a characteristic that likely applies generally to other miRNAs. Using stringent selection criteria, we identified 53 novel miR-144/451 target mRNAs. One of these, Cox10, facilitates the assembly of mitochondrial electron transport complex IV. Loss of miR-144/451 caused increased Cox10 mRNA and protein, accumulation of complex IV, and increased mitochondrial membrane potential with no change in mitochondrial mass. Thus, miR-144/451 represses mitochondrial respiration during erythropoiesis by inhibiting the production of Cox10.

Transcriptional Response of White Adipose Tissue to Withdrawal of Vitamin B3.

ScopeDistinct markers for mild vitamin B3 deficiency are lacking. To identify these, the molecular responses of white adipose tissue (WAT) to vitamin B3 withdrawal are examined.Methods and resultsA dietary intervention is performed in male C57BL/6JRccHsd mice, in which a diet without nicotinamide riboside (NR) is compared to a diet with NR at the recommended vitamin B3 level. Both diets contain low but adequate level of tryptophan. Metabolic flexibility and systemic glucose tolerance are analyzed and global transcriptomics, qRT‐PCR, and histology of epididymal WAT (eWAT) are performed. A decreased insulin sensitivity and a shift from carbohydrate to fatty acid oxidation in response to vitamin B3 withdrawal are observed. This is consistent with molecular changes in eWAT, including an activated MEK/ERK signaling, a lowering of glucose utilization markers, and an increase in makers of fatty acid catabolism, possibly related to the consistent lower expression of mitochondrial electron transport complexes. The synthesis pathway of tetrahydropteridine (BH4), an essential cofactor for neurotransmitter synthesis, is transcriptionally activated. Genes marking these processes are technically validated.ConclusionThe downregulation of Anp32a, Tnk2 and the upregulation of Mapk1, Map2k1, Qdpr, Mthfs, and Mthfsl are proposed as a WAT transcriptional signature marker for mild vitamin B3 deficiency.

Flura-seq identifies organ-specific metabolic adaptations during early metastatic colonization.

Metastasis-initiating cells dynamically adapt to the distinct microenvironments of different organs, but these early adaptations are poorly understood due to the limited sensitivity of in situ transcriptomics. We developed fluorouracil-labeled RNA sequencing (Flura-seq) for in situ analysis with high sensitivity. Flura-seq utilizes cytosine deaminase (CD) to convert fluorocytosine to fluorouracil, metabolically labeling nascent RNA in rare cell populations in situ for purification and sequencing. Flura-seq revealed hundreds of unique, dynamic organ-specific gene signatures depending on the microenvironment in mouse xenograft breast cancer micrometastases. Specifically, the mitochondrial electron transport Complex I, oxidative stress and counteracting antioxidant programs were induced in pulmonary micrometastases, compared to mammary tumors or brain micrometastases. We confirmed lung metastasis-specific increase in oxidative stress and upregulation of antioxidants in clinical samples, thus validating Flura-seq's utility in identifying clinically actionable microenvironmental adaptations in early metastasis. The sensitivity, robustness and economy of Flura-seq are broadly applicable beyond cancer research.

Simvastatin Inhibits L-Type Ca2+-Channel Activity Through Impairment of Mitochondrial Function.

Plasma membrane ion channels and mitochondrial electron transport complexes (mETC) are recognized "off targets" for certain drugs. Simvastatin is one such drug, a lipophilic statin used to treat hypercholesterolemia, but which is also associated with adverse effects like myopathy and increased risk of glucose intolerance. Such myopathy is thought to arise through adverse actions of simvastatin on skeletal muscle mETC and mitochondrial respiration. In this study, we investigated whether the glucose intolerance associated with simvastatin is also mediated via adverse effects on mETC in pancreatic beta-cells because mitochondrial respiration underlies insulin secretion from these cells, an effect in part mediated by promotion of Ca2+ influx via opening of voltage-gated Ca2+ channels (VGCCs). We used murine pancreatic beta-cells to investigate these ideas. Mitochondrial membrane potential, oxygen consumption, and ATP-sensitive-K+-channel activity were monitored as markers of mETC activity, respiration, and cellular ATP/ADP ratio respectively; Ca2+ channel activity and Ca2+ influx were also measured. In intact beta-cells, simvastatin inhibited oxidative respiration (IC50 approximately 3 µM) and mETC (1 < IC50 < 10 µM), effects expected to impair VGCC opening. Consistent with this idea simvastatin > 0.1 µM reversed activation of VGCCs by glucose but had no significant effect in the sugar's absence. The VGCC effects were mimicked by rotenone which also decreased respiration and ATP/ADP. This study demonstrates modulation of beta-cell VGCC activity by mitochondrial respiration and their sensitivity to mETC inhibitors. This reveals a novel outcome for the action of drugs like simvastatin for which mETC is an "off target".

