Normoxic Controls Research Articles

Changes in expression of genes related to glucose metabolism in liver and skeletal muscle of rats exposed to acute hypoxia

The aim of this study was to determine changes in gene expression associated with glucose metabolism in the liver and soleus muscles of rats exposed to hypoxia to improve work capacity under high altitude conditions. Rats were divided into normobaric normoxia (control) and normobaric hypoxia (hypoxia) groups (n = 7 each), and the hypoxia group was exposed to 10.5% oxygen for 90 min. Glucose metabolism-related gene expression was examined by real-time polymerase chain reaction. In the liver, the expression levels of the glucose utilization-related genes solute carrier family 2 member 1, glucokinase, and liver-type phosphofructokinase and the gluconeogenesis-related gene phosphoenolpyruvate carboxykinase 1 (Pck1) were significantly increased upon hypoxic exposure. In contrast, gene expression in the soleus was unchanged, with the exception of Pck1. The results suggest that under hypoxia, both glucose utilization and gluconeogenesis are accelerated in the liver, and liver glycogen is degraded to maintain blood glucose level.

The Effects Of Exercise Training In Hyperoxia Compared To Normoxia On Cardiorespiratory Fitness: A Meta-analysis

PURPOSE: Previous studies have indicated that supplemental oxygen (i.e. hyperoxia) may acutely enhance exercise performance, providing benefits for patients with limited physical capacity. However, the evidence for superior long-term adaptations of regular hyperoxic exercise in clinical populations is limited. Therefore, this systematic review and meta-analysis aimed to evaluate the effects of chronic exercise in hyperoxia compared to normoxia in patients diagnosed with chronic diseases. METHODS: Databases were systematically searched for randomized controlled trials in accordance with PRISMA until May 20th, 2019. Eligibility criteria included adult patients with various chronic diseases (i.e. cardiovascular, pulmonary, neurological, metabolic, musculoskeletal or cancer) engaging in regular supervised exercise training in hyperoxia compared to an exercising normoxic control group. The outcome of interest included maximal power output (PO), peak oxygen consumption (VO2peak) and maximal distance in the 6-minute walk test (6MWD). Standardized mean differences (SMD) were calculated and a random-effects model was used to pool effect sizes using R (3.6.1). RESULTS: Out of the identified 4038 studies, 11 articles were deemed eligible. A total of 132 patients (64.3 ± 3.2 yrs., 36% women) and 131 patients (64.9 ± 3.6 yrs., 33% women) were included in hyperoxia and normoxia groups, respectively. The majority of patients were diagnosed with chronic obstructive pulmonary disease (COPD, 94.1%) while 18 patients were diagnosed with coronary artery disease. The average duration of interventions was 8.9 ± 5.1 weeks. The observed effects for PO (SMD -0.33; 95% CI -0.67, 0.01; p = 0.06), VO2peak (SMD -0.24; 95% CI -0.54, 0.05; p = 0.11) and 6MWD (SMD -0.21; 95% CI -0.78, 0.36; p = 0.46) showed no statistical significant difference between the two conditions. CONCLUSION: There is no evidence for beneficial chronic adaptations in cardiorespiratory fitness, when exercise is performed in hyperoxia. However, the studies to date have only examined patients diagnosed with COPD and coronary artery disease. Future studies should identify the optimal dose-response mechanisms to bridge the gap between acute responses and chronic adaptations in hyperoxic exercise.

Mechanisms of opioid-induced respiratory depression in sleep apnoea: new insights for anaesthesiology.

Mechanisms of opioid-induced respiratory depression in sleep apnoea: new insights for anaesthesiology.

Remifentanil preconditioning protects against hypoxia-induced senescence and necroptosis in human cardiac myocytes in vitro.

Remifentanil and other opioids are suggested to be protective against ischemia-reperfusion injury in animal models and coronary artery bypass surgery patients, however the molecular basis of such protection is far from being understood. In the present study, we have used a model of human cardiomyocytes treated with the hypoxia-mimetic agent cobalt chloride to investigate remifentanil preconditioning-based adaptive responses and underlying mechanisms. Hypoxic conditions promoted oxidative and nitrosative stress, p21-mediated cellular senescence and the activation of necroptotic pathway that was accompanied by a 2.2-, 9.6- and 8.2-fold increase in phosphorylation status of mixed lineage kinase domain-like pseudokinase (MLKL) and release of pro-inflammatory cytokine IL-8 and cardiac troponin I, a marker of myocardial damage, respectively. Remifentanil preconditioning was able to lower hypoxia-mediated protein carbonylation and limit MLKL-based signaling and pro-inflammatory response to almost normoxic control levels, and decrease hypoxia-induced pro-senescent activity of about 21% compared to control hypoxic conditions. In summary, we have shown for the first time that remifentanil can protect human cardiomyocytes against hypoxia-induced cellular senescence and necroptosis that may have importance with respect to the use of remifentanil to diminish myocardial ischemia and reperfusion injury in patients undergoing cardiac surgery.

