Mitochondrial capacity and reactive oxygen species production during hypoxia and reoxygenation in the ocean quahog, Arctica islandica

Oxygen fluctuations are common in marine waters, and hypoxia/reoxygenation (H/R) stress can negatively affect mitochondrial metabolism. The long-lived ocean quahog, Arctica islandica, is known for its hypoxia tolerance associated with metabolic rate depression, yet the mechanisms that sustain mitochondrial function during oxygen fluctuations are not well understood. We used top-down metabolic control analysis (MCA) to determine aerobic capacity and control over oxygen flux in the mitochondria of quahogs exposed to short-term hypoxia (24 h <0.01% O­2) and subsequent reoxygenation (1.5 h 21% O­2) compared to normoxic control animals (21% O­2). We demonstrated that flux capacities of the substrate oxidation and proton leak subsystems were not affected by hypoxia, while the capacity of the phosphorylation subsystem was enhanced during hypoxia associated with a depolarization of the mitochondrial membrane. Reoxygenation decreased oxygen flux capacities of all three mitochondrial subsystems. Control over oxidative phosphorylation (OXPHOS) respiration was mostly exerted by substrate oxidation regardless of H/R stress, whereas the control of the proton leak subsystem over LEAK respiration increased during hypoxia and returned to normoxic level during reoxygenation. During hypoxia, reactive oxygen species (ROS) efflux was elevated in the LEAK state, while suppressed in the OXPHOS state. Mitochondrial ROS efflux returned to normoxic control levels during reoxygenation. Thus, mitochondria of A. islandica appear robust to hypoxia by maintaining stable substrate oxidation and upregulating phosphorylation capacity, but remain sensitive to reoxygenation. This mitochondrial phenotype might reflect adaptation of A. islandica to environments with unpredictable oxygen fluctuations and its behavioural preference for low oxygen levels.

The Metabolomic Response of Crucian Carp (Carassius carassius) to Anoxia and Reoxygenation Differs between Tissues and Hints at Uncharacterized Survival Strategies

The anoxia-tolerant crucian carp (Carassius carassius) has been studied in detail for numerous years, with particular focus on unravelling the underlying physiological mechanisms of anoxia tolerance. However, relatively little work has been focused on what occurs beyond anoxia, and often the focus is a single organ or tissue type. In this study, we quantified more than 100 metabolites by capillary electrophoresis-mass spectrometry (CE-MS) in brain, heart, liver, and blood plasma from four experimental groups, being normoxic (control) fish, anoxia-exposed fish, and two groups that had been exposed to anoxia followed by reoxygenation for either 3 h or 24 h. The heart, which maintains cardiac output during anoxia, unexpectedly, was slower to recover compared to the brain and liver, mainly due to a slower return to control concentrations of the energy-carrying compounds ATP, GTP, and phosphocreatine. Crucian carp accumulated amino acids in most tissues, and also surprisingly high levels of succinate in all tissues investigated during anoxia. Purine catabolism was enhanced, leading to accumulation of uric acid during anoxia and increasing urea formation that continued into 24 h of reoxygenation. These tissue-specific differences in accumulation and distribution of the metabolites may indicate an intricate system of transport between tissues, opening for new avenues of investigation of possible mechanisms aimed at reducing the generation of reactive oxygen species (ROS) and resultant tissue damage during reoxygenation.

Hyperoxygenation With Cardiopulmonary Resuscitation and Targeted Temperature Management Improves Post–Cardiac Arrest Outcomes in Rats