Hypoxically Induced Nitric Oxide: Potential Role as a Vasodilator in Mytilus edulis Gills.

Intertidal Mytilus edulis experience rapid transgression to hypoxia when they close their valves during low tide. This induces a physiological stress response aiming to stabilize tissue perfusion against declining oxygen partial pressure in shell water. We hypothesized that nitric oxide (NO) accumulation supports blood vessel opening in hypoxia and used live imaging techniques to measure NO and superoxide anion () formation in hypoxia-exposed gill filaments. Thirty minutes of moderate (7 kPa pO2) and severe hypoxia (1 kPa pO2) caused 1.6- and 2.4-fold increase, respectively, of NO accumulation in the endothelial muscle cells of the hemolymphatic vessels of the gill filaments. This led to a dilatation of blood vessel diameter by 43% (7 kPa) and 56% (1 kPa), which facilitates blood flow. Experiments in which we applied the chemical NO-donor Spermine NONOate (concentrations ranging from 1 to 6 mM) under normoxic conditions corroborate the dilatational effect of NO on the blood vessel. The formation of within the filament epithelial cells increased 1.5 (7 kPa) and 2-fold (1 kPa) upon treatment. Biochemical analysis of mitochondrial electron transport complexes in hypoxia-exposed gill tissue indicates decreased activity of complexes I and III in both hypoxic conditions; whereas complex IV (cytochrome-c oxidase) activity increased at 7 kPa and decreased at 1 kPa compared to normoxic exposure conditions. This corresponds to the pattern of pO2-dependent gill respiration rates recorded in ex-vivo experiments. Severe hypoxia (1 kPa) appears to have a stabilizing effect on NO accumulation in gill cells, since less O2 is available for NO oxidation to nitrite/nitrate. Hypoxia thus supports the NO dependent inhibition of complex IV activity, a mechanism that could fine tune mitochondrial respiration to the local O2 availability in a tissue. Our study highlights a basal function of NO in improving perfusion of hypoxic invertebrate tissues, which could be a key mechanism of tolerance toward environmental O2 variations.

Anti-aging Effect of Ganoderma (Lingzhi) with Health and Fitness.

Although Ganoderma (Lingzhi in Chinese) has been used as an elixir for thousands of years, its anti-aging effects still need to be clarified. Aging is related to immunoregulation, oxidation stress, and free radical product. Till now, Ganoderma exert life span elongation activities by inhibiting ROS production, lipid peroxidation, and advanced oxidation protein products; increasing production of mitochondrial electron transport complexes, SOD, CAT, GSH and GSH-Px, DPPH, and ABTS radical scavenger activities; and having immunomodulatory and antioxidant activity by increasing radical scavenging activity and ferric reducing antioxidant power. Ganoderma's anti-aging effect on human remains a mystery, and its potential mechanisms underlying anti-aging effect for its clinical application still need to be elucidated.

Parenteral L-Arginine Improves Mitochondrial Function in Children with Sickle Cell Disease Admitted for Vaso-Occlusive Pain Episodes