Intermittent hypercapnic hypoxia induces respiratory hypersensitivity to fentanyl accompanied by tonic respiratory depression by endogenous opioids.

Sleep apnoea increases susceptibility to opioid-induced respiratory depression (OIRD). Endogenous opioids are implicated as a contributing factor in sleep apnoea. Rats exposed to sleep-phase chronic intermittent hypercapnic hypoxia (CIHH) for 7days exhibited exaggerated OIRD to systemic fentanyl both while anaesthetized and artificially ventilated and while conscious and breathing spontaneously, implicating heightened CNS inhibitory efficacy of fentanyl. CIHH also induced tonic endogenous opioid suppression of neural inspiration. Sleep-related episodes of hypercapnic hypoxia, as in sleep apnoea, promote hypersensitivity to OIRD, with tonic respiratory depression by endogenous opioids implicated as a potential underlying cause. Sleep apnoea (SA) increases opioid-induced respiratory depression (OIRD) and lethality. To test the hypothesis that this results from chronic intermittent bouts of hypercapnic hypoxia (CIHH) accompanying SA, we compared OIRD across continuously normoxic control rats and rats exposed to sleep-phase (8h/day) CIHH for 1week. OIRD sensitivity was first assessed in anaesthetized (urethane/α-chloralose), vagotomized and artificially ventilated rats by recording phrenic nerve activity (PNA) to index neural inspiration and quantify PNA burst inhibition to graded doses (0, 2, 20, 50μgkg-1 , i.v.) of the synthetic opioid fentanyl. Fentanyl dose-dependently reduced PNA burst frequency (P=0.0098-0.0001), while increasing the duration of burst quiescence at 50μgkg-1 (P<0.0001, n=5-6/group/dose). CIHH shifted the fentanyl dose-phrenic burst frequency response curve to the left (P=0.0163) and increased the duration of burst quiescence (P<0.0001). During fentanyl recovery, PNA burst width was increased relative to baseline in normoxic and CIHH rats. Systemic naloxone (1mgkg-1 , i.v.) reversed fentanyl-induced PNA arrest in both groups (P=0.0002), and increased phrenic burst amplitude above baseline (P=0.0113) in CIHH rats only. Differential sensitivity to anaesthesia as a cause of CIHH-related OIRD hypersensitivity was excluded by observing in conscious spontaneously breathing rats that fentanyl at 20μgkg-1 (i.v.), which silenced PNA in anaesthetized rats, differentially increased breathing variability in normoxic versus CIHH rats (P=0.0427), while significantly reducing breathing frequency (P<0.0001) and periodicity (P=0.0003) in CIHH rats only. Findings indicate that CIHH increased OIRD sensitivity, with tonic inspiratory depression by endogenous opioids as a likely contributing cause.

Chronic intermittent hypoxia vs chronic continuous hypoxia: Effects on vascular endothelial function and myocardial contractility.

Both chronic intermittent hypoxia (CIH) and chronic continuous hypoxia (CCH) are risk factors for cardiovascular disease, which are associated with cardiac systolic function and associated with dysfunction of endothelia and coagulation-fibrinolysis system in the vasculature. However, the different effects of these two hypoxic models are not fully understood. In our study, we systemically compared the effects of CIH and CCH on cardiac function and related factor levels in serum using rat model. Forty-five male Sprague-Dawley rats were randomly divided into the normoxia control (NC), CIH and CCH groups. The rat CIH and CCH models were established, then the blood and tissue samples were collected to analyze the function of endothelium and the coagulation-fibrinolysis system. Also, the ultrasound cardiogram was performed to directly assess myocardial contractility. Both CIH and CCH significantly decreased the NO, eNOS, P-eNOS and AT-III levels in the rat serum but significantly increased the levels of ET-1, vWF, COX-2, NF-κB, FIB, FVIII and PAI-1 in the rat serum (P < 0.05). The expression of ET-1, VWF and ICAM-1 in CIH group were higher than CCH group (P < 0.05), however, the expression of CD62p was increased in CCH group but not in CIH group. The expression of t-PA in CIH group were lower than CCH group (P < 0.05), but there were no significant differences in CCH group and NC group (P > 0.05). Using transmission electron microscope, we found that the mitochondrial ultrastructure of thoracic aorta endothelial cells in CIH and CCH group were damaged. Moreover, the myocardial contractility in CIH and CCH group were significantly decreased compared with NC group. Our results suggested that CIH and CCH could cause endothelial dysfunction, dysfunction of the coagulation-fibrinolysis system and decreasing of myocardial contractility. Compared with CCH, CIH has greater effect on vasoconstriction and adhesion of vascular endothelial cells, and stronger procoagulant effect.

Quantitative proteomics reveal an altered pattern of protein expression in saliva of hypobaric hypoxia-induced rat model

Hypobaric hypoxia (HH), a pathophysiological condition caused by an ascent to a high altitude. It occurs due to a deficiency of oxygen at the tissue level. Saliva is an advantageous biological sample because of easy, low cost and minimally invasive sample collection and processing. Although few reports have documented the effect of long term exposure to hypobaric hypoxia on salivary proteome but short term proteome based study remains uninvestigated. In this study, age-matched male SD rats were exposed to simulated hypobaric hypoxia (~ 25,000 ft, 7620 m) for 1 day (HD1), 3 days (HD3) and 7 days (HD7) followed by iTRAQ based LCMS/MS comparison with normoxic controls. Oxidative stress being the main event of hypobaric hypoxia suggested an initial increase in levels of oxidative stress parameters such as reactive oxygen species (ROS) and lipid peroxidation; and decrease in antioxidant enzymes such as the activity of catalase and reduced glutathione. Based on iTRAQ, out of 67 differentially expressed proteins, 36 (22 up-regulated and 14 down-regulated), 45 (24 up-regulated and 21 down-regulated), and 45 (19 up-regulated and 26 down-regulated) were modulated in HD1, HD3, and HD7 group respectively. Few proteins such as BPI fold-containing family A member 2, cystatin, and carbonic anhydrase 1 showed many folds differential expression in exposed groups as compared to normoxia. Through Ingenuity Pathways analysis, we have observed glucocorticoid receptor signaling and MSP-RON pathway to be most significantly affected. Glucocorticoid receptor signaling and MSP-RON signalling being the most significant pathways involved modulated proteins such as annexin 1, hsp90, keratin, actin-beta, and kallikrein 1. These proteins were found to aid in acclimatization and survival during hypobaric hypoxia. Taken together, this study provided the proof of concept for footprints of hypobaric hypoxia-mediated events during short term exposure through rat salivary proteome.

Blood and urine biomarkers associated with long-term respiratory dysfunction following neonatal hyperoxia exposure: Implications for prematurity and risk of SIDS

Former preterm infants, many of whom required supplemental O2 support, exhibit sleep disordered breathing and attenuated ventilatory responses to acute hypoxia (HVR) beyond their NICU stay. There is an increasing awareness that early detection of biomarkers in biological fluids may be useful predictors/identifiers of short- and long-term morbidities. In the present study, we identified serotonin (5-HT), dopamine (DA) and hyaluronan (HA) as three potential biomarkers that may be increased by neonatal hyperoxia and tested whether they would be associated with an impaired HVR in a rat model of supplemental O2 exposure. Neonatal rats (postnatal age (P) 6 days, P6) exposed to hyperoxia (40% FIO2, 24 h/day between P1–P5 days of age) exhibited an attenuated early (1 min), but not the late (4−5 min) phase of the HVR compared to normoxia control rats; the attenuated early phase HVR was associated with increased levels of DA (urine and serum), 5-HT (platelet poor plasma only, PPP), and HA (serum only). At P21, both the early and late phases of the HVR were attenuated, but serum and urine levels of all 3 biomarkers were similar to age-matched control rats. These data indicate that changes in several serum and/or urine biomarkers (5-HT, DA, and HA) following short-term (days) neonatal hyperoxia can signify long-term (weeks) respiratory control dysfunction. Further studies are needed to determine whether early detection of similar biomarkers could be convenient predictors of increased risk of abnