Mitochondrial Aconitase Activity Research Articles

A systematic analysis reveals an essential role for high-affinity iron uptake system, haemolysin and CFEM domain-containing protein in iron homoeostasis and virulence in Candida glabrata.

Iron is an essential nutrient for all living organisms and human pathogens employ a battery of factors to scavenge iron from the high-affinity iron-binding host proteins. In the present study, we have elucidated, via a candidate gene approach, major iron acquisition and homoeostatic mechanisms operational in an opportunistic human fungal pathogen Candida glabrata. Phenotypic, biochemical and molecular analysis of a set of 13 C. glabrata strains, deleted for proteins potentially implicated in iron metabolism, revealed that the high-affinity reductive iron uptake system is required for utilization of alternate carbon sources and for growth under both in vitro iron-limiting and in vivo conditions. Furthermore, we show for the first time that the cysteine-rich CFEM (common in fungal extracellular membranes) domain-containing cell wall structural protein, CgCcw14, and a putative haemolysin, CgMam3, are essential for maintenance of intracellular iron content, adherence to epithelial cells and virulence. Consistent with their roles in iron homoeostasis, mitochondrial aconitase activity was lower and higher in mutants disrupted for high-affinity iron transport, and haemolysin respectively. Additionally, we present evidence that the mitochondrial frataxin, CgYfh1, is pivotal to iron metabolism. Besides yielding insights into major in vitro and in vivo iron acquisition strategies, our findings establish high-affinity iron uptake mechanisms as critical virulence determinants in C. glabrata.

Effects of methylglyoxal and pyridoxamine in rat brain mitochondria bioenergetics and oxidative status.

Advanced glycation end products (AGEs) and methylglyoxal (MG), an important intermediate in AGEs synthesis, are thought to contribute to protein aging and to the pathogenesis of age-and diabetes-associated complications. This study was intended to investigate brain mitochondria bioenergetics and oxidative status of rats previously exposed to chronic treatment with MG and/or with pyridoxamine (PM), a glycation inhibitor. Brain mitochondrial fractions were obtained and several parameters were analyzed: respiratory chain [states 3 and 4 of respiration, respiratory control ratio (RCR), and ADP/O index] and phosphorylation system [transmembrane potential (ΔΨm), ADP-induced depolarization, repolarization lag phase, and ATP levels]; hydrogen peroxide (H2O2) production levels, mitochondrial aconitase activity, and malondialdehyde levels as well as non-enzymatic antioxidant defenses (vitamin E and glutathione levels) and enzymatic antioxidant defenses (glutathione disulfide reductase (GR), glutathione peroxidase (GPx), and manganese superoxide dismutase (MnSOD) activities). MG treatment induced a statistical significant decrease in RCR, aconitase and GR activities, and an increase in H2O2 production levels. The administration of PM did not counteract MG-induced effects and caused a significant decrease in ΔΨm. In mitochondria from control animals, PM caused an adaptive mechanism characterized by a decrease in aconitase and GR activities as well as an increase in both α-tocopherol levels and GPx and MnSOD activities. Altogether our results show that high levels of MG promote brain mitochondrial impairment and PM is not able to reverse MG-induced effects.

Pre-endurance training prevents acute alcoholic liver injury in rats through the regulation of damaged mitochondria accumulation and mitophagy balance.

The aim of this study is to investigate the effect of pre-endurance training on the prevention of alcohol-induced acute hepatic injury and on hepatic mitophagy. Forty-eight male Sprague-Dawley rats were randomly divided into four groups: (1) control group, (2) 12-week exercise training group, (3) 5-day alcohol intake group, and (4) 12-week exercise training plus 5-day alcohol intake group. The rats were examined to determine the following: BCL2/adenovirus E1B 19kDa protein-interacting protein 3 (BNIP3), hypoxia-inducible factor-1α (HIF-1α), cytochrome P450 2E1 (CYP2E1), alcohol dehydrogenase (ADH), microtubule-associated protein 1 light chain 3 (LC3II), Beclin1 mRNA and protein expressions, mitochondrial reactive oxygen species (ROS) production, mitochondrial thiobarbituric acid-reactive substances (TBARS) level, aconitase and ATP synthase activities, mitochondrial inner membrane potential, NADH/NAD(+) ratio, triglyceride (TG), the number of mtDNA and mitochondrial respiration functions in liver tissue, and serum ALT and AST. Pre-endurance training attenuated acute alcohol treatment-induced increase in mitochondrial TBARS, ROS production, NADH/NAD(+) ratio, state 4 respiration rate, TG, serum ALT and AST, as well as BNIP3, HIF-1α, LC3II, and Beclin 1 mRNA and protein levels, however, CYP2E1 and ADH mRNA and protein levels unchanged. Meanwhile, it attenuated the acute alcohol intake-induced decrease in aconitase activity, inner mitochondrial membrane potential (Δψ), ATP synthase activity, state 3 respiration rate, respiratory control ratio, and the number of mtDNA. Pre-endurance training can decrease acute alcohol intake-induced damaged mitochondria accumulation and reduced acute alcohol intake-induced mitophagy, which built a new balance between mitophagy and damaged mitochondria accumulation.