Background Oxygen plays a pivotal role in cardiopulmonary resuscitation (CPR) and postresuscitation intervention for cardiac arrest. However, the optimal method to reoxygenate patients has not been determined. This study investigated the effect of timing of hyperoxygenation on neurological outcomes in cardiac arrest/CPR rats treated with targeted temperature management. Methods and Results After induction of ventricular fibrillation, male Sprague‐Dawley rats were randomized into 4 groups (n=16/group): (1) normoxic control; (2) O 2 _CPR, ventilated with 100% O 2 during CPR; (3) O 2 _CPR+postresuscitation, ventilated with 100% O 2 during CPR and the first 3 hours of postresuscitation; and (4) O 2 _postresuscitation, ventilated with 100% O 2 during the first 3 hours of postresuscitation. Targeted temperature management was induced immediately after resuscitation and maintained for 3 hours in all animals. Postresuscitation hemodynamics, neurological recovery, and pathological analysis were assessed. Brain tissues of additional rats undergoing the same experimental procedure were harvested for ELISA‐based quantification assays of oxidative stress–related biomarkers and compared with the sham‐operated rats (n=6/group). We found that postresuscitation mean arterial pressure and quantitative electroencephalogram activity were significantly increased, whereas astroglial protein S100B, degenerated neurons, oxidative stress–related biomarkers, and neurologic deficit scores were significantly reduced in the O 2 _CPR+postresuscitation group compared with the normoxic control group. In addition, 96‐hour survival rates were significantly improved in all of the hyperoxygenation groups. Conclusions In this cardiac arrest/CPR rat model, hyperoxygenation coupled with targeted temperature management attenuates ischemia/reperfusion‐induced injuries and improves survival rates. The beneficial effects of high‐concentration oxygen are timing and duration dependent. Hyperoxygenation commenced with CPR, which improves outcomes when administered during hypothermia.

Intermittent hypoxia promotes functional neuroprotection from retinal ischemia in untreated first-generation offspring

ABSTRACTEnvironmental stimuli can promote short- or long-lasting changes in phenotype through epigenetics. Under certain circumstances, induced phenotypes can be passed through the germline to subsequent generations, providing a novel mechanistic basis for disease heritability. In the present study, we tested the hypothesis that repetitively exposing parents to a nonharmful epigenetic stimulus can promote disease resilience in offspring. Male and female mice were mated following brief exposures to mild systemic hypoxia every other day for 16 weeks. Electroretinographic determinations of postischemic function in response to transient unilateral retinal ischemia in their 5-month-old F1 progeny revealed significant resilience to injury relative to animals derived from normoxic control parents. Mass spectrometry identified hundreds of differentially expressed proteins between protected and injured retinae; bioinformatic analyses of the pathways and networks these proteins comprise provided specific mechanistic insights into the molecular manifestation of this injury-resilient phenotype. Thus, epigenetics can modify heritability to promote disease resilience.

Effects of Exogenous Neuroglobin (Ngb) on retinal inflammatory chemokines and microglia in a rat model of transient hypoxia

Neuroglobin is an endogenous neuroprotective protein. We determined the safety of direct delivery of Neuroglobin in the rat retina and its effects on retinal inflammatory chemokines and microglial during transient hypoxia. Exogenous Neuroglobin protein was delivered to one eye and a sham injection to the contralateral eye of six rats intravitreally. Fundus photography, Optical Coherence Topography, electroretinogram, histology and Neuroglobin, chemokines level were determined on days 7 and 30. Another 12 rats were subjected to transient hypoxia to assess the effect of Neuroglobin in hypoxia exposed retina by immunohistochemistry, retinal Neuroglobin concentration and inflammatory chemokines. Intravitreal injection of Neuroglobin did not incite morphology or functional changes in the retina. Retinal Neuroglobin protein was reduced by 30% at day 7 post hypoxia. It was restored to normoxic control levels with intravitreal exogenous Neuroglobin injections and sustained up to 30 days. IL-6, TNFα, IL-1B, RANTES, MCP-1 and VEGF were significantly decreased in Neuroglobin treated hypoxic retinae compared to non-treated hypoxic controls. This was associated with decreased microglial activation in the retina. Our findings provide proof of concept suggesting intravitreal Neuroglobin injection is non-toxic to the retina and can achieve the functional level to abrogate microglial and inflammatory chemokines responses during transient hypoxia.

OR-026 Exercise induces HIF-1α redistribution in the small intestine

Objective Intestinal epithelial cells are positioned between an anaerobic lumen and a highly metabolic lamina propria, affected by reduced blood flow and tissue hypoxia. Exercise induces blood flow redistribution, leading to hypoperfusion and gastrointestinal (GI) compromise. The hypoxia-inducible factor (HIF) 1α is pivotal in the transcriptional response to oxygen flux. In this study, we hypothesized that exercise induces GI system hypoxia and accumulates HIF-1α. Methods (1) ROSA26 ODD-Luc/+ mouse model (ODD-Luc) was used to detect HIF-1α expression in the intestine (female, 8-week, n=6/group). ODD-Luc mice were randomized into 4 groups: stayed in 21% O2 as the normoxic control (C), exercise (E), injected HIF-1α inhibitor PX-478 before swimming (PS), placed in the chamber containing 9% O2 for 4 hours as the positive control (PC). (2) Exercise models were conducted by volume: Moderate Exercise (ME): mice voluntarily swam for 30 min; Heavy-intensity Exercise (HE): mice swam for 1.5 hours with 5% body weight loads attached to their tails; Long-time Exercise (LE): mice voluntarily swam for 3 hours or till fatigue. Results (1) Exercise increased HIF-1α in the abdominal area. The luciferase activities boosted after exercise, compared to the controls (ME v.s. C, P<0.05; HE v.s. C, P<0.05; LE v.s. C, P<0.05) but no differences among three exercise groups (ME v.s. HE, P>0.99; ME v.s. LE, P>0.99; HE v.s. LE, P>0.99); (2) Exercise altered HIF-1α distribution in the small intestine in a time-dependent manner. The expression of HIF-1α was significantly increased after exercise and gradually reduced to the rest level. The photons increased at the 0th hour after exercise compared to that of the normoxic control (P<0.01). The level of photons then reduced over time, while the 2nd, 4th and 6th hour post-exercise were still greater than that of the normoxic control (2nd hour v.s. C, P<0.01; 4th hour v.s. C, P<0.01; 6th hour v.s. C, P<0.05), and returned to normal after 24 hours (24th hour v.s. C, P>0.99). Conclusions Exercise induced the distribution of HIF-1α in the small intestine. The expression of HIF-1α is shown in a time-dependent manner after exercise.

Oxygen Sensing By Succinate Dehydrogenase Regulates Transcriptional Response to Hypoxia in Monocytes

Monocytes are recruited from peripheral blood to inflamed tissues during infections and wound healing. Monocyte adaptation to hypoxia is essential in inflamed tissues which are characterized by low oxygen tensions. Monocytes respond to hypoxia by widespread changes in transcript levels. We have also recently described that hypoxia induces site-specific C>U RNA editing of hundreds of genes in monocytes, which is mediated by the innate antiviral restriction factor APOBEC3A (A3A) cytidine deaminase. However, mechanisms regulating hypoxia-sensing and signaling in monocytes are poorly understood. Here we show that inhibition of mitochondrial complex II (succinate dehydrogenase; SDH) by the ubiquinone-analogs atpenin A5 (AA5) or TTFA induces hypoxic gene expression and A3A-mediated RNA editing in normoxic cultures of monocytes. RNA seq and RNA editing analyses show that normoxic treatment of monocytes with AA5 closely mimics the hypoxia effects in inducing transcriptome-scale changes (Figure 1). RT-qPCR validation experiments confirm that normoxic treatment of monocytes with AA5 induces A3A-mediated RNA editing and causes upregulation of many hypoxia-related and pro-angiogenic genes including VEGFA, CXCL8 (IL8) and CXCR4. Treatment of monocytes with AA5 in normoxia further enhances the interferon-induced cytidine deaminase enzyme activity of A3A. AA5 does not inhibit A3A-mediated RNA editing or transcript induction in hypoxic monocytes. In contrast, myxothiazol, a complex III inhibitor, does not induce A3A-mediated RNA editing in normoxia and inhibits it in hypoxia. Thus, monocyte response to hypoxia is specifically triggered by inactivation of mitochondrial complex II. Previous studies on familial paragangliomas, pseudohypoxic highly vascularized neuroendocrine tumors which are associated with germ line SDH mutations, have suggested stabilization of HIF1 alpha in mediating the hypoxia-induced gene expression. In contrast, we find that normoxic treatment with AA5 does not stabilize HIF-1 alpha or HIF-2 alpha in monocytes or in 293T embryonic kidney cells (Figure 2). Moreover, forced stabilization of HIF-1 alpha by treatment of monocytes with DMOG blocks hypoxic induction of A3A-mediated RNA editing. Treatment of monocytes with small molecule inhibitor of HIF-1 alpha (CAY10585) or with an HIF-2 alpha antagonist (SML0883) does not inhibit hypoxic induction of A3A-mediated RNA editing. Taken together, our findings identify complex II inhibitor AA5 as a hypoxia mimetic that enhances the interferon-inducible RNA editing activity of the antiviral enzyme A3A in monocytes and suggest that oxygen sensing by SDH controls transcriptional responses to hypoxia through a distinct signaling pathway that does not involve HIF-1 alpha or HIF-2 alpha. Figure 1. Heat map shows that the most differentially expressed genes (listed on the right) between normoxic control (c1,c2,c3) and hypoxic (1% O2) monocytes (h1,h2,h3) (defined as FDR<0.05, log2 fold change >3 and average expression >6 cpm) also show similar changes in normoxic monocytes exposed to AA5 (a1, a2, a3). (green high/red low expression). Figure 1. Heat map shows that the most differentially expressed genes (listed on the right) between normoxic control (c1,c2,c3) and hypoxic (1% O2) monocytes (h1,h2,h3) (defined as FDR<0.05, log2 fold change >3 and average expression >6 cpm) also show similar changes in normoxic monocytes exposed to AA5 (a1, a2, a3). (green high/red low expression). Figure 2. Western blots show that in contrast to DMOG (1 mM) and DFO (0.5 mM), which are known to stabilize HIF-1 alpha, atpenin A5 (1 μM) does not stabilize HIF-1 alpha in normoxia in 293T cells. Actin bands are loading controls. Figure 2. Western blots show that in contrast to DMOG (1 mM) and DFO (0.5 mM), which are known to stabilize HIF-1 alpha, atpenin A5 (1 μM) does not stabilize HIF-1 alpha in normoxia in 293T cells. Actin bands are loading controls. Disclosures No relevant conflicts of interest to declare.

217 THE EXPRESSION OF β-OXIDATION RELATING GENES UNDER HYPOXIC STRESS IN HUMAN PLACENTAL CHORIOCARCINOMA CELL (BeWo)

Preeclampsia (PE) is thought in many cases to be caused by a shallowly implanted placenta that becomes hypoxic. Hypoxia can result from a failure at any stage in the delivery of oxygen to the cells. In peripheral tissues, oxygen diffuses down a pressure gradient into cells and moves into their mitochondria, where it is used to produce energy in conjunction with the breakdown of glucose, fats, and some amino acids. The aim of the study was to investigate the question of whether hypoxic stress is involved in β-oxidation of human placental BeWo cells. One of the β-oxidation related genes, ACADVL was detected by gene-fishing technology using the preeclamptic placenta of human. We conducted in vitro experiments to confirm a preliminary study by inducing hypoxic stress in the human placental BeWo cell. BeWo cells were cultured at 37°C in a 20% O2, 5% CO2 humidified tissue culture incubator. And then we induced hypoxic stress in BeWo cell cultured under 1% O2, 5% CO2, and balanced with N2. The BeWo cells were divided into three groups: normoxia, hypoxia 24 h, and hypoxia 48 h. The expression of β-oxidation related genes (ACADVL, EHHADH, HADH, ACAA) were observed under hypoxic condition in BeWo cells by using real-time RT-PCR. Relative quantification with HPRT1 was based on the comparison of CT at a constant fluorescent intensity. The amount of transcript is inversely related to the observed CT, and for every 2-fold dilution in the transcript, CT is expected to increase by 1. Relative expression was calculated using the equation R = 2–(CTsample – CTcontrol). Data were analysed with a nonparametric one-way ANOVA, followed by Tukey's test for multiple comparisons. All statistical analyses were performed using GraphPad Prism software (v 4.0, GraphPad Software, La Jolla, CA, USA). P-values <0.05 were considered statistically significant. The expression of a gene known as a biomarker for hypoxia, HIF-1a, was significantly increased in BeWo cells which induced preeclampsia. The elevated level of HIF-1a indicates that our experimental conditions closely mimicked preeclampsia. The β-oxidation related genes, ACADVL, EHHADH, and HADH expressions were significantly increased under hypoxic condition in BeWo compared with normoxic control. These results indicate that changes of β-oxidation related genes observed under hypoxic BeWo cells are similar to ones associated with preeclampsia, and the expression of β-oxidation related genes were up-regulated by hypoxic stress. They might be involved in pathogenesis of preeclampsia during gestation. Taken together, increase of β-oxidation-related genes under hypoxic stress may cause a compensation of energy metabolism deficiency through lipid metabolism.

Hypobaric hypoxia causes deleterious effects on spermatogenesis in rats

The study was conducted to explore the effects of hypobaric hypoxia on spermatogenesis in rats. Adult male Wistar rats were randomly divided into four groups: three hypoxia-exposed groups and one normoxic control group. Rats in the normoxic control group were raised at an altitude of 300 m, while rats in the 5-, 15-, and 30-day hypoxic groups were raised in a hypobaric chamber simulating a high altitude of 5000 m for 5, 15, and 30 days respectively. Flow cytometry was used to detect the DNA content of testicular spermatogenic cells in rats. The apoptosis of germ cells in testis was analyzed by using TUNEL assay. Spermatogenesis was also evaluated by morphology. Flow cytometry analysis revealed that 5–30 days of hypobaric hypoxia exposure significantly reduced the percentage of tetraploid cell population in rat testis. After rats were exposed to hypobaric hypoxia for 30 days, the ratio of haploid and diploid cell populations in testis reduced significantly. Seminiferous tubules with apoptotic germ cell increased after exposure to hypoxia. Most apoptotic germ cells were spermatogonia and spermatocytes. Hypoxia also caused decrease of cellularity of seminiferous epithelium, degeneration and sloughing of seminiferous epithelial cells occasionally. The data suggest that hypobaric hypoxia inhibits the spermatogenesis in rats. Decrease of tetraploid spermatogenic cells (primary spermatocytes) induced by hypoxia is an important approach to suppress spermatogenesis. The apoptosis of primary spermatocytes and spermatogonia may contribute to the loss of tetraploid cell populations.

Genomic assessment of a multikinase inhibitor, sorafenib, in a rodent model of pulmonary hypertension

Pulmonary hypertension (PH) and cancer pathology share growth factor- and MAPK stress-mediated signaling pathways resulting in endothelial and smooth muscle cell dysfunction and angioproliferative vasculopathy. In this study, we assessed sorafenib, an antineoplastic agent and inhibitor of multiple kinases important in angiogenesis [VEGF receptor (VEGFR)-1–3, PDGF receptor (PDGFR)-β, Raf-1 kinase] as a potential PH therapy. Two PH rat models were used: a conventional hypoxia-induced PH model and an augmented PH model combining dual VEGFR-1 and -2 inhibition (SU-5416, single 20 mg/kg injection) with hypoxia. In addition to normoxia-exposed control animals, four groups were maintained at 10% inspired O2fraction for 3.5 wk (hypoxia/vehicle, hypoxia/SU-5416, hypoxia/sorafenib, and hypoxia/SU-5416/sorafenib). Compared with normoxic control animals, rats exposed to hypoxia/SU-5416 developed hemodynamic and histological evidence of severe PH while rats exposed to hypoxia alone displayed only mild elevations in hemodynamic values (pulmonary vascular and right ventricular pressures). Sorafenib treatment (daily gavage, 2.5 mg/kg) prevented hemodynamic changes and demonstrated dramatic attenuation of PH-associated vascular remodeling. Compared with normoxic control rats, expression profiling (Affymetrix platform) of lung RNA obtained from hypoxia [false discovery rate (FDR) 6.5%]- and hypoxia/SU-5416 (FDR 1.6%)-challenged rats yielded 1,019 and 465 differentially regulated genes (fold change >1.4), respectively. A novel molecular signature consisting of 38 differentially expressed genes between hypoxia/SU-5416 and hypoxia/SU-5416/sorafenib (FDR 6.7%) was validated by either real-time RT-PCR or immunoblotting. Finally, immunoblotting studies confirmed the upregulation of the MAPK cascade in both PH models, which was abolished by sorafenib. In summary, sorafenib represents a novel potential treatment for severe PH with the MAPK cascade a potential canonical target.