Introduction: Vaso-occlusive pain episodes (VOE) are the leading cause of emergency department visits and hospitalization in patients with sickle cell disease (SCD). There is currently no standard therapy for treating the underlying causes of VOE; supportive care & symptomatic pain relief with parenteral opioids is the mainstay of therapy. Prior work by Morris & colleagues has demonstrated that patients in VOE show depleted concentrations of plasma L-arginine (Arg), and that supplementation with Arg significantly decreases total parenteral opioid use and improves pain scores during hospitalization in these patients (Morris et al 2013, Haematologica). The mechanisms by which Arg mediates these positive effects remain unclear, but are potentially due to its ability to modulate metabolism through increased Arg transport or affect vascular signaling through the stimulation of nitric oxide production. The Shiva lab has previously demonstrated that platelet mitochondrial function is altered in patients with SCD compared to healthy controls (Cardenas et al 2014, Blood). This alteration is manifested as a decrease in mitochondrial electron transport Complex V (ATP Synthase) activity, which leads to decreased respiration and increased detrimental oxidant production. Here we hypothesized that treatment of SCD patients in VOE with Arg increases mitochondrial function.Methods: In a pharmacokinetic study, 10 subjects with Hb-SS or Sb0-thalassemia hospitalized for VOE (age 14±3 years, 70% male, 80% Hb-SS) were randomized to treatment utilizing one of 3 dosing schemes of L-arginine: 1) 100mg/kg IV three times/day (n=3); 2) loading dose (200mg/kg) then 100mg/kg three times/day (n=4); or 3) loading dose (200mg/kg) followed by continuous infusion (300mg/kg/day) until discharge (n=3). Platelet rich plasma was isolated & stored at baseline & discharge for each subject & mitochondrial electron transport complex activities, protein expression, as well as protein carbonyls were measured.Results: Compared to a cohort of SCD patients in steady state, all subjects in VOE had a significantly decreased complex V activity (Fig 1A). Notably, complex V activity was increased at discharge in subjects with VOE treated with Arg in all three dosing schemes. However, the increase in complex V activity was greatest when utilizing a loading dose. (Fig 1B, p<0.001). While complex IV & citrate synthase activities were not changed in platelets from subjects with VOE compared to SCD patients in steady state (data not shown), the activities of these enzymes were also significantly increased in VOE subjects after Arg treatment when utilizing a loading dose( Fig 1C-D, p<0.01). These changes with Arg do not appear to be due to increased mitochondrial number as quantification of mitochondrial DNA before and after Arg was not different (Fig 1E). Complex V protein expression was also unchanged after Arg treatment (data not shown). However, Arg treatment did significantly decrease levels of protein carbonyls in the platelet rich plasma across all treatment dosing schemes (Fig 1F, p<0.01 in all dosing groups), suggesting a decrease in oxidative stress.Conclusion: These data demonstrate for the first time that Arg supplementation increases mitochondrial activity and decreases oxidative stress in children with SCD and VOE. Our prior study showed that complex V inhibition leads to increased mitochondrial oxidant production in platelets from SCD patients. The improvement in complex V function & decreased oxidative markers observed with Arg treatment are consistent with Arg-induced decrease in mitochondrial oxidant generation. However, Arg treatment potentially regulates other oxidant sources and could decrease overall oxidative stress in SCD patients. Arg therapy has been reported to improve symptoms associated with MELAS syndrome (mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes) suggesting an impact of arginine on mitochondrial activity that may be beneficial for patients with SCD. With the recent FDA approval of L-glutamine, which is an Arg prodrug, this is also a potential consequence of glutamine that warrants further study. This increase in mitochondrial function may mechanistically contribute to decreased pain and ultimately less opioid requirement after IV Arg treatment in SCD, as well as have further implications for improved metabolism and oxidative signaling in these patients. DisclosuresDampier:Pfizer: Research Funding. Morris:Pfizer: Consultancy; MAST Therapeutics: Research Funding; UCSF-Benioff Children's Hospital Oakland: Patents & Royalties: For nutritional supplement licensed to Lifetrients; Calithera: Consultancy; FDA: Research Funding; NIH/NHLBI: Research Funding.

The role of decreased cardiolipin and impaired electron transport chain in brain damage due to cardiac arrest

Ischemic brain damage is the major cause of mortality in cardiac arrest (CA). However, the molecular mechanism responsible for brain damage is not well understood. We previously found that mitochondrial state-3 respiration, which had been decreased following CA, was recovered in the kidney and liver, but not in the brain following cardiopulmonary bypass (CPB) resuscitation. Examination of mitochondria from these tissues may shed light on why the brain is the most vulnerable. In this study, adult male Sprague-Dawley rats were subjected to asphyxia-induced CA for 30 min or 30 min followed by 60 min CPB resuscitation. Mitochondria were then isolated from brain, heart, kidney, and liver tissues for examination using spectrophotometry and mass spectrometry to measure the activities of mitochondrial electron transport complexes and the cardiolipin content. We found significantly decreased complex I activity in mitochondria isolated from all four organs following CA, while complex III and IV activities remained intact. Following CPB resuscitation, complex I activity was normalized in kidney and liver, but unrecovered in brain and heart mitochondria. In addition, complex III activity in brain mitochondria was decreased by 22% with a concomitant decrease in cardiolipin following CPB resuscitation. These results suggest that of the tissues tested only brain mitochondria suffer reperfusion injury in addition to ischemic alterations, resulting in diminished overall mitochondrial respiration following resuscitation.

PDSS2 Deficiency Induces Hepatocarcinogenesis by Decreasing Mitochondrial Respiration and Reprogramming Glucose Metabolism.

Glucose metabolic reprogramming from oxidative phosphorylation to glycolysis is one of the hallmarks of cancer development. Coenzyme Q10 (CoQ10) is essential for electron transport in the mitochondrial respiratory chain and for antioxidant defense. Here, we investigated the role of a key factor in CoQ10 synthesis, prenyldiphosphate synthase subunit 2 (PDSS2), in hepatocellular carcinoma (HCC) tumorigenesis. PDSS2 was frequently downregulated in HCC tissues and was significantly associated with poorer HCC prognosis (P = 0.027). PDSS2 downregulation was a prognostic factor independent of T status and stage (P = 0.028). Downregulation of CoQ10 was significantly correlated with downregulation of PDSS2 in HCC tumor tissues (R = 0.414; P < 0.001). Of the six different splicing isoforms of PDSS2, the five variants other than full-length PDSS2 showed loss of function in HCC. Reintroduction of full-length PDSS2 into HCC cells increased CoQ10 and mitochondrial electron transport complex I activity and subsequently induced a metabolic shift from aerobic glycolysis to mitochondrial respiration in cells. Reintroduction of PDSS2 also inhibited foci formation, colony formation in soft agar, and tumor formation in nude mice. Knockdown of PDSS2 induced chromosomal instability in the MIHA immortalized human liver cell line. Furthermore, knockdown of PDSS2 in MIHA induced malignant transformation. Overall, our findings indicate that PDSS2 deficiency might be a novel driving factor in HCC development.Significance: Downregulation of PDSS2 is a driving factor in hepatocellular carcinoma tumorigenesis. Cancer Res; 78(16); 4471-81. ©2018 AACR.

Neutrophil extracellular trap formation requires OPA1-dependent glycolytic ATP production

Optic atrophy 1 (OPA1) is a mitochondrial inner membrane protein that has an important role in mitochondrial fusion and structural integrity. Dysfunctional OPA1 mutations cause atrophy of the optic nerve leading to blindness. Here, we show that OPA1 has an important role in the innate immune system. Using conditional knockout mice lacking Opa1 in neutrophils (Opa1N∆), we report that lack of OPA1 reduces the activity of mitochondrial electron transport complex I in neutrophils. This then causes a decline in adenosine-triphosphate (ATP) production through glycolysis due to lowered NAD+ availability. Additionally, we show that OPA1-dependent ATP production in these cells is required for microtubule network assembly and for the formation of neutrophil extracellular traps. Finally, we show that Opa1N∆ mice exhibit a reduced antibacterial defense capability against Pseudomonas aeruginosa.

Depressed mitochondrial function and electron transport Complex II-mediated H2O2 production in the cortex of type 1 diabetic rodents

AimsAbnormalities in mitochondrial function under diabetic conditions can lead to deficits in function of cortical neurons and their support cells exhibiting a pivotal role in the pathogenesis of several neurodegenerative disorders, including Alzheimer's disease. We aimed to assess mitochondrial respiration rates and membrane potential or H2O2 generation simultaneously and expression of proteins involved in mitochondrial dynamics, ROS scavenging and AMPK/SIRT/PGC-1α pathway activity in cortex under diabetic conditions. MethodsCortical mitochondria from streptozotocin (STZ)-induced type 1 diabetic rats or mice, and aged-matched controls were used for simultaneous measurements of mitochondrial respiration rates and mitochondrial membrane potential (mtMP) or H2O2 using OROBOROS oxygraph. Measurements of enzymatic activities of respiratory complexes were performed using spectophotometry. Protein levels in cortical mitochondria and homogenates were determined by Western blotting. ResultsMitochondrial coupled respiration rates and FCCP-induced uncoupled respiration rates were significantly decreased in mitochondria of cortex of STZ-diabetic rats compared to controls. The mtMP in the presence of ADP was significantly depolarized and succinate-dependent respiration rates and H2O2 were significantly diminished in cortical mitochondria of diabetic animals compared to controls, accompanied with reduced expression of CuZn- and Mn-superoxide dismutase. The enzymatic activities of Complex I, II, and IV and protein levels of certain components of Complex I and II, mitofusin 2 (Mfn2), dynamin-related protein 1 (DRP1), P-AMPK, SIRT2 and PGC-1α were significantly diminished in diabetic cortex. ConclusionDeficits in mitochondrial function, dynamics, and antioxidant capabilities putatively mediated through sub-optimal AMPK/SIRT/PGC-1α signaling, are involved in the development of early sub-clinical neurodegeneration in the cortex under diabetic conditions.

Potential mitochondrial dysfunction in skeletal muscle of mouse models of Osteogenesis imperfecta.

Osteogenesis imperfecta (OI), also referred to as brittle bone disease, is a heritable connective tissue disorder characterized by short stature, fragile bones, craniofacial deformity and muscle weakness. OI is most often the result of autosomal dominant gene defects in the type I collagen genes: COL1A1 and COL1A2. The remaining rare autosomal recessive forms result from defects in gene products responsible for modification and packaging of type I collagen. Muscle weakness not only impacts a patient's quality of life, but likely negatively affects bone health via altered bone‐muscle crosstalk.The oim/oim mouse models severe human type III OI with reduced body mass, skeletal fragility, and reduced muscle mass and function compared to their wildtype (WT) counterparts. Whereas, the G610C OI mouse models the less severe human type I/IV OI with skeletal weakness, but near normal muscle function. The differential presence of inherent muscle pathology between oim/oim and G610C mice suggest mutation specific mechanisms.To investigate the mechanisms underlying the muscle pathology, we examined citrate synthase activity, a known marker of mitochondrial activity and determined, in the mixed fiber type gastrocnemius muscle, that citrate synthase activity was significantly increased only in oim/oim mice relative to WT mice, despite evidence of reduced muscle function; whereas G610C mice did not have altered citrate synthase activity. The unexpected increase in citrate synthase activity led to the question of whether or not mitochondria play a role in the muscle weakness observed in OI. To assess mitochondrial health, we examined biomarkers of mitogenesis and mitophagy, and found increased mitogenesis in oim/oim mice relative to WT as determined by PGC1α and TFAM protein content in the mixed gastrocnemius muscle. Additionally, measurement of mitochondrial electron transport complex proteins also suggest altered mitochondria in the oim/oim mice. These data suggest that oim/oim mice have increased mitogenesis and altered mitochondrial function accompanying decreased muscle function.Support or Funding InformationSupported by March of Dimes, National Institutes of Health/National Institute of Arthritis and Musculoskeletal and Skin Diseases (RO1 AR055907), the Leda J. Sears Trust, the Kansas City Area Life Sciences Institute Patton Trust Research, Inc., University of Missouri Research Board Grant, and in part by the Department of Veterans Affairs (VA‐Merit Grant I01BX003271‐01). A portion of this work was also supported by resources and the use of facilities at the Harry S. Truman Memorial Veterans Hospital in Columbia, MO.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

MTA1 is a novel regulator of autophagy that induces tamoxifen resistance in breast cancer cells

ABSTRACTTamoxifen is commonly used to treat patients with ESR/ER-positive breast cancer, but its therapeutic benefit is limited by the development of resistance. Recently, alterations in macroautophagy/autophagy function were demonstrated to be a potential mechanism for tamoxifen resistance. Although MTA1 (metastasis-associated 1) has been implicated in breast tumorigenesis and metastasis, its role in endocrine resistance has not been studied. Here, we report that the level of MTA1 expression was upregulated in the tamoxifen resistant breast cancer cell lines MCF7/TAMR and T47D/TR, and knockdown of MTA1 sensitized the cells to 4-hydroxytamoxifen (4OHT). Moreover, knockdown of MTA1 significantly decreased the enhanced autophagy flux in the tamoxifen resistant cell lines. To confirm the role of MTA1 in the development of tamoxifen resistance, we established a cell line, MCF7/MTA1, which stably expressed MTA1. Compared with parental MCF7, MCF7/MTA1 cells were more resistant to 4OHT-induced growth inhibition in vitro and in vivo, and showed increased autophagy flux and higher numbers of autophagosomes. Knockdown of ATG7 or cotreatment with hydroxychloroquine, an autophagy inhibitor, restored sensitivity to 4OHT in both the MCF7/MTA1 and tamoxifen resistant cells. In addition, AMP-activated protein kinase (AMPK) was activated, probably because of an increased AMP:ATP ratio and decreased expression of mitochondrial electron transport complex components. Finally, publicly available breast cancer patient datasets indicate that MTA1 levels correlate with poor prognosis and development of recurrence in patients with breast cancer treated with tamoxifen. Overall, our findings demonstrated that MTA1 induces AMPK activation and subsequent autophagy that could contribute to tamoxifen resistance in breast cancer.

In rats gestational iron deficiency does not change body fat or hepatic mitochondria in the aged offspring.

Mitochondrial dysfunction and resulting changes in adiposity have been observed in the offspring of animals fed a high fat (HF) diet. As iron is an important component of the mitochondria, we have studied the offspring of female rats fed complete (Con) or iron-deficient (FeD) rations for the duration of gestation to test for similar effects. The FeD offspring were ~12% smaller at weaning and remained so because of a persistent reduction in lean tissue mass. The offspring were fed a complete (stock) diet until 52 weeks of age after which some animals from each litter were fed a HF diet for a further 12 weeks. The HF diet increased body fat when compared with animals fed the stock diet, however, prenatal iron deficiency did not change the ratio of fat:lean in either the stock or HF diet groups. The HF diet caused triglyceride to accumulate in the liver, however, there was no effect of prenatal iron deficiency. The activity of the mitochondrial electron transport complexes was similar in all groups including those challenged with a HF diet. HF feeding increased the number of copies of mitochondrial DNA and the prevalence of the D-loop mutation, however, neither parameter was affected by prenatal iron deficiency. This study shows that the effects of prenatal iron deficiency differ from other models in that there is no persistent effect on hepatic mitochondria in aged animals exposed to an increased metabolic load.

Abstract 1490: Role of sub-cellular specific reactive oxygen species in heart regeneration after cancer therapy

Abstract Introduction: Cardiovascular disease and cancer are the two leading causes of morbidity and mortality worldwide. As advancements in radiation therapy (RT) have significantly increased the number of cancer survivors, the risk of radiation-induced cardiovascular disease in this group is a growing concern. However, the molecular mechanism of radiation-induced heart failure is still elusive. Recently, it has been discovered that the reactive oxygen species (ROS)-mediated oxidative DNA damage is the primary upstream mechanism that prevents cardiomyocyte proliferation. Therefore, elucidating the spatial and temporal aspects of ROS production will lead to the development of countermeasures to prevent heart injury following chest radiotherapy. Methods: We have generated GSH redox potential (Grx-roGFP) ROS probe targeted to cytoplasm, chromatin, nucleolus, telomere, nuclear inner membrane and heterochromatin. These probes have been inserted into cardiomyocytes specific AAV9 vectors which were used to infect cardiomyocytes both in vitro an in vivo. For in vivo study, we infected three months old mice with high-titer AAV particles via tail vein injection and then exposed to chest-only radiation (5-10 Gy). At different post-radiation times, fresh heart slices of 300-500 µM thickness were either mock- or treated with mitochondrial electron transport complex (ETC) inhibitors and then subjected to live tissue imaging using a confocal microscope. Results: Interestingly, our results showed that the distribution of basal ROS levels is not uniform in different sub-nuclear compartments. Significantly, upon the induction of oxidative stress, the ROS levels was elevated in all the cellular compartments, but the extent of ROS level was significantly higher in the cytoplasm. Similarly, radiation altered ROS levels in all the cellular compartments; however the effect of radiation on the ROS levels was sub-nuclear compartment-specific. Intriguingly, we found that the complex IV of the ETC was critical for the maintenance of ROS levels in different cellular compartments as compared with complexes I and II. Conclusion and Future Directions: Our data clearly indicate that the spatial and temporal levels of ROS are not uniform across the cell and the mitochondrial ETC plays a major role in regulation ROS levels in different sub-nuclear compartments. The results obtained from this study can be utilized to develop sub-cellular compartment specific targeted both genetic and pharmacological ROS scavengers that will help to regenerate adult heart by re-activating the proliferative capacity of cardiomyocytes following cancer therapy. Finally, our novel approach can be applied to assess alterations in ROS levels in different cancers. Funding: This work was supported by the NASA (NNX13AD57G/NNX15AE06G) CPRIT (RP160520) and NIH R01AG053341 grants. Citation Format: Salim Abdisalaam, Souparno Bhattacharya, Kalayarasan Srinivasan, Shibani Mukherjee, Hesham A. Sadek, Aroumougame Asaithamby. Role of sub-cellular specific reactive oxygen species in heart regeneration after cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1490. doi:10.1158/1538-7445.AM2017-1490

Faster Recovery after Exercise with Phytochemicals Aimed at Mitochondrial Energy Turnover - A Double Blind Randomized Placebo Control Study in College Female Soccer Players

In order to replenish energy stores after exercise we developed a nutraceutical containing ingredients that precisely fuel the ATP generating mitochondria thus resulting in a faster recovery after workout.To test the working hypothesis 18 healthy female college soccer players (mean age 18) enrolled in a short but extreme workout on the treadmill for 12 min as demonstrated in their rise in lactic acid. The volunteers were given an energy drink containing Q10-H2 (ubiquinol), NADH (nicotinamide-adenine-dinucleotide-hydride or Q1), ribose, eleutherosides B &amp; E, Vit B2 &amp; B3, passion flower and Vit E over a period of one week. Following a washout of 4 weeks a similar looking and tasting placebo solution was given in a double blind randomized crossover design. Heart rate recovery rate and systolic blood pressure demonstrated a significant faster return to base line than after placebo within the first 1-5 minutes following exercise. In addition, ATP regeneration capacity in leucocytes following washout of sodiumazide, a specific inhibitor at the mitochondrial electron transport complex V, was statistically higher (p &lt; 0.05) in subjects after verum.The composition consisting of mitochondrial active substances, results in a higher synthesis rate of ATP, which coincided with a faster heart rate recovery rate after workout.

Functional and structural evaluation of bovine heart cytochrome c oxidase incorporated into bicelles.

Bilayered long- and short-chain phospholipid assemblies, known as bicelles, have been widely used as model membranes in biological studies. However, to date, there has been no demonstration of structural or functional viability for the fundamental mitochondrial electron transport complexes reconstituted into or interacting with bicelles. In the present work, bicelles were formed from the mixture of long- and short-chain phospholipids, specifically 14:0 and 6:0 phosphatidylcholines (1,2-dimyristoyl-sn-glycero-3-phosphocholine, (DMPC) and 1,2-dihexanoyl-sn-glycero-3-phosphocholine, (DHPC)). Isolated from bovine heart, cytochrome c oxidase was successfully incorporated into bicelles. Bicelles and cytochrome c oxidase incorporated into bicelles ("proteobicelles") were characterized by absorption spectroscopy, dynamic light scattering, atomic force microscopy, sedimentation velocity and differential scanning calorimetry. It was demonstrated that at total concentration of phospholipids CL=24mM and the molar ratio (q) of long-chain DMPC over short-chain DHPC equal to 0.4, the diameter of bicelles formed at neutral pH is in the range of 30-60nm with the thickness of bicelles of about 4nm. Adding cytochrome c oxidase to bicelles unified the size of the resulting proteobicelles to about 160nm. Cytochrome c oxidase in bicelles was fully reducible by artificial donors of electrons, exhibited "normal" reaction with external ligands, and was fully active. Both, sedimentation velocity analysis and temperature-induced denaturation indicated that enzyme in bicelles is monomeric. We concluded that cytochrome c oxidase in bicelles maintains its structural and functional integrity, and that bicelles can be used for more comprehensive investigation of cytochrome c oxidase and most likely other mitochondrial electron transfer complexes.

Effect of polyphenolic phytochemicals on ectopic oxidative phosphorylation in rod outer segments of bovine retina.

The rod outer segments (OS) of the retina are specialized organelles where phototransduction takes place. The mitochondrial electron transport complexes I-IV, cytochrome c and Fo F1 -ATP synthase are functionally expressed in the OS disks. Here, we have studied the effect of some polyphenolic compounds acting as inhibitors of mitochondrial ATPase/synthase activity on the OS ectopic Fo F1 - ATP synthase. The mechanism of apoptosis in the OS was also investigated studying the expression of cytochrome c, caspase 9 and 3 and Apaf-1. We prepared OS from fresh bovine retinae. Semi-quantitative Western blotting, confocal and electron microscopy, and cytofluorimetry were used along with biochemical analyses such as oximetry, ATP synthesis and hydrolysis. Resveratrol and curcumin plus piperine inhibited ATP synthesis and oxygen consumption in the OS. Epigallocatechin gallate and quercetin inhibited ATP hydrolysis and oxygen consumption in the OS. Malondialdehyde and hydrogen peroxide were produced in respiring OS in the presence of substrates. Cytochrome c was located inside the disk membranes. Procaspase 9 and 3, as well as Apaf-1 were expressed in the OS. These polyphenolic phytochemicals modulated the Fo F1 -ATP synthase activity of the the OS reducing production of reactive oxygen intermediates by the OS ectopic electron transport chain. Polyphenols decrease membrane peroxidation and cytochrome c release from disks, preventing the induction of caspase-dependent apoptosis in the OS Such effects are relevant in the design of protection against functional impairment of the OS following oxidative stress from exposure to intense illumination.

Molecular transitions from papillomavirus infection to cervical precancer and cancer: Role of stromal estrogen receptor signaling

To study the multistep process of cervical cancer development, we analyzed 128 frozen cervical samples spanning normalcy, increasingly severe cervical intraepithelial neoplasia (CIN1- CIN3), and cervical cancer (CxCa) from multiple perspectives, revealing a cascade of progressive changes. Compared with normal tissue, expression of many DNA replication/repair and cell proliferation genes was increased in CIN1/CIN2 lesions and further sustained in CIN3, consistent with high-risk human papillomavirus (HPV)-induced tumor suppressor inactivation. The CIN3-to-CxCa transition showed metabolic shifts, including decreased expression of mitochondrial electron transport complex components and ribosomal protein genes. Significantly, despite clinical, epidemiological, and animal model results linking estrogen and estrogen receptor alpha (ERα) to CxCa, ERα expression declined >15-fold from normalcy to cancer, showing the strongest inverse correlation of any gene with the increasing expression of p16, a marker for HPV-linked cancers. This drop in ERα in CIN and tumor cells was confirmed at the protein level. However, ERα expression in stromal cells continued throughout CxCa development. Our further studies localized stromal ERα to FSP1+, CD34+, SMA- precursor fibrocytes adjacent to normal and precancerous CIN epithelium, and FSP1-, CD34-, SMA+ activated fibroblasts in CxCas. Moreover, rank correlations with ERα mRNA identified IL-8, CXCL12, CXCL14, their receptors, and other angiogenesis and immune cell infiltration and inflammatory factors as candidates for ERα-induced stroma-tumor signaling pathways. The results indicate that estrogen signaling in cervical cancer has dramatic differences from ERα+ breast cancers, and imply that estrogen signaling increasingly proceeds indirectly through ERα in tumor-associated stromal fibroblasts.

Abstract 484: Platelet Specific Deletion of Opa-1 Reveals Gender Dimorphic Effects on Platelet Dysfunction and Thrombus Formation in Mice

Diabetes Mellitus and its complications remains a major public health burden. Cardiovascular disease (CVD) is the leading cause of death amongst diabetics and disproportionally affects women. Thrombosis represents one of the primary underlying pathologies in CVD. However, the mechanisms by which platelet dysfunction develops in diabetics are unclear. Preliminary studies revealed enrichment in platelet-derived transcripts encoding the mitochondrial dynamics proteins optic atrophy (OPA)-in females and mitofusin-2, dynamin-related protein-1, and mitochondrial fission 1 protein in both genders collected from the Framingham Offspring cohort, suggesting that platelet dysfunction in the diabetic milieu might be related to abnormal mitochondrial dynamics. Thus to define the relationship between mitochondrial dynamics and platelet dysfunction/thrombosis we generated mice with platelet-specific deletion of OPA1, a protein that regulates mitochondrial fusion, electron transport complex (ETC) assembly and apoptosis. Deletion of OPA-1 in platelets reduced mitochondrial oxygen consumption by approximately 50 % and reduced mitochondrial ETC subunit expression in both genders. Hydrogen peroxide levels were reduced by 40% in females but increased by 20% in males. Unexpectedly, females exhibited a 10-fold increase in expression of the NADPH oxidase (NOX) subunits gp91phox and p47phox, whereas NOX was unchanged in males. The absence of OPA1 was associated with longer time to stable occlusion in females but with decreased time to stable occlusion in males as assessed in vivo by rose Bengal photochemical injury. Also, male OPA1 deficient mice, were more prone to develop a thrombus (14/15 knockouts vs. 4/8 controls) following permanent ligation of the inferior vena cava, but this increase in venous thrombosis was not observed in females mice. Taken together, these data suggest that increased OPA1 expression observed in females with diabetes could represent a critical pathophysiologic mechanism in diabetic females that could contribute to their disproportional increased relative risk of thrombosis. We also identify for the first time, an essential role for OPA1 in regulating platelet mitochondrial function, platelet activation and thrombosis.