Wolfberries potentiate mitophagy in the liver of obese mice (372.3)

Objective: The aim is to investigate whether AMP‐activated protein kinase α2 (AMPKα2) is essential for wolfberry protective effects on mitochondrial dysfunction and subsequent hepatic steatosis in mice.Methods: Six‐week‐old male AMPKα2 knockout (KO) mice and genetic background C57BL/6J (B6) mice were fed the control, high‐fat (HD, 45% [kcal] fat), and/or HD with 5% (kcal) wolfberry diets for 18 weeks. At termination, blood and liver tissues were sampled for analysis by ELISA, HPLC, microscopy, real‐time PCR, and Western blot. Results: HD lowered hepatic lutein and zeaxanthin contents, inhibited β,β‐carotene 9,10 oxygenase 2 (BCO2) protein expression, induced mitochondrial oxidative stress as indicated by an increased reactive oxygen species level and the decreased mitochondrial membrane potential, respiratory rate, and aconitase activity. HD also suppressed mitophagy and mitochondrial biogenesis as determined by accumulation of p62, inhibited phosphorylation of Unc‐51‐like kinase 1 on Ser555, and declined expression of peroxisome proliferator‐activated receptor γ co‐activator 1 α, resulting in hepatic steatosis in B6 and KO mice. Dietary wolfberry elevated the xanthophyll concentrations and enhanced BCO2 expression, attenuated mitochondrial oxidative stress, activated AMPKα2, potentiated mitophagy and mitochondrial biogenesis, and enhanced lipid oxidation and secretion in the liver of B6 mice. Conclusion: Dietary wolfberry selectively activated AMPKα2, which resulted in enhanced mitochondrial biogenesis and potentiated mitophagy, leading to the prevention of hepatic steatosis in obese mice.Grant Funding Source: Supported by NIH NCRR Grant P20‐RR‐017686

Modulation of cardiac mitochondrial permeability transition and apoptotic signaling by endurance training and intermittent hypobaric hypoxia

BackgroundModulation of the mitochondrial permeability transition pore (MPTP) and inhibition of the apoptotic signaling are critically associated with the cardioprotective phenotypes afforded by both intermittent hypobaric-hypoxia (IHH) and endurance-training (ET). We recently proposed that IHH and ET improve cardiac function and basic mitochondrial capacity, although without showing addictive effects. Here we investigate whether a combination of IHH and ET alters cardiac mitochondrial vulnerability to MPTP and related apoptotic signaling. MethodsMale Wistar rats were divided into normoxic-sedentary (NS), normoxic-exercised (NE, 1h/day/5week treadmill-running), hypoxic-sedentary (HS, 6000m, 5h/day/5weeks) and hypoxic-exercised (HE) to study susceptibility to calcium-induced cardiac MPTP opening. Mitochondrial cyclophilin D (CypD), adenine nucleotide translocator (ANT), Bax and Bcl-2 protein contents were semi-quantified by Western blotting. Cardiac caspase 3-, 8- and 9-like activities were measured. Mitochondrial aconitase and superoxide dismutase (MnSOD) activity and malondialdehyde (MDA) and sulphydryl group (–SH) content were determined. ResultsSusceptibility to MPTP decreased in NE and HS vs. NS and even further in HE. The ANT content increased in HE vs. NS. Bcl-2/Bax ratio increased in NE and HS compared to NS. Decreased activities in tissue caspase 3-like (HE vs. NS) and caspase 9-like (HS and HE vs. NS) were observed. Mitochondrial aconitase increased in NE and HS vs. NS. No alterations between groups were observed for caspase 8-like activity, MnSOD, CypD, MDA and –SH. ConclusionsData confirm that IHH and ET modulate cardiac mitochondria to a protective phenotype characterized by decreased MPTP induction and apoptotic signaling, although without visible addictive effects as initially hypothesized.

On the molecular relationships between high-zinc tolerance and aconitase (Aco1) in Saccharomyces cerevisiae

Zinc is an essential metal for all organisms, as it participates in the structure and/or function of many proteins. However, zinc excess is as deleterious to cells as zinc deficiency. A genome-wide study of the transcriptomic response to high zinc in S. cerevisiae performed in our laboratory allowed the identification of a zinc hyper-tolerant deletion mutant (pif1Δ), which lacks the Pif1 DNA helicase. Further molecular characterization of this strain phenotype revealed that the lack of Pif1 leads to increased iron accumulation, redistribution of the aconitase protein to mitochondria, and also a loss of aconitase activity, despite normal Aco1 protein levels being present, probably due to the epistasis in protecting mtDNA between PIF1 and ACO1. The results presented in this work focus now on the characterization of different features related to the Aco1 protein and activity in yeast and the tolerance to high zinc. Hence, multiple phenotypic traits related to metal metabolism, namely Aco1 protein content and activity levels, succinate dehydrogenase activity, citrate levels, metal content, BPS influence in cultures, and the range of transcription of some iron metabolism related genes, have been analyzed for several strains, some of them constructed to this end, including BY4741, the deletants pif1Δ and aco1Δ, and the aco1 mutants aco1Δ-d4, aco1-C448S, aco1-R476S and aco1-R668S. Overall, lack of Aco1 enzymatic activity in mitochondria, citrate accumulation and lack of activity of [Fe-S] enzymes, e.g. succinate dehydrogenase, appear to be direct molecular indicators of increased zinc tolerance in S. cerevisiae.

Changes to mitochondrial ultrastructure in optic nerve vulnerable to secondary degeneration in vivo are limited by irradiation at 670 nm

BackgroundTraumatic injury to the central nervous system results in damage to tissue beyond the primary injury, termed secondary degeneration. Key events thought to be associated with secondary degeneration involve aspects of mitochondrial function which may be modulated by red/near-infrared irradiation therapy (R/NIR-IT), but precisely how mitochondria are affected in vivo has not been investigated. Secondary degeneration was modelled by transecting the dorsal aspect of the optic nerve in adult rats and mitochondrial ultrastructure in intact ventral optic nerve vulnerable to secondary degeneration investigated with transmission electron microscopy.ResultsDespite reported increases in fission following central nervous system injury, we saw no change in mitochondrial densities in optic nerve vulnerable to secondary degeneration in vivo. However, in axons, frequency distributions of mitochondrial profile areas showed higher cumulative probabilities of smaller mitochondrial profiles at day 1 after injury. Glial mitochondrial profiles did not exhibit changes in area, but a more elliptical mitochondrial shape was observed at both day 1 and 7 following injury. Importantly, mitochondrial autophagic profiles were observed at days 1 and 7 in optic nerve vulnerable to secondary degeneration in vivo. Citrate synthase activity was used as an additional measure of mitochondrial mass in ventral optic nerve and was decreased at day 7, whereas mitochondrial aconitase activity increased at day 1 and day 28 after injury in optic nerve vulnerable to secondary degeneration. R/NIR-IT has been used to treat the injured central nervous system, with reported improvements in oxidative metabolism suggesting mitochondrial involvement, but ultrastructural information is lacking. Here we show that R/NIR-IT of injured animals resulted in distributions of mitochondrial areas and shape not significantly different from control and significantly reduced mitochondrial autophagic profiles. R/NIR-IT also resulted in decreased citrate synthase activity (day 7) and increased aconitase activity (day 1) in optic nerve vulnerable to secondary degeneration.ConclusionsThese findings suggest that mitochondrial structure and activity of enzymes of the citric acid cycle are dynamically altered during secondary degeneration in vivo and R/NIR-IT may protect mitochondrial structure.

Mitochondrial susceptibility in a model of paraquat neurotoxicity

Paraquat is a highly toxic herbicide capable of generating oxidative stress and producing brain damage after chronic exposure. The aim of this research was to investigate the contribution of mitochondria to the molecular mechanism of apoptosis in an in vivo experimental model of paraquat neurotoxicity. Sprague-Dawley adult female rats received paraquat (10 mg/kg i.p.) or saline once a week during a month. Paraquat treatment increased cortical and striatal superoxide anion levels by 45% and 18%, respectively. As a consequence, mitochondrial aconitase activity was significantly inhibited in cerebral cortex and striatum. Paraquat treatment increased cortical and striatal lipid peroxidation levels by 16% and 28%, respectively, as compared with control mitochondria Also, cortical and striatal cardiolipin levels were decreased by 13% and 49%, respectively. Increased Bax and Bak association to mitochondrial membranes was observed after paraquat treatment in cerebral cortex and striatum. Also, paraquat induced cytochrome c and AIF release from mitochondria.These findings support the conclusion that a weekly dose of paraquat during four weeks induces oxidative damage that activates mitochondrial pathways associated with molecular mechanisms of cell death. The release of apoptogenic proteins from mitochondria to cytosol after paraquat treatment would be the consequence of an alteration in mitochondrial membrane permeability due to the presence of high superoxide anion levels. Also, our results suggest that under chronic exposure, striatal mitochondria were more sensitive to paraquat oxidative damage than cortical mitochondria. Even in the presence of a high oxidative stress in striatum, equal levels of apoptosis were attained in both brain areas.

Mitochondrial Complex Enzyme Activities and Cytochrome c Expression Changes in Multiple Sclerosis

Blood platelets have been widely proposed as biomarkers in studies of mitochondrial function and aging-related and neurodegenerative diseases. Defects in mitochondrial function were found not only in the substantia nigra of Parkinson's disease patients but also in their blood platelets. Similarly, it has also been described in the blood platelet mitochondria of Alzheimer's disease patients. To study mitochondrial aerobic metabolism function and protein expression in platelets of multiple sclerosis (MS) patients and control subjects, mitochondrial aconitase, mitochondrial superoxide dismutases 1 and 2 (SOD1 and SOD2), and respiratory complex enzyme activities in platelets of MS patients and control subjects were determined. Likewise, mitochondrial lipid peroxidation and mitochondrial SOD1 and cytochrome c expressions were investigated. Mitochondrial aconitase activity was higher in MS patients than in controls (P < 0.05). A significant increase on all respiratory complex activities in MS patients was observed (P < 0.05). Mitochondrial lipid peroxidation was significantly higher in MS patients than in controls (P < 0.05). Significant changes of cytochrome c and mitochondrial SOD1 expressions were detected (P < 0.05), with a decrease of 44 ± 5 % and an increase of 46 ± 6 %, respectively. Our study reveals that significant changes in mitochondrial aerobic metabolism function and mitochondrial SOD1 and cytochrome c expressions are produced in platelets of MS patients.

Postresuscitation Cyclosporine Treatment Attenuates Myocardial and Cardiac Mitochondrial Injury in Newborn Piglets with Asphyxia-Reoxygenation*

Cardiovascular dysfunction occurs in the majority of asphyxiated neonates and has been suggested to be a major cause of neonatal morbidity and mortality. We previously demonstrated that cyclosporine A treatment during resuscitation can significantly improve cardiovascular performance in asphyxiated newborn piglets. However, the mechanisms through which cyclosporine elicits its protective effect in neonates have not yet been fully characterized. We hypothesized that cyclosporine A treatment would attenuate myocardial and cardiac mitochondrial injury during the resuscitation of asphyxiated newborn piglets. After acute instrumentation, piglets received normocapnic alveolar hypoxia (10% to 15% oxygen) for 2 hours followed by reoxygenation with 100% oxygen (0.5 hr) and then 21% oxygen (3.5 hr). At 4 hours of reoxygenation, plasma troponin level, left ventricle myocardial levels of lipid hydroperoxides, cytochrome-c, and mitochondrial aconitase activity were determined. Neonatal asphyxia and reoxygenation. Twenty-four newborn (1-4 days old) piglets. Piglets were randomized to receive an IV bolus of cyclosporine A (10 mg/kg) or normal saline (placebo, control) at 5 minutes of reoxygenation (n=8/group). Sham-operated piglets (n=8) underwent no asphyxia-reoxygenation. Asphyxiated piglets treated with cyclosporine had lower plasma troponin and myocardial lipid hydroperoxides levels (vs. controls, both p<0.05, analysis of variance). Cyclosporine treatment also improved mitochondrial aconitase activity and attenuated the rise in cytosol cytochrome-c level (vs. controls, all p<0.05). The improved mitochondrial function significantly correlated with cardiac output (p<0.05, Spearman rank-correlation test). We demonstrate that the postresuscitation administration of cyclosporine attenuates myocardial and cardiac mitochondrial injury in asphyxiated newborn piglets following resuscitation.

Angiotensin Receptor Blockade Reduces Oxidative Stress while Improving Redox Signaling and Mitochondrial Function in the Heart of Diet‐induced Obese Insulin Resistant Rats

The contributions of angiotensin receptor type 1 (AT1) activation to NADPH oxidase (Nox)‐derived oxidative stress during hypertension and diabetes are well established. The role of AT1 in modulating redox signaling, mitochondrial function and oxidative stress in the heart during metabolic syndrome, however, remains elusive. To test the hypothesis that AT1 activation increases oxidative stress while impairing mitochondrial function and redox signaling in the heart during metabolic syndrome, diet‐induced obese, insulin resistant (OLETF) rats were treated with the AT1 blocker (ARB) Olmesartan for 6 weeks. Cardiac Nox2 expression increased 40% in OLETF compared to age‐matched lean (LETO) rats while expression of the H2O2‐producing Nox4 increased 40%. ARB treatment prevented the increase in Nox2 without altering Nox4. ARB treatment also normalized increased protein and lipid oxidation and increased the redox‐sensitive transcription factor Nrf2 30%, and antioxidant enzymes by 50–70%. Mitochondrial aconitase activity increased 60% in OLETF compared to LETO whereas succinate decreased 35%; ARB treatment normalized both. These data demonstrate that AT1 activation contributes to increased Nox2‐derived oxidative stress and to impaired redox signaling and mitochondrial activity, and suggests that, besides its anti‐hypertensive effects, ARB treatment may improve myocardial function during metabolic syndrome.

Mitochondria‐targeted antioxidant promotes recovery of skeletal muscle mitochondrial function after burn trauma assessed byin vivo31P nuclear magnetic resonance and electron paramagnetic resonance spectroscopy

Burn injury causes a major systemic catabolic response that is associated with mitochondrial dysfunction in skeletal muscle. We investigated the effects of the mitochondria-targeted peptide antioxidant Szeto-Schiller 31 (SS-31) on skeletal muscle in a mouse burn model using in vivo phosphorus-31 nuclear magnetic resonance ((31)P NMR) spectroscopy to noninvasively measure high-energy phosphate levels; mitochondrial aconitase activity measurements that directly correlate with TCA cycle flux, as measured by gas chromatography mass spectrometry (GC-MS); and electron paramagnetic resonance (EPR) to assess oxidative stress. At 6 h postburn, the oxidative ATP synthesis rate was increased 5-fold in burned mice given a single dose of SS-31 relative to untreated burned mice (P=0.002). Furthermore, SS-31 administration in burned animals decreased mitochondrial aconitase activity back to control levels. EPR revealed a recovery in redox status of the SS-31-treated burn group compared to the untreated burn group (P<0.05). Our multidisciplinary convergent results suggest that SS-31 promotes recovery of mitochondrial function after burn injury by increasing ATP synthesis rate, improving mitochondrial redox status, and restoring mitochondrial coupling. These findings suggest use of noninvasive in vivo NMR and complementary EPR offers an approach to monitor the effectiveness of mitochondrial protective agents in alleviating burn injury symptoms.

Nitric Oxide Promotes Survival of Cerebral Cortex Neurons with Simultaneous Addition of [Fe(II)(β-Citryl-L-glutamate)] Complex in Primary Culture

It has been reported that the activity of mitochondrial aconitase (m-aconitase) is rapidly inhibited in a variety of cells when exposed to nitric oxide (NO). In present study, we found that NO significantly increased the number of surviving neurons via enhanced mitochondrial functions with simultaneous addition of the [Fe(II)(β-citryl-L-glutamate; β-CG)] complex. In vitro, a variety of aconitase-inhibitors, such as fluorocitrate, cyanide ion, ferricyanide ([Fe(CN)6]), and various oxidants including superoxide anion, inhibited the activity of m-aconitase even in the presence of Fe(II), whereas a NO-donor, nitroprusside (SNP) ([Fe(CN)5NO]), was the only agent that significantly increased activity of that enzyme. Therefore, it is reasonable to assume that NO released from SNP promotes Fe-dependent activation of aconitase. All other tested NO-donors, including 3-morpholino-sydnonimine (SIN), Deta NONOate (NOC18), and NaNO2, also promoted activation of m-aconitase in time- and dose-dependent manners in the presence of Fe(II). The promoting effects of the NO-donors on activation disappeared with the addition of NO-scavengers. In intact mitochondria, all tested NO-donors promoted reactivation of aconitase in a dose-dependent manner in the presence of Fe(II), whereas that was not seen in its absence. These findings suggest that NO released from NO-donors promotes Fe-dependent activation of aconitase. In mixed neuronal and glial cultures, NO-donors except for SNP enhanced mitochondrial activity at low concentrations. Furthermore, simultaneous addition of the [Fe(II)(β-CG)] complex significantly enhanced those activities and greatly increased the number of surviving neurons. Thus, NO can carry Fe ions into m-aconitase via the guide of the tag of β-CG addressed to the enzyme.

Respiratory complex I deficiency results in low nitric oxide levels, induction of hemoglobin and upregulation of fermentation pathways

The cytoplasmic male-sterile (CMS) mutant of Nicotiana sylvestris which lacks NAD7, one of the subunits of respiratory complex I (NADH: ubiquinone oxidoreductase, EC 1.6.5.3), is characterized by very low (~10 times lower as compared to the wild type plants) emissions of nitric oxide (NO) under hypoxic conditions. The level of the non-symbiotic class 1 hemoglobin, as shown by Western blotting, is increased compared to the wild type plants not only under hypoxia but this protein reveals its marked expression in the CMS mutant even under normoxic conditions. The activity of aconitase (EC 4.2.1.3) is low in the CMS mutant, especially in the mitochondrial compartment, which indicates the suppression of the tricarboxylic acid cycle. The CMS mutant exhibits the severalfold higher activities of alcohol dehydrogenase (EC 1.1.1.1) and lactate dehydrogenase (EC 1.1.1.27) under the normoxic conditions as compared to the wild type plants. It is concluded that the lack of functional complex I results in upregulation of the pathways of hypoxic metabolism which include both fermentation of pyruvate and scavenging of NO by the non-symbiotic hemoglobin.

Macrophage migration inhibitory factor antagonizes pressure overload-induced cardiac hypertrophy

Macrophage migration inhibitory factor (MIF) functions as a proinflammatory cytokine when secreted from the cell, but it also exhibits antioxidant properties by virtue of its intrinsic oxidoreductase activity. Since increased production of ROS is implicated in the development of left ventricular hypertrophy, we hypothesized that the redox activity of MIF protects the myocardium when exposed to hemodynamic stress. In a mouse model of myocardial hypertrophy induced by transverse aortic coarctation (TAC) for 10 days, we showed that growth of the MIF-deficient heart was significantly greater by 32% compared with wild-type (WT) TAC hearts and that fibrosis was increased by fourfold (2.62 ± 0.2% vs. 0.6 ± 0.1%). Circulating MIF was increased in TAC animals, and expression of MIF receptor, CD74, was increased in the hypertrophic myocardium. Gene expression analysis showed a 10-fold increase (P < 0.01) in ROS-generating mitochondrial NADPH oxidase and 2- to 3-fold reductions (P < 0.01) in mitochondrial SOD2 and mitochondrial aconitase activities, indicating enhanced oxidative injury in the hypertrophied MIF-deficient ventricle. Hypertrophic signaling pathways showed that phosphorylation of cytosolic glycogen synthase kinase-3α was greater (P < 0.05) at baseline in MIF-deficient hearts than in WT hearts and remained elevated after 10-day TAC. In the hemodynamically stressed MIF-deficient heart, nuclear p21(CIP1) increased sevenfold (P < 0.01), and the cytosolic increase of phospho-p21(CIP1) was significantly greater than in WT TAC hearts. We conclude that MIF antagonizes myocardial hypertrophy and fibrosis in response to hemodynamic stress by maintaining a redox homeostatic phenotype and attenuating stress-induced activation of hypertrophic signaling pathways.

Novel Frataxin Isoforms May Contribute to the Pathological Mechanism of Friedreich Ataxia

Friedreich ataxia (FRDA) is an inherited neurodegenerative disease caused by frataxin (FXN) deficiency. The nervous system and heart are the most severely affected tissues. However, highly mitochondria-dependent tissues, such as kidney and liver, are not obviously affected, although the abundance of FXN is normally high in these tissues. In this study we have revealed two novel FXN isoforms (II and III), which are specifically expressed in affected cerebellum and heart tissues, respectively, and are functional in vitro and in vivo. Increasing the abundance of the heart-specific isoform III significantly increased the mitochondrial aconitase activity, while over-expression of the cerebellum-specific isoform II protected against oxidative damage of Fe-S cluster-containing aconitase. Further, we observed that the protein level of isoform III decreased in FRDA patient heart, while the mRNA level of isoform II decreased more in FRDA patient cerebellum compared to total FXN mRNA. Our novel findings are highly relevant to understanding the mechanism of tissue-specific pathology in FRDA.

Insulin-induced recurrent hypoglycemia exacerbates diabetic brain mitochondrial dysfunction and oxidative imbalance

Intensive insulin therapy can prevent or slow the progression of long-term diabetes complications but, at the same time, it increases the risk for episodes of severe hypoglycemia. In our study, we used a protocol intended to mimic the levels of blood glucose that occur in type 1 diabetic patients under an intensive insulin therapy. Streptozotocin (STZ)-induced diabetic rats were treated subcutaneously with twice-daily insulin injections for 2weeks to induce hypoglycemic episodes. Brain cortical and hippocampal mitochondria were isolated and mitochondrial bioenergetics (respiratory chain and phosphorylation system) and oxidative status parameters (malondialdehyde (MDA) levels, mitochondrial aconitase activity and enzymatic and non-enzymatic antioxidant defenses) were analyzed. The protein levels of synaptophysin, a marker of synaptic integrity, and caspase 9 activity were also evaluated in cortical and hippocampal homogenates. Brain cortical mitochondria isolated from hyper- and recurrent hypoglycemic animals presented higher levels of MDA and α-tocopherol together with an increased glutathione disulfide reductase activity, lower manganese superoxide dismutase (MnSOD) activity and glutathione-to-glutathione disulfide (GSH/GSSG) ratio. No significant alterations were found in cortical mitochondrial respiratory chain and oxidative phosphorylation system. Hippocampal mitochondria from both experimental groups presented an impaired oxidative phosphorylation system characterized by a decreased mitochondrial energization potential and ATP levels and higher repolarization lag phase. In addition, higher MDA levels and decreased GSH/GSSG, α-tocopherol levels, and aconitase, glutathione peroxidase and MnSOD activities were observed in both groups of animals. Hippocampal mitochondria from recurrent hypoglycemic animals also showed an impairment of the respiratory chain characterized by a lower state 3 of respiration, respiratory control ratio and ADP/O index, and a higher state 4 of respiration. Additionally, a non-statistically significant decrease in synaptophysin protein levels was observed in cortical homogenates from recurrent hypoglycemic rats as well as in hippocampal homogenates from hyperglycemic and recurrent hypoglycemic rats. An increase in caspase 9 activity was also observed in hippocampal homogenates from hyperglycemic and recurrent hypoglycemic animals. Our results show that mitochondrial dysfunction induced by long-term hyperglycemic effects is exacerbated by recurrent hypoglycemia, which may compromise the function and integrity of brain cells.

Streptozotocin-Induced Cytotoxicity, Oxidative Stress and Mitochondrial Dysfunction in Human Hepatoma HepG2 Cells

Streptozotocin (STZ) is an antibiotic often used in the treatment of different types of cancers. It is also highly cytotoxic to the pancreatic beta-cells and therefore is commonly used to induce experimental type 1 diabetes in rodents. Resistance towards STZ-induced cytotoxicity in cancer cells has also been reported. Our previous studies have reported organ-specific toxicity and metabolic alterations in STZ-induced diabetic rats. STZ induces oxidative stress and metabolic complications. The precise molecular mechanism of STZ-induced toxicity in different tissues and carcinomas is, however, unclear. We have, therefore, investigated the mechanism of cytotoxicity of STZ in HepG2 hepatoma cells in culture. Cells were treated with different doses of STZ for various time intervals and the cytotoxicity was studied by observing the alterations in oxidative stress, mitochondrial redox and metabolic functions. STZ induced ROS and RNS formation and oxidative stress as measured by an increase in the lipid peroxidation as well as alterations in the GSH-dependent antioxidant metabolism. The mitochondria appear to be a highly sensitive target for STZ toxicity. The mitochondrial membrane potential and enzyme activities were altered in STZ treated cells resulting in the inhibition of ATP synthesis. ROS-sensitive mitochondrial aconitase activity was markedly inhibited suggesting increased oxidative stress in STZ-induced mitochondrial toxicity. These results suggest that STZ-induced cytotoxicity in HepG2 cells is mediated, at least in part, by the increase in ROS/RNS production, oxidative stress and mitochondrial dysfunction. Our study may be significant for better understanding the mechanisms of STZ action in chemotherapy and drug induced toxicity.

Implications of Altered Glutathione Metabolism in Aspirin-Induced Oxidative Stress and Mitochondrial Dysfunction in HepG2 Cells

We have previously reported that acetylsalicylic acid (aspirin, ASA) induces cell cycle arrest, oxidative stress and mitochondrial dysfunction in HepG2 cells. In the present study, we have further elucidated that altered glutathione (GSH)-redox metabolism in HepG2 cells play a critical role in ASA-induced cytotoxicity. Using selected doses and time point for ASA toxicity, we have demonstrated that when GSH synthesis is inhibited in HepG2 cells by buthionine sulfoximine (BSO), prior to ASA treatment, cytotoxicity of the drug is augmented. On the other hand, when GSH-depleted cells were treated with N-acetyl cysteine (NAC), cytotoxicity/apoptosis caused by ASA was attenuated with a significant recovery in oxidative stress, GSH homeostasis, DNA fragmentation and some of the mitochondrial functions. NAC treatment, however, had no significant effects on the drug-induced inhibition of mitochondrial aconitase activity and ATP synthesis in GSH-depleted cells. Our results have confirmed that aspirin increases apoptosis by increased reactive oxygen species production, loss of mitochondrial membrane potential and inhibition of mitochondrial respiratory functions. These effects were further amplified when GSH-depleted cells were treated with ASA. We have also shown that some of the effects of aspirin might be associated with reduced GSH homeostasis, as treatment of cells with NAC attenuated the effects of BSO and aspirin. Our results strongly suggest that GSH dependent redox homeostasis in HepG2 cells is critical in preserving mitochondrial functions and preventing oxidative stress associated complications caused by aspirin treatment.

Effects of peroxisomal catalase inhibition on mitochondrial function

Peroxisomes produce hydrogen peroxide as a metabolic by-product of their many oxidase enzymes, but contain catalase that breaks down hydrogen peroxide in order to maintain the organelle’s oxidative balance. It has been previously demonstrated that, as cells age, catalase is increasingly absent from the peroxisome, and resides instead as an unimported tetrameric molecule in the cell cytosol; an alteration that is coincident with increased cellular hydrogen peroxide levels. As this process begins in middle-passage cells, we sought to determine whether peroxisomal hydrogen peroxide could contribute to the oxidative damage observed in mitochondria in late-passage cells. Early-passage human fibroblasts (Hs27) treated with aminotriazole (3-AT), an irreversible catalase inhibitor, demonstrated decreased catalase activity, increased levels of cellular hydrogen peroxide, protein carbonyls, and peroxisomal numbers. This treatment increased mitochondrial reactive oxygen species levels, and decreased the mitochondrial aconitase activity by ∼85% within 24 h. In addition, mitochondria from 3-AT treated cells show a decrease in inner membrane potential. These results demonstrate that peroxisome-derived oxidative imbalance may rapidly impair mitochondrial function, and considering that peroxisomal oxidative imbalance begins to occur in middle-passage cells, supports the hypothesis that peroxisomal oxidant release occurs upstream of, and contributes to, the mitochondrial damage observed in aging cells.