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Duchenne muscular dystrophy (DMD) is a severe childhood disease characterised by progressive muscle wasting caused by widespread myofibre necrosis. Implicated in the pathology of DMD is oxidative stress, caused by excessive generation of reactive oxygen and nitrogen species (RONS). One consequence of RONS exposure is post-translational oxidative modifications to proteins, which can cause loss of protein function. This study used the dystrophic mdx mouse model for DMD to visualise the precise location of different oxidative modifications to proteins in dystrophic muscles, including both reversible (protein thiol oxidation and s-nitrosylation) and irreversible (carbonylation and dityrosine formation) oxidation at various stages of dystrophic muscle necrosis and regeneration. High levels of protein oxidation were observed in mdx myofibres undergoing degeneration and immune cell infiltration (myonecrosis). Since irreversible protein oxidation, especially dityrosine formation, was only colocalised to areas of myonecrosis, we suggest that this specific measurement could be a useful biomarker of myonecrosis. To test this we quantified dityrosines in muscle homogenates; this analysis showed significantly higher levels of dityrosines in mdx (compared with control normal) mice aged 23 days, an age when acute onset of extensive myonecrosis occurs in mdx muscles. These results indicate a major localised role of immune cells in RONS generation in dystrophic muscle, and strongly support a role for protein oxidation in myonecrosis and associated dystropathology. Consequently, the measurement of protein oxidation (specifically dityrosines) in dystrophic muscles may be a useful biomarker for indirectly quantifying myonecrosis in research studies using mdx mice and other animal models for DMD.

Isotope-coded affinity tags (ICATs) are valuable tools for mass spectrometry-based quantitative proteomics, in particular, for comparison of protein (cysteine-residue) thiol oxidation state in normal, stressed, and diseased tissue. However, the iodoacetamido electrophile used in most commercial ICATs suffers from poor thiol-selectivity and modest rates of adduct formation, which can lead to spurious results. Hence, we designed and synthesized three ICATs containing thiol-selective N-alkylmaleimide electrophiles (isotope-coded maleimide affinity tags = ICMATs) and assessed these as mass spectrometry probes for ratiometric analysis of lysozyme and muscle proteomes. Two ICMAT pairs containing butylene/D8-butylene linkers were effective MS probes, but not ideal for typical proteomics workflows, because peptides bearing these tags frequently did not coelute with HPLC. A switch to a phenylene/13C6-phenylene linker solved this issue without compromising the efficiency of adduct formation.

Duchenne muscular dystrophy (DMD) is a lethal, X-chromosome linked muscle-wasting disease affecting about 1 in 3500-6000 boys worldwide. Myofibre necrosis and subsequent loss of muscle mass are due to several molecular sequelae, such as inflammation and oxidative stress. We have recently shown increased neutrophils, highly reactive oxidant hypochlorous acid (HOCl) generation by myeloperoxidase (MPO), and associated oxidative stress in muscle from the GRMD dog and mdx mouse models for DMD. These findings have led us to hypothesise that generation of HOCl by myeloperoxidase released from neutrophils has a significant role in dystropathology. Since access to muscle from DMD patients is limited, the aim of this study was to develop methods to study this pathway in urine. Using immunoblotting to measure markers of protein oxidation, we show increased labelling of proteins with antibodies to dinitrophenylhydrazine (DNP, oxidative damage) and DiBrY (halogenation by reactive oxidants from myeloperoxidase) in GRMD and mdx urine. A strong positive correlation was observed between DiBrY labelling in dog urine and muscle. A strong positive correlation was also observed when comparing DNP and DiBrY labelling (in muscle and urine) to markers of dystropathology (plasma creatine kinase) and neutrophil presence (muscle MPO). Our results indicate the presence of neutrophil mediated oxidative stress in both models, and suggest that urine is a suitable bio-fluid for the measurement of such biomarkers. These methods could be employed in future studies into the role of neutrophil mediated oxidative stress in DMD and other inflammatory pathologies.

Duchenne muscular dystrophy (DMD) is a lethal genetic muscle wasting disorder, which currently has no cure. Supplementation with the drug taurine has been shown to offer therapeutic benefit in the mdx model for DMD, however the mechanism by which taurine protects dystrophic muscle is not fully understood. Mdx muscle is deficient in taurine, however it is not known if this deficiency occurs in the extracellular space, in other cells present in the tissue (such as immune cells) or in the myofibre itself. Likewise, the tissue location of taurine enrichment in taurine treated mdx muscle is not known. In this study we applied X-ray absorption near edge spectroscopy (XANES) at the sulfur K-edge in an imaging format to determine taurine distribution in muscle tissue. XANES is the only technique currently capable of imaging taurine directly in muscle tissue, at a spatial resolution approaching myocyte cell size (20-50 μm). Using a multi-modal approach of XANES imaging and histology on the same tissue sections, we show that in mdx muscle, it is the myofibres that are deficient in taurine, and taurine supplementation ameliorates this deficiency. Increasing the taurine content of mdx myofibres was associated with a decrease in myofibre damage (as shown by the percentage of intact myofibres) and inflammation. These data will help drive future studies to better elucidate the molecular mechanisms through which taurine protects dystrophic muscle; they also support the continued investigation of taurine as a therapeutic intervention for DMD.

Duchenne muscular dystrophy (DMD) is a lethal, X-linked disease that causes severe loss of muscle mass and function in young children. Promising therapies for DMD are being developed, but the long lead times required when using clinical outcome measures are hindering progress. This progress would be facilitated by robust molecular biomarkers in biofluids, such as blood and urine, which could be used to monitor disease progression and severity, as well as to determine optimal drug dosing before a full clinical trial. Many candidate DMD biomarkers have been identified, but there have been few follow-up studies to validate them. This Review describes the promising biomarkers for dystrophic muscle that have been identified in muscle, mainly using animal models. We strongly focus on myonecrosis and the associated inflammation and oxidative stress in DMD muscle, as the lack of dystrophin causes repeated bouts of myonecrosis, which are the key events that initiate the resultant severe dystropathology. We discuss the early events of intrinsic myonecrosis, along with early regeneration in the context of histological and other measures that are used to quantify its incidence. Molecular biomarkers linked to the closely associated events of inflammation and oxidative damage are discussed, with a focus on research related to protein thiol oxidation and to neutrophils. We summarise data linked to myonecrosis in muscle, blood and urine of dystrophic animal species, and discuss the challenge of translating such biomarkers to the clinic for DMD patients, especially to enhance the success of clinical trials., Summary: This Review discusses biomarkers in blood and urine linked to myonecrosis, inflammation and oxidative stress, to enhance development of therapies for DMD, and the challenges to be overcome for clinical translation.

Oxidative stress, caused by reactive oxygen and nitrogen species (RONS), is important in the pathophysiology of many diseases. A key target of RONS is the thiol group of protein cysteine residues. Because thiol oxidation can affect protein function, mechanistic information about how oxidative stress affects tissue function can be ascertained by identifying oxidized proteins. The probes used must be specific and sensitive, such as maleimides for the alkylation of reduced cysteine thiols. However, we find that maleimide-alkylated peptides (MAPs) are oxidized and hydrolyzed under sample preparation conditions common for proteomic studies. This can result in up to 90% of the MAP signal being converted to oxidized or hydrolyzed MAPs, decreasing the sensitivity of the analysis. A substantial portion of these modifications were accounted for by Coomassie "blue silver" staining (∼14%) of gels and proteolytic digestion buffers (∼20%). More than 40% of the MAP signal can be retained with the use of thioglycolic acid during gel electrophoresis, trichloroethanol-UV protein visualization in gels, and proteolytic digestion buffer of pH 7.0 TRIS. This work demonstrates that it is possible to decrease modifications to MAPs through changes to the sample preparation workflow, enhancing the potential usefulness of maleimide in identifying oxidized peptides.

Duchenne Muscular Dystrophy (DMD) is a fatal skeletal muscle wasting disease presenting with excessive myofibre necrosis and increased inflammation and oxidative stress. In the mdx mouse model of DMD, homeostasis of the amino acid taurine is altered, and taurine administration drastically decreases muscle necrosis, dystropathology, inflammation and protein thiol oxidation. Since the severe pathology of the Golden Retriever Muscular Dystrophy (GRMD) dog model more closely resembles the human DMD condition, we aimed to assess the generation of oxidants by inflammatory cells and taurine metabolism in this species. In muscles of 8 month GRMD dogs there was an increase in the content of neutrophils and macrophages, and an associated increase in elevated myeloperoxidase, a protein secreted by neutrophils that catalyses production of the highly reactive hypochlorous acid (HOCl). There was also increased chlorination of tyrosines, a marker of HOCl generation, increased thiol oxidation of many proteins and irreversible oxidative protein damage. Taurine, which functions as an antioxidant by trapping HOCl, was reduced in GRMD plasma; however taurine was increased in GRMD muscle tissue, potentially due to increased muscle taurine transport and synthesis. These data indicate a role for HOCl generated by neutrophils in the severe dystropathology of GRMD dogs, which may be exacerbated by decreased availability of taurine in the blood. These novel data support continued research into the precise roles of oxidative stress and taurine in DMD and emphasise the value of the GRMD dogs as a suitable pre-clinical model for testing taurine as a therapeutic intervention for DMD boys., Graphical abstract fx1, Highlights • We investigated oxidative stress in muscle from the GRMD dog model of DMD. • Excess neutrophils were associated with hypochlorous acid generation in GRMD muscle. • GRMD muscle exhibited oxidative damage to proteins, and protein thiol oxidation. • These data imply a role of immune cell generated oxidative stress in GRMD pathology.

Oxidative stress, caused by excess reactive oxygen species (ROS), has been hypothesized to cause or exacerbate skeletal muscle wasting in a number of diseases and chronic conditions. ROS, such as hydrogen peroxide, have the potential to affect signal transduction pathways such as the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3 K)/Akt pathway that regulates protein synthesis. Previous studies have found contradictory outcomes for the effect of ROS on the PI3K/Akt signaling pathway, where oxidative stress can either enhance or inhibit Akt phosphorylation. The apparent contradictions could reflect differences in experimental cell types or types of ROS treatments. We replicate both effects in myotubes of cultured skeletal muscle C2C12 cells, and show that increased oxidative stress can either inhibit or enhance Akt phosphorylation. This differential response could be explained: thiol oxidation of Akt, but not the phosphatases PTEN or PP2A, caused a decline in Akt phosphorylation; whereas the thiol oxidation of Akt, PTEN and PP2A increased Akt phosphorylation. These observations indicate that a more complete understanding of the effects of oxidative stress on a signal transduction pathway comes not only from identifying the proteins susceptible to thiol oxidation, but also their relative sensitivity to ROS.

Duchenne Muscular Dystrophy (DMD) is a fatal muscle wasting disease manifested in young boys, for which there is no current cure. We have shown that the amino acid taurine is safe and effective at preventing dystropathology in the mdx mouse model for DMD. This study aimed to establish if treating growing mdx mice with a higher dose of taurine was more effective at improving strength and reducing inflammation and oxidative stress. Mice were treated with a dose of taurine estimated to be 16 g/kg/day, in drinking water from 1-6 weeks of age, after which in vivo and ex vivo muscle strength was assessed, as were measures of inflammation, oxidative stress and taurine metabolism. While the dose did decrease inflammation and protein oxidation in dystrophic muscles, there was no improvement in muscle strength (in contrast with benefits observed with the lower dose) and growth of the young mice was significantly restricted. We present novel data that a high taurine dose increases the cysteine content of both mdx liver and plasma, a possible result of down regulation of the taurine synthesis pathway in the liver (which functions to dispose of excess cysteine, which is toxic). These data caution that a high dose of taurine can have adverse effects and may be less efficacious than lower taurine doses. Therefore, monitoring of taurine dosage needs to be considered in future pre-clinical trials, in anticipation of using taurine as a clinical therapy for growing DMD boys (and other conditions).

Oxidative stress caused by reactive oxygen species is proposed to cause age related muscle wasting (sarcopenia). Reversible oxidation of protein thiols by reactive oxygen species can affect protein function, so we evaluated whether muscle wasting in normal aging was associated with a pervasive increase in reversible oxidation of protein thiols or with an increase in irreversible oxidative damage to macromolecules. In gastrocnemius muscles of C57BL/6J female mice aged 3, 15, 24, 27, and 29 months there was no age related increase in protein thiol oxidation. In contrast, there was a significant correlation (R 2 = 0.698) between increasing protein carbonylation, a measure of irreversible oxidative damage to proteins, and loss of mass of gastrocnemius muscles in aging female mice. In addition, there was an age-related increase in lipofuscin content, an aggregate of oxidised proteins and lipids, in quadriceps limb muscles in aging female mice. However, there was no evidence of an age-related increase in malondialdehyde or F2-isoprostanes levels, which are measures of oxidative damage to lipids, in gastrocnemius muscles. In summary, this study does not support the hypothesis that a pervasive increase in protein thiol oxidation is a contributing factor to sarcopenia. Instead, the data are consistent with an aging theory which proposes that molecular damage to macromolecules leads to the structural and functional disorders associated with aging.

Iron chelators and antioxidants have been shown to prevent hypothermia-induced apoptosis in hepatocytes. This study examined whether iron chelation and antioxidants could also prevent hypothermia-induced necrosis. Isolated rat hepatocytes were incubated at 4 degrees C for 6 hours and then rewarmed at 37 degrees C for 18 hours with or without the iron chelator deferoxamine and a selection of antioxidants. There was no evidence of increased cell death or adenosine triphosphate depletion during hypothermic incubation. After hypothermia and rewarming, the majority of rat hepatocytes died of necrosis as indicated by the absence of DNA fragmentation, caspase 3 activity, and apoptotic bodies. Cell death was significantly reduced if deferoxamine or a selection of antioxidants were present during hypothermia and rewarming. Deferoxamine was more effective in preventing cell death when added prior to hypothermia, indicating cell death processes were likely initiated during hypothermia.

The c-Jun N-terminal kinases (JNKs) have been the subject of intense interest since their discovery in the early 1990s. Major research programs have been directed to the screening and/or design of JNK-selective inhibitors and testing their potential as drugs. We begin this review by considering the first commercially-available JNK ATP-competitive inhibitor, SP600125. We focus on recent studies that have evaluated the actions of SP600125 in lung, brain, kidney and liver following exposure to a range of stress insults including ischemia/reperfusion. In many but not all cases, SP600125 administration has proved beneficial. JNK activation can also follow infection, and we next consider recent examples that demonstrate the benefits of SP600125 administration in viral infection. Additional ATP-competitive JNK inhibitors have now been described following high throughput screening of small molecule libraries, but information on their use in biological systems remains limited and thus these inhibitors will require further evaluation. Peptide substrate-competitive ATP-non-competitive inhibitors of JNK have also now been described, and we discuss the recent advances in the use of JNK inhibitory peptides in the treatment of neuronal death, diabetes and viral infection. We conclude by raising a number of questions that should be considered in the quest for JNK-specific inhibitors.

Oxidative stress during cold preservation has been identified as a significant cause of cell injury but the process by which injury occurs is poorly understood. We examined loss of lysosomal integrity as a possible cause of cell injury during extended cold storage of isolated rat hepatocytes. After 21 h of hypothermia, there was a marked decline in lysosomal integrity, which was correlated with an increase in lipid peroxidation. When lipid peroxidation was prevented with the antioxidant Trolox (a vitamin E analog) or the iron chelator desferrioxamine, lysosomal integrity was preserved. In contrast, increasing lysosomal iron with ferric chloride caused an increase in lipid peroxidation and decreased lysosomal integrity. Loss of lysosomal integrity during cold preservation in this experimental model was consistent with iron-initiated oxidative stress. The progressive loss of lysosomal integrity during hypothermic incubation has the potential to affect liver function after transplantation.

Peptide inhibitors of c-Jun N-terminal kinase (JNK) have been shown to potently protect against cerebral ischemia. The protective effect has been ascribed to prevention of apoptosis, but cell death following cerebral ischemia is a consequence of both apoptotic and necrotic cell death. We evaluated whether a peptide inhibitor (TAT-TIJIP) of JNK could prevent necrotic cell death in an in vitro model of excitotoxic neuronal death. We find that TAT-TIJIP effectively prevented cell death by interfering with several processes which have been identified as leading to cell death by necrosis. In particular, reactive oxygen species production was reduced, as indicated by an 88% decrease in the rate of dihydroethidium fluorescence in the presence of TAT-TIJIP. Furthermore, TAT-TIJIP attenuated the increase in cytosolic calcium following the excitotoxic insult. The potent neuroprotective properties of JNK peptide inhibitors likely reflects their abilities to prevent cell death by necrosis as well as apoptosis.

We sought to understand the effect of a transient exposure of cardiac myocytes to H 2 O 2 at a concentration that did not induce apoptosis. Myocytes were exposed to 30 μmol/L H 2 O 2 for 5 minutes followed by 10 U/mL catalase for 5 minutes to degrade the H 2 O 2 . Cellular superoxide was measured using dihydroethidium. Transient exposure to H 2 O 2 caused a 66.4% increase in dihydroethidium signal compared with controls exposed to only catalase, without activation of caspase 3 or evidence of necrosis. The increase in dihydroethidium signal was attenuated by the mitochondrial inhibitors myxothiazol or carbonyl cyanide p -(trifluoromethoxy)phenyl-hydrazone and when calcium uptake by the mitochondria was inhibited with Ru360. We investigated the L-type Ca 2+ channel ( I Ca-L ) as a source of calcium influx. Nisoldipine, an inhibitor of I Ca-L , attenuated the increase in superoxide. Basal channel activity increased from 5.4 to 8.9 pA/pF. Diastolic calcium was significantly increased in quiescent and contracting myocytes after H 2 O 2 . The response of I Ca-L to β-adrenergic receptor stimulation was used as a functional reporter because decreasing intracellular H 2 O 2 alters the sensitivity of I Ca-L to isoproterenol. H 2 O 2 increased the K 0.5 required for activation of I Ca-L by isoproterenol from 5.8 to 27.8 nmol/L. This effect and the increase in basal current density persisted for several hours after H 2 O 2 . We propose that extracellular H 2 O 2 is associated with an increase in superoxide from the mitochondria caused by an increase in Ca 2+ influx from I Ca-L . The effect persists because a positive feedback exists among increased basal channel activity, elevated intracellular calcium, and superoxide production by the mitochondria.

Oxidative stress has been implicated in cell death in range of disease states including ischemia/reperfusion injury of the heart and heart failure. Here we have investigated the mechanisms of cell death following chronic exposure of cardiac myocytes to oxidative stress initiated by hydrogen peroxide. This exposure induced a delayed form of cell death with ultrastructural changes typical of necrosis, and that was accompanied by the release of lactate dehydrogenase and increased lipid peroxidation. However, this delayed death was not accompanied by the loss of mitochondrial membrane potential or caspase-3 activation. Furthermore, we could demonstrate that this delayed necrosis was at least partially prevented by pre-treatment with the hypertrophic stimuli endothelin-1 or leukemic inhibitory factor. Our results suggest that this delayed form necrosis may also comprise an ordered series of events involving pathways amenable to therapeutic modulation.

We previously reported that cyclophilin A protein is up-regulated in cortical neuronal cultures following several preconditioning treatments. In the present study, we have demonstrated that adenoviral-mediated over-expression of cyclophilin A in rat cortical neuronal cultures can protect neurons from oxidative stress (induced by cumene hydroperoxide) and in vitro ischemia (induced by oxygen glucose deprivation). We subsequently demonstrated that cultured neurons, but not astrocytes, express the recently identified putative cyclophilin A receptor, CD147 (also called neurothelin, basigin and EMMPRIN), and that administration of purified cyclophilin A protein to neuronal cultures induces a rapid but transient phosphorylation of the extracellular signal-regulated kinase (ERK) 1/2. Furthermore, administration of purified cyclophilin A protein to neuronal cultures protects neurons from oxidative stress and in vitro ischemia. Interestingly, we detected up-regulation of cyclophilin A mRNA, but not protein in the hippocampus following a 3-min period of sublethal global cerebral ischemia in the rat. Despite our in vivo findings, our in vitro data show that cyclophilin A has both intracellular- and extracellular-mediated neuroprotective mechanisms. To this end, we propose cyclophilin A's extracellular-mediated neuroprotection occurs via CD147 receptor signalling, possibly by activation of ERK1/2 pro-survival pathways. Further characterization of cyclophilin A's neuroprotective mechanisms may aid the development of a neuroprotective therapy.

We previously reported that peroxiredoxin 2 (PRDX2) and Cu/Zn superoxide dismutase 1 (SOD1) proteins are up-regulated in rat primary neuronal cultures following erythropoietin (EPO) preconditioning. In the present study, we have demonstrated that adenovirally mediated overexpression of PRDX2 in cortical neuronal cultures can protect neurons from in vitro ischemia (oxygen-glucose deprivation) and an oxidative insult (cumene hydroperoxide) but not glutamate excitotoxicity. We have also demonstrated that adenovirally mediated overexpression of SOD1 in cortical neuronal cultures protected neurons only against the oxidative insult. Interestingly, we did not detect up-regulation of PRDX2 or SOD1 protein in the rat hippocampus following exposure to either 3 min or 8 min of global cerebral ischemia. Further characterization of PRDX2's neuroprotective mechanisms may aid in the development of a neuroprotective therapy.

The amino acid taurine is essential for the function of skeletal muscle and administration is proposed as a treatment for Duchenne Muscular Dystrophy (DMD). Taurine homeostasis is dependent on multiple processes including absorption of taurine from food, endogenous synthesis from cysteine and reabsorption in the kidney. This study investigates the cause of reported taurine deficiency in the dystrophic mdx mouse model of DMD. Levels of metabolites (taurine, cysteine, cysteine sulfinate and hypotaurine) and proteins (taurine transporter [TauT], cysteine deoxygenase and cysteine sulfinate dehydrogenase) were quantified in juvenile control C57 and dystrophic mdx mice aged 18 days, 4 and 6 weeks. In C57 mice, taurine content was much higher in both liver and plasma at 18 days, and both cysteine and cysteine deoxygenase were increased. As taurine levels decreased in maturing C57 mice, there was increased transport (reabsorption) of taurine in the kidney and muscle. In mdx mice, taurine and cysteine levels were much lower in liver and plasma at 18 days, and in muscle cysteine was low at 18 days, whereas taurine was lower at 4: these changes were associated with perturbations in taurine transport in liver, kidney and muscle and altered metabolism in liver and kidney. These data suggest that the maintenance of adequate body taurine relies on sufficient dietary intake of taurine and cysteine availability and metabolism, as well as retention of taurine by the kidney. This research indicates dystrophin deficiency not only perturbs taurine metabolism in the muscle but also affects taurine metabolism in the liver and kidney, and supports targeting cysteine and taurine deficiency as a potential therapy for DMD.

SUMMARY 1 Quantitative proteomics is a technique that allows for large-scale comparison of the levels of individual proteins present in a biological sample. This technique has not previously been applied to examine the response of skeletal muscle proteins to an acute bout of exercise. 2 In the present study, quantitative proteomics was applied to investigate whether the levels of individual skeletal muscle proteins are acutely affected by a short bout of high-intensity exercise. 3 Gastrocnemius muscle was sampled from fasted rats either at rest, immediately following 3 min of high-intensity exercise or after 30 min of recovery. Muscle samples were submitted to two-dimensional gel electrophoresis and 61 of the resulting protein spots were selected for quantitative analysis. 4 It was found that skeletal muscle protein levels were generally not acutely affected by a short bout of high-intensity exercise, with only four of the 61 proteins selected for analysis being significantly altered. These altered proteins were identified using liquid chromatography electrospray ionization–tandem mass spectrometry as creatine kinase, troponin T and a combination of heat shock 20 kDa protein and adenylate kinase 1. 5 In conclusion, quantitative proteomics is sensitive enough to detect acute changes in skeletal muscle protein levels in response to exercise. We have found that the levels of most individual skeletal muscle proteins are not immediately altered in response to a short bout of high-intensity exercise and recovery in fasted rats.

In this study we confirmed the presence of the erythropoietin (EPO) receptor on both cultured cortical neurons and PC12 cells and showed that EPO can induce changes in p38, ERK, and JNK signaling molecules in these cells. We induced EPO preconditioning in cortical neuronal cultures that protected neurons from a subsequent in vitro ischemic insult (transient oxygen-glucose deprivation). To investigate downstream changes in protein expression in EPO-preconditioned cortical neuronal cultures, we used two-dimensional gel electrophoresis. Overall, EPO preconditioning resulted in protein up-regulation, and, from 84 of the most differentially expressed proteins selected for identification, the proteins or tentative proteins were identified in 57 cases, representing 40 different proteins. Different protein spots representing the same or closely related protein(s) occurred for 13 of the identified proteins and are likely to represent posttranslational modifications or proteolytic fragments of the protein. Two proteins (78-kD glucose-regulated protein and tropomyosin, fibroblast isoform 1) were detected in control neuronal cultures, but not following EPO preconditioning treatment, whereas one protein (40S ribosomal protein SA) was detected only following EPO preconditioning. Most of the other proteins identified had not previously been associated with EPO preconditioning and will aid in the understanding of EPO's neuroprotective response and possibly the development of new therapeutic interventions to inhibit neuronal death in acute and chronic neurodegenerative diseases. © 2006 Wiley-Liss, Inc.

Proteomic analyses are being increasingly used to identify protein changes accompanying changes in cellular function. An advantage of this approach is that it is largely unbiased by prior assumptions on the importance of each protein in the process under investigation. Here we have evaluated the protein changes that accompany the enlargement, or hypertrophy, of cardiomyocytes in culture. We have taken the additional step of comparing the changes that accompany a concentric hypertrophic phenotype stimulated by endothelin-1 exposure and an eccentric hypertrophic phenotype stimulated by leukemic inhibitory factor exposure. Following separation of the protein extracts by two-dimensional gel electrophoresis and staining with colloidal Coomassie Brilliant Blue, we identified 15 protein spots representing 12 proteins that changed in response to endothelin-1. In comparison, 17 protein spots representing 17 proteins changed in response to leukemic inhibitory factor, and 35 protein spots representing 28 proteins did not change under these conditions. Importantly the well established marker of cardiac pathology, atrial natriuretic factor, was identified as a protein up-regulated by both endothelin-1 and leukemic inhibitory factor (2.4+/-0.8- and 2.2+/-0.3-fold, respectively). However, nine of the observed protein changes occurred for only endothelin-1, whereas 11 of the changes occurred only with leukemic inhibitory factor exposure. These two different stimuli are therefore able to elicit unique changes in the protein expression profile of cardiac myocytes. This is consistent with the differences in morphologies noted as well as the different signaling pathways utilized by these different stimuli.

Background —The aim of the present study was to establish whether isolated neonatal mammalian cardiomyocytes were capable of downregulating energy-using processes other than contraction while maintaining metabolic stability when oxygen availability was reduced. Methods and Results —Metabolic response of cardiomyocytes was investigated under moderate (5 to 6 μmol/L) and severe (2 to 3 μmol/L) forms of hypoxia. Cells exposed to oxygen concentrations of 5 to 6 μmol/L exhibited rates of oxygen consumption, which were decreased to 64% of normoxic rates. Rates of cellular energy usage were decreased because this reduced rate of oxygen consumption was not associated with either decreased intracellular ATP and phosphocreatine concentrations or a compensatory switch to glycolysis. When cells were exposed to oxygen concentrations of 2 to 3 μmol/L, rates of oxygen consumption decreased to 9% of normoxic rates. This decreased rate of oxygen consumption was associated with energetic stress, because a significant switch to glycolysis occurred and intracellular phosphocreatine concentrations were decreased by 40%. Rates of cellular energy usage were further decreased as indicated by stable intracellular ATP concentrations. Conclusions —Our results suggest that isolated cardiomyocytes are capable of downregulating energy-consuming processes other than contraction when oxygen supply is decreased. Regions of myocardial tissue are also capable of downregulating metabolic activity during ischemia by shutting down contractile activity (myocardial hibernation). We suggest that metabolic downregulation associated with myocardial hibernation may not be exclusively due to reduced rates of contractile activity. Other energy-using processes (eg, protein synthesis, mRNA synthesis, ion channel activity, and proton leak) may also be shut down.

Cell culture preparations now play a significant and essential role in physiological and biochemical studies of cell biology. However, the fuels offered in cell culture media are only glucose and glutamine, plus whatever might be in the added sera. It is currently difficult to find a rational way forward on this problem, as there are few data on what fuels cells use in vivo or even in an in vitro physiological situation. A recent study on human platelets redressed the situation somewhat by finding that 75% of ATP turnover could be accounted for by aerobic glycolysis, and by the oxidation of glucose, hydroxybutyrate, acetate, glutamine, palmitate and oleate. In the present study we used a similar strategy to investigate fuel choices by trout thymocytes, cells with a similar function but from a different phylogenetic group. When these cells were presented with a physiological medium, we found that aerobic glycolysis accounted for 9% of total ATP turnover, glucose and glutamine oxidation made a combined contribution of 2.3%, oleate and palmitate oxidation accounted for 15%, and 74% was unaccounted for. These patterns of fuel use are very different from that in human platelets. They demonstrate the cell- and animal-specific nature of cellular metabolism and again expose the inadequacy of the fuel component in culture media.

An enzymatic assay for the determination of nonesterified fatty acid concentrations in milk and plasma is described. The procedure is semiautomated for use with a plate luminometer or plate spectrophotometer and enables routine batch processing of large numbers of small samples (or =5 microL). Following the activation of nonesterified fatty acids (NEFA) by acylCoA synthetase, the current assay utilizes UDP-glucose pyrophosphorylase to link inorganic pyrophosphate to the production of NADH through the reactions catalyzed by phosphoglucomutase and glucose-6-phosphate 1-dehydrogenase. With this assay sequence the formation of NADH from NEFA is complete within 50 min at 37 degrees C. Enzymatic spectrophotometric techniques were unsuitable for NEFA determination in human milk due to the opacity of the sample. The use of the NADH-luciferase system has overcome this problem, allowing the enzymatic determination of NEFA in human milk. Sample collection and treatment procedures for milk and plasma have been developed to prevent enzymatic lipolysis and to limit interference from enzymes present in milk. The recovery of palmitic acid added to milk and plasma samples was 94.9+/-2.9 and 100+/-4.5%, respectively. There was no difference (P = 0.13) in plasma NEFA concentrations determined by the current method and a commercially available enzymatic spectrophotometric technique (Wako NEFA-C kit). Plasma NEFA concentrations determined by gas chromatography were 28% higher compared to both the Wako NEFA-C kit and the current method.

A highly sensitive and simplified method for the luminometric determination of plasma metabolites has been developed. Furthermore, the technique has been automated for the Dynatech ML2250 Microtiter Plate Luminometer and can be applied to the measurement of any plasma metabolite which may be coupled to a reaction involving the reduction of NAD+. Assays are described for lactose/galactose, beta-hydroxybutyrate and D-lactate, and have been validated with plasma samples. The assays require 1-2 microliters of plasma, and are capable of detecting concentrations below 5 mumol/l. Since luminometry is based on the kinetics of the luciferase/oxidoreductase enzyme system, components of complex biological samples may interfere with the rate of the reaction; necessitating the use of internal standards for individual samples. However, the need for internal standards to account for sample to sample variation in the luminescent response, has been eliminated with the present technique.

Reactive oxygen species (ROS) are not only a cause of oxidative stress in a range of disease conditions but are also important regulators of physiological pathways in vivo. One mechanism whereby ROS can regulate cell function is by modification of proteins through the reversible oxidation of their thiol groups. An experimental challenge has been the relative lack of techniques to probe the biological significance of protein thiol oxidation in complex multicellular tissues and organs. We have developed a sensitive and quantitative fluorescence labeling technique to detect and localize protein thiol oxidation in histological tissue sections. In our technique, reduced and oxidized protein thiols are visualized and quantified on two consecutive tissue sections and the extent of protein thiol oxidation is expressed as a percentage of total protein thiols (reduced plus oxidized). We tested the application of this new technique using muscles of dystrophic (mdx) and wild-type C57Bl/10Scsn (C57) mice. In mdx myofibers, protein thiols were consistently more oxidized (19 ± 3%) compared with healthy myofibers (10 ± 1%) in C57 mice. A striking observation was the localization of intensive protein thiol oxidation (70 ± 9%) within myofibers associated with necrotic damage. Oxidative stress is an area of active investigation in many fields of research, and this technique provides a useful tool for locating and further understanding protein thiol oxidation in normal, damaged, and diseased tissues.

To evaluate whether desferrioxamine decreases ischemia and perfusion injury aggravated by cold storage (CS) in a rat liver perfusion model.Isolated rat livers were kept in CS in University of Wisconsin Solution for 20 h at 4 °C, then exposed to 25 min of warm ischemia (WI) at 37 °C followed by 2 h of warm perfusion (WP) at 37 °C with oxygenated (95% oxygen and 5% carbon dioxide) Krebs-Henseleit buffer. Desferrioxamine (DFO), an iron chelator, was added at different stages of storage, ischemia and perfusion: in CS only, in WI only, in WP only, in WI and perfusion, or in all stages. Effluent samples were collected after CS and after WI. Perfusate samples and bile were collected every 30 min (0, 0.5, 1, 1.5 and 2 h) during liver perfusion. Cellular injury was assessed by the determination of lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) in the effluent and perfusate samples. Total iron was analysed in the perfusate samples. After WP, the liver was collected for the determination of liver swelling (wet to dry ratio) and liver morphological examination (hematoxylin and eosin staining).Increased CS time caused increased liver dysfunction during WP. After 2 h of WP, liver injury was indicated by increased release of AST (0.5 h CS: 9.4 ± 2.2 U/g liver vs 20 h CS: 45.9 ± 10.8 U/g liver, P0.05) and LDH (0.5 h CS: 59 ± 14 U/g liver vs 20 h CS: 297 ± 71 U/g liver, P0.05). There was an associated increase in iron release into the perfusate (0.5 h CS: 0.11 ± 0.03 μmoL/g liver vs 20 h CS: 0.58 ± 0.10 μmoL/g liver, P0.05) and reduction in bile flow (0.5 h CS: 194 ± 12 μL/g vs 20 h CS: 71 ± 8 μL/g liver, P0.05). When DFO was added during WI and WP following 20 h of CS, release of iron into the perfusate was decreased (DFO absent 0.58 ± 0.10 μmoL/g liver vs DFO present 0.31 ± 0.06 μmoL/g liver, P0.05), and liver function substantially improved with decreased release of AST (DFO absent 45.9 ± 10.8 U/g liver vs DFO present 8.1 ± 0.9 U/g liver, P0.05) and LDH (DFO absent 297 ± 71 U/g liver vs DFO present 56 ± 7 U/g liver, P0.05), and increased bile flow (DFO absent 71 ± 8 μL/g liver vs DFO present 237 ± 36 μL/g liver, P0.05). DFO was also shown to improve liver morphology after WP. Cellular injury (the release of LDH and AST) was significantly reduced with the addition of DFO in CS medium but to a lesser extent compared to the addition of DFO in WP or WI and perfusion. There was no effect on liver swelling or bile flow when DFO was only added to the CS medium.DFO added during WI and perfusion decreased liver perfusion injury aggravated by extended CS.

Oxidative stress has been implicated in the pathology of the lethal skeletal muscle disease Duchenne muscular dystrophy (DMD), and various antioxidants have been investigated as a potential therapy. Recently, treatment of the mdx mouse model for DMD with the antioxidant and cysteine and glutathione (GSH) precursor n-acetylcysteine (NAC) was shown to decrease protein thiol oxidation and improve muscle pathology and ex vivo muscle strength. This study further investigates the mechanism for the benefits of NAC on dystrophic muscle by administering l-2-oxothiazolidine-4-carboxylate (OTC) which also upregulates intracellular cysteine and GSH, but does not directly function as an antioxidant. We observed that OTC, like NAC, decreases protein thiol oxidation, decreases pathology and increases strength, suggesting that the both NAC and OTC function via increasing cysteine and GSH content of dystrophic muscle. We demonstrate that mdx muscle is not deficient in either cysteine or GSH and that these are not increased by OTC treatment. However, we show that dystrophic muscle of 12 week old mdx mice is deficient in taurine, a by-product of disposal of excess cysteine, a deficiency that is ameliorated by OTC treatment. These data suggest that in dystrophic muscles, apart from the strong association of increased oxidative stress and protein thiol oxidation with dystropathology, another major issue is an insufficiency in taurine that can be corrected by increasing the availability of cysteine. This study provides new insight into the molecular mechanism underlying the benefits of NAC in muscular dystrophy and supports the use of OTC as an alternative drug for potential clinical applications to DMD.

This study was designed to investigate 1) whether a protocol employing a gradual reduction in O2 availability to submaximally contracting muscle results in relatively minor disturbances in intracellular homeostasis and 2) the interaction between tissue oxygenation and the proposed regulators of muscle respiration, metabolism, and force production. O2 delivery to isolated submaximally contracting [isometric contractions at 3 Hz; approximately 50% of peak O2 uptake (VO2)] in situ canine gastrocnemius (n = 6) was manipulated by decreasing arterial PO2 (hypoxemia; H) or muscle blood flow (ischemia; I) during three separate periods in each muscle [control (C), H, or I; each separated by 45 min of rest]. O2 delivery was reduced gradually in small steps every 3 min by H or I during two of the contraction periods (6 steps for a total of 21 min; O2 delivery reduced by 67% by the end of 21 min), whereas C was at normal O2 delivery for a 15-min period. Muscle VO2 was maintained at control levels for the first two O2 delivery reduction steps for the H and I conditions and then fell proportionally with O2 delivery to approximately 35% of the initial value by the end of the 21-min contraction period. Muscle force development generally fell in parallel with VO2. There was no significant changes from the values obtained during C contractions in intracellular concentrations of ATP, phosphocreatine, NH3, calculated free ADP, lactate, and redox state ratios as the O2 delivery was reduced, even with the severe decline in VO2 and developed force. These results demonstrated that when O2 availability was reduced gradually to contracting skeletal muscle, 1) developed force (ATP utilization) was reduced through a tight coupling with aerobic ATP supply, such that there was little additional disruption of intracellular homeostasis, and 2) there was an apparent dissociation of some of the proposed regulators of cell respiration and force development from the control of these processes.

Automated bioluminescent assays for lactate, oxoglutarate, pyruvate, ammonia, NAD, glutamate, ATP, phosphocreatine, creatine, and NADH were developed from existing spectrophotometric and fluorometric assays. A key feature was the development of a luminescent reagent that was not only relatively cheap and stable, but also could be stored frozen. With this reagent it was possible to measure NADH concentrations as low as 0.1 nM in a 25-microliters sample. The sensitivity of the other assays was limited by contamination of the enzymes and cofactors used in the assays and ranged from 0.05 to 25 microM with sample volumes of 0.75 - 10 microliters.

The muscular dystrophies comprise more than 30 clinical disorders that are characterized by progressive skeletal muscle wasting and degeneration. Although the genetic basis for many of these disorders has been identified, the exact mechanism for pathogenesis generally remains unknown. It is considered that disturbed levels of reactive oxygen species (ROS) contribute to the pathology of many muscular dystrophies. Reactive oxygen species and oxidative stress may cause cellular damage by directly and irreversibly damaging macromolecules such as proteins, membrane lipids and DNA; another major cellular consequence of reactive oxygen species is the reversible modification of protein thiol side chains that may affect many aspects of molecular function. Irreversible oxidative damage of protein and lipids has been widely studied in Duchenne muscular dystrophy, and we have recently identified increased protein thiol oxidation in dystrophic muscles of the mdx mouse model for Duchenne muscular dystrophy. This review evaluates the role of elevated oxidative stress in Duchenne muscular dystrophy and other forms of muscular dystrophies, and presents new data that show significantly increased protein thiol oxidation and high levels of lipofuscin (a measure of cumulative oxidative damage) in dysferlin-deficient muscles of A/J mice at various ages. The significance of this elevated oxidative stress and high levels of reversible thiol oxidation, but minimal myofibre necrosis, is discussed in the context of the disease mechanism for dysferlinopathies, and compared with the situation for dystrophin-deficient mdx mice.

Ceroid and lipofuscin are autofluorescent granules thought to be generated as a consequence of chronic oxidative stress. Because ceroid and lipofuscin are persistent in tissue, their measurement can provide a lifetime history of exposure to chronic oxidative stress. Although ceroid and lipofuscin can be measured by quantification of autofluorescent granules, current methods rely on subjective assessment. Furthermore, there has not been any evaluation of variables affecting quantitative measurements. The article describes a simple statistical approach that can be readily applied to quantitate ceroid and lipofuscin. Furthermore, it is shown that several factors, including magnification tissue thickness and tissue level, can affect precision and sensitivity. After optimizing for these factors, the authors show that ceroid and lipofuscin can be measured reproducibly in the skeletal muscle of dystrophic mice (ceroid) and aged mice (lipofuscin).

Oxidative stress is implicated as a factor that increases necrosis of skeletal muscles in Duchenne Muscular Dystrophy (DMD) and the dystrophic mdx mouse. Consequently, drugs that minimize oxidative stress are potential treatments for muscular dystrophy. This study examined the in vivo benefits to mdx mice of an antioxidant treatment with the cysteine precursor N-acetylcysteine (NAC), administered in drinking water. NAC was completely effective in preventing treadmill exercise-induced myofibre necrosis (assessed histologically) and the increased blood creatine kinase levels (a measure of sarcolemma leakiness) following exercise were significantly lower in the NAC treated mice. While NAC had no effect on malondialdehyde level or protein carbonylation (two indicators of irreversible oxidative damage), treatment with NAC for one week significantly decreased the oxidation of glutathione and protein thiols, and enhanced muscle protein thiol content. These data provide in vivo evidence for protective benefits of NAC treatment on dystropathology, potentially via protein thiol modifications.

The extent of muscle pathology in sedentary adult mdx mice is very low and treadmill exercise is often used to increase myofibre necrosis; however, the early events in dystrophic muscle and blood in response to treadmill exercise (leading to myofibre necrosis) are unknown. This study describes in detail two standardised protocols for the treadmill exercise of mdx mice and profiles changes in molecular and cellular events after a single 30 min treadmill session (Protocol A) or after 4 weeks of (twice weekly) treadmill exercise (Protocol B). Both treadmill protocols increased multiple markers of muscle damage. We conclude that a single 30 min treadmill exercise session is a sufficient and conveniently fast screening test and could be used in 'proof-of-concept' studies to evaluate the benefits of pre-clinical drugs in vivo. Myofibre necrosis, blood serum CK and oxidative stress (specifically the ratio of oxidised to reduced protein thiols) are reliable markers of muscle damage after exercise; many parameters demonstrated high biological variation including changes in mRNA levels for key inflammatory cytokines in muscle. The sampling (sacrifice and tissue collection) time after exercise for these parameters is critical. A more precise understanding of the changes in dystrophic muscle after exercise aims to identify biomarkers and new potential therapeutic drug targets for Duchenne Muscular Dystrophy.

Ethanol decreases protein synthesis and mitochondrial respiration of the heart. We developed a new time-saving, non-radioactive, small-scale method to determine ethanol-induced alteration in cellular protein content of the cells cultivated in a 96-well plate. To verify the utility of our small-scale method, we used endothelin-1 (ET-1) as a positive control.Neonatal rat cardiomyocytes were cultivated in a 96-well plate and exposed to ethanol (10, 50 and 100 mM) or ET-1 (100 nM). The protein content per cell was determined by measuring both the DNA (Hoechst 33258) and protein amounts of the same well with fluorometric and spectrometric plate readers. We also examined ultrastructure, mitochondrial membrane potential (deltapsim) using JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide), anaerobic glycolysis measured by lactate amount released to the medium, and superoxide production using dihydroethidium.As expected, ethanol dose- and time-dependently decreased protein content per cell of cardiomyocytes at 24 and 48 h, while ET-1 time-dependently increased it. Ultrastructurally, ethanol decreased rough endoplasmic reticulum at 24 h, whereas ET-1 increased it as well as Golgi apparatus. Among ethanol groups, only 100 mM ethanol affected deltapsim: hyperpolarization just after exposure but slight depolarization at 24 h. ET-1 significantly depolarized deltapsim at 24 h, but the depolarization was within the physiological fluctuation. Ethanol did not change the lactate release at 3 and 24 h. Ethanol did not increase superoxide production at 24 h, but rather 10 mM ethanol significantly decreased it.Our small-scale method was able to demonstrate that ethanol and ET-1 induced expected alterations in protein content per cell with dose- and time-dependencies and statistically significant differences compared with the control, thereby validating the practical use of this method. Ultrastructural alterations supported the biochemical results. The ethanol-decreased cellular protein content might partly result from energy shortage attributable to reduced mitochondrial respiration, which was not compensated by anaerobic glycolysis.

Oxidative stress can result in the reversible oxidation of protein thiols. Because the activity of numerous proteins is sensitive to thiol oxidation, this has the potential to affect many cellular functions. We describe a highly sensitive, quantitative labeling technique that measures global and specific protein thiol oxidative state in skeletal muscle tissue. The technique involves labeling the reduced and oxidized protein thiols with different fluorescent dyes. The resulting sample is assayed using a 96-well plate fluorimeter, or individual protein bands are separated using SDS-PAGE. We show that artifactual oxidation during sample preparation and analysis has the potential to confound results, and techniques to prevent this are described. We tested the technique by analyzing the muscles of mdx and c57 mice and found that the muscles of mdx mice were significantly (p

The capacity of mitochondria to respond to changes in oxygen delivery has the potential to affect the ability of the heart to tolerate decreased oxygen delivery. Respiration by mitochondria is typically regarded as independent of oxygen tension (pO(2)) until critically low oxygen concentrations limit the activity of cytochrome oxidase. Paradoxically, there is evidence that cellular and mitochondrial oxygen consumption (respiration) can decline at oxygen tensions well above this critical pO(2). We tested the hypothesis that oxygen sensitive decreases in mitochondrial hydrogen peroxide production can decrease cardiac mitochondrial respiration rate. Consistent with previous work, an acute decline in pO(2) from 146 mm Hg to 10-13 mm Hg in less than 10 min did not affect mitochondrial respiration rate. In contrast, sustained incubation of mitochondria at a pO(2) of 10-13 mm Hg for 30 min caused a 50% decrease in mitochondrial respiration rate. This decrease in mitochondrial respiration rate was mimicked by incubation with the hydrogen peroxide scavenger catalase and the decrease in mitochondrial respiration rate was fully reversible by reintroducing oxygen or by adding hydrogen peroxide. Incubation at low pO(2) was also associated with a decreased rate of mitochondrial reactive oxygen species production. These findings indicate that oxygen-dependent decreases in the rate of mitochondrial hydrogen peroxide production can decrease cardiac mitochondrial respiration.

SummaryChanges in the concentrations of glucose and galactose were measured in the peripheral blood of ten piglets after they had ingested milk during a natural sucking. In addition, the mild stress associated with the experimental procedure was determined by sampling nine fasted piglets over a period of 9 to 12 min. During this period there was a significant increase in the concentration of glucose in the blood of the piglets but no change in the concentration of galactose. After milk ingestion during a natural sucking the concentrations of both glucose and galactose increased from 5·7 mM and 19 μM to reach peak values of 7·7 mM and 122 μM, respectively, by 30 to 35 min. The concentrations of glucose and galactose returned to initial values in 60–80 min and 80–100 min, respectively, after sucking. Since the change in the concentration of galactose in the peripheral blood was much lower than the change in the concentration of glucose, we conclude that galactose was rapidly removed by the livers of sucking piglets. However, after the ingestion of milk the percentage increase (from initial to peak values) in the concentration of galactose in the blood was much larger (650%) than the increase in the concentration of glucose (43%). Thus, we propose that the determination of galactose in the peripheral blood may provide a qualitative method for monitoring the digestion and absorption of milk lactose in sucking piglets.

Activation of the ERK mitogen-activated protein kinase pathway has been implicated in pro-survival and cellular protective mechanisms, so that chronic ERK activation may be a useful therapeutic strategy. Here, we further explored the consequences of prolonged ERK activation following expression of constitutively active form of MEK, MEK-EE, in cardiac myocytes. We confirmed that chronic MEK-EE overexpression halved myocyte death following glucose deprivation, but surprisingly this was not associated with preserved intracellular ATP levels. Whilst activities of a number of antioxidant enzymes were not altered upon MEK-EE expression, paradoxically Cu/Zn superoxide dismutase activity was almost halved upon MEK-EE expression. When we then exposed myocytes to the superoxide generator menadione, we observed significantly higher death of MEK-EE expressing myocytes. Pre-incubation with U0126 inhibited menadione-induced death. Our results are the first to show that MEK-ERK signalling can act to increase or decrease cell survival, the outcome depending on the form of stress stimulus encountered.

In order to investigate protein function in rat primary cortical neuronal cultures, we modified an adenoviral vector expression system and assessed the strength and specificity of the cytomegalovirus (CMV), rous sarcoma virus (RSV), and rat and human synapsin 1 (SYN1) promoters to drive DsRed-X expression. We also incorporated the woodchuck post-transcriptional regulatory element (WPRE) and a CMV promoter-enhanced green fluorescent protein (EGFP) reporter cassette. We observed that the RSV promoter activity was strong in neurons and moderate in astrocytes, while the CMV promoter activity was weak-to-moderate in neurons and very strong in astrocytes. The rat and human SYN1 promoters exhibited similar but weak activity in neurons, despite inclusion of the WPRE. We confirmed that the WPRE enhanced RSV promoter-mediated DsRed-X expression in a time-dependent fashion. Interestingly, we observed very weak SYN1-mediated DsRed-X expression in astrocytes and HEK293 cells suggesting incomplete neuronal-restrictive behavior for this promoter. Finally, using our adenoviral expression system, we demonstrated that RSV promoter-mediated Bcl-X(L) overexpression attenuated neuronal death caused by in vitro ischemia and oxidative stress.

Steroid receptor RNA activator (SRA), the only known RNA coactivator, augments transactivation by nuclear receptors (NRs). We identified SLIRP (SRA stem-loop interacting RNA binding protein) binding to a functional substructure of SRA, STR7. SLIRP is expressed in normal and tumor tissues, contains an RNA recognition motif (RRM), represses NR transactivation in a SRA- and RRM-dependent manner, augments the effect of Tamoxifen, and modulates association of SRC-1 with SRA. SHARP, a RRM-containing corepressor, also binds STR7, augmenting repression with SLIRP. SLIRP colocalizes with SKIP (Chr14q24.3), another NR coregulator, and reduces SKIP-potentiated NR signaling. SLIRP is recruited to endogenous promoters (pS2 and metallothionein), the latter in a SRA-dependent manner, while NCoR promoter recruitment is dependent on SLIRP. The majority of the endogenous SLIRP resides in the mitochondria. Our data demonstrate that SLIRP modulates NR transactivation, suggest it may regulate mitochondrial function, and provide mechanistic insight into interactions between SRA, SLIRP, SRC-1, and NCoR.

The role of NAD(P)H oxidase in regulating cellular production of reactive oxygen species (ROS) and the L-type Ca2+ channel during acute hypoxia was examined in adult ventricular myocytes from guinea pig.The fluorescent indicator dihydroethidium (DHE) was used to detect superoxide and the response of the L-type Ca2+ channel to beta-adrenergic receptor stimulation was used as a functional reporter since hypoxia increases the sensitivity of the L-type Ca2+ channel (I(Ca-L)) to isoproterenol (Iso).Hypoxia caused a 41.2+/-5.2% decrease in the rate of the DHE signal (n=21; p0.01). Of the classical NAD(P)H oxidase inhibitors, DPI but not apocynin mimicked the effect of hypoxia on the sensitivity of I(Ca-L) to Iso. However, the potent NAD(P)H oxidase agonist angiotensin II had no effect on cellular superoxide or the sensitivity of I(Ca-L) to Iso. Although DPI inhibits NAD(P)H oxidase, it also decreased superoxide in isolated mitochondria in a concentration-dependent manner. Partial inhibition of mitochondrial function with nanomolar concentrations of FCCP or myxothiazol mimicked the effect of hypoxia on cellular superoxide and the sensitivity of I(Ca-L) to Iso. In addition, hypoxia caused a 69.3+/-0.8% decrease in superoxide in isolated mitochondria (n=4; p0.01), providing direct evidence for a role for the mitochondria.Our data suggest that mitochondria appear to be involved in oxygen sensing, regulation of cellular ROS, and the function of I(Ca-L) during acute hypoxia in cardiac myocytes and NAD(P)H oxidase does not appear to contribute substantially.

Preconditioning describes a variety of treatments that induce neurons to become more resistant to a subsequent ischemic insult. How preconditioned neurons adapt to subsequent ischemic stress is not fully understood, but is likely to involve multiple protective mechanisms. We hypothesized hypoxic preconditioning induces protection by a coordinated up-regulation of antioxidant enzyme activity. To test this hypothesis, we developed two in vitro models of ischemia/reperfusion, involving oxygen-glucose deprivation (OGD) where neuronal cell death was predominantly by necrosis (necrotic model) or programmed cell death (PCD model). Hypoxic preconditioning 24 h prior to OGD significantly reduced cell death from 83% to 22% in the necrotic model and 68% to 11% in the PCD model. Consistent with the hypothesis, the activity of the antioxidant enzymes glutathione peroxidase, glutathione reductase, and Mn superoxide dismutase were significantly increased by 54%, 73% and 32%, respectively, in neuronal cultures subjected to hypoxic preconditioning. Furthermore, superoxide and hydrogen peroxide concentrations following OGD were significantly lower in the PCD model that had been subjected to hypoxic preconditioning.

It has been widely hypothesized that neurons reduce cellular energy use in response to periods of energy deprivation. To test this hypothesis, we measured rates of energy use under normoxia and anoxia in immature (6 days in vitro) and mature (13 days in vitro) neuronal cultures. During anoxic incubation immature and mature cultures reduced cellular energy use by 80% and 45%, respectively. Reduced cellular energy use dramatically affected ATP depletion in neuronal cultures under anoxia. Intracellular ATP stores were expected to deplete within 3 min of anoxia. However, ATP was maintained at decreased but stabilized concentrations for at least 3 h. The capacity of neuronal cultures to reduce cellular energy use during anoxia correlated with their sensitivity towards simulated ischemia. Immature cultures, with the largest capacity to reduce cellular energy use, survived simulated ischemia 2.5 times longer than mature cultures. The addition of glutamate receptor antagonists to mature cultures further decreased cellular energy use during anoxia and significantly extended their survival time under simulated ischemia. This study verifies that primary cortical neuronal cultures reduce cellular energy use during energy deprivation. Additionally, we show that maturation of glutamate receptor activity increases non-depressible energy demand in neuronal cultures.

In cardiac myocytes, hypoxia inhibits the basal L-type Ca 2+ current ( I Ca-L ) and increases the sensitivity of I Ca-L to β-adrenergic receptor stimulation. We investigated whether hydrogen peroxide (H 2 O 2 ) is involved in the hypoxic response. Guinea pig ventricular myocytes were dialyzed with catalase, which specifically catalyzes the conversion of H 2 O 2 to H 2 O and oxygen, and then I Ca-L was recorded during exposure to isoproterenol (Iso). Catalase decreased the K 0.5 for activation of I Ca-L by Iso from 2.7±0.3 nmol/L (in cells dialyzed with heat-inactivated catalase) to 0.4±0.1 nmol/L. The increase in sensitivity to Iso by catalase may be attenuated when cells are preexposed to H 2 O 2 . A significant increase in sensitivity of I Ca-L to Iso was recorded when mitochondrial function was inhibited with myxothiazol or FCCP, suggesting that the source of H 2 O 2 was from the mitochondria. Prior exposure of cells to H 2 O 2 attenuated the inhibition of basal I Ca-L during hypoxia and the increase in sensitivity of I Ca-L to Iso during hypoxia. Additionally, extracellularly applied catalase mimicked the effect of hypoxia on basal I Ca-L . Measurement of the rate of production of hydrogen peroxide using 5- (and 6-)chloromethyl-2′, 7′-dichlorodihydrofluorescein diacetate acetyl ester indicated that hypoxia was associated with a significant decrease in the production of hydrogen peroxide in the cells. These data suggest that hypoxia mediates changes in channel activity through a lowering in H 2 O 2 levels and that H 2 O 2 is a key intermediate in modifying basal channel activity and the β-adrenergic responsiveness of the channel during hypoxia.

The aim was to identify energy-consuming processes, other than contraction, downregulated during moderate hypoxia (≈5 μmol/L, 0.5% O 2 ) and severe hypoxia (2 ) in isolated neonatal cardiomyocytes. The metabolic response of cardiomyocytes to moderate and severe hypoxia was assessed by measuring rates of energy consumption and energetic status of cells maintained under these conditions. We found that the rates of energy production were decreased during both forms of hypoxia. Decreased rates of energy production under moderate hypoxia were associated with reduced energy wastage through a downregulation of proton leak in the mitochondria. Cellular protein synthesis and RNA synthesis, major energy-consuming pathways, were downregulated only during severe hypoxia, when oxygen concentrations were low enough to induce energetic stress (quantitatively defined as being any situation in which phosphocreatine concentrations had fallen by ≥40%). Our results suggest that energetic stress is the signal responsible for this downregulation.

Oxygen conformance can be described as the ability to reduce energy demand, and hence oxygen consumption, in response to a decline in oxygen availability without a decrease in the concentration of ATP. It has been proposed that oxygen conformance may enhance cellular survival at low oxygen concentrations. We demonstrate that non-contracting C2C12 cells, a mouse skeletal muscle cell line, are capable of oxygen conformance. Typically, we found oxygen consumption to decline by 30-40% as the concentration of oxygen was reduced from 100 microM to 10 microM. Unexpectedly, the rate of protein synthesis, a major energy consumer in the cell, did not decrease significantly during oxygen conformance. Unlike oxygen conformance, severe hypoxia (0.5 microM) caused a 36% decline in the concentration of PCr, and under these conditions of energy stress, the rate of protein synthesis declined by 43%. We conclude that there are two distinct metabolic responses to declines in oxygen concentration in non-contracting C2C12 cells.

Many cellular metabolites can be measured with high sensitivity using bioluminescent techniques. These metabolites are coupled to an appropriate enzyme to produce NAD(P)H, which can then be coupled to the bioluminescent reactions. The sensitivity of bioluminescence cannot be readily applied to methods in which cellular metabolites consume NAD(P)H because of the difficulty in measuring, with sufficient sensitivity, decreases in the concentration of NAD(P)H against a high background NAD(P)H concentration. We have overcome these technical difficulties by developing a bioluminescent reagent to measure the production of NAD(P) + . Assays for creatine/creatine phosphate, pyruvate, and succinate, as well as the kinetic measurement of lactate, are described for a range of biological material. The assays are highly sensitive, quantitative, and reproducible and show no sample-specific inhibition. The range of assays and the diverse biological material tested suggests that NAD(P) + bioluminescence has a wide potential for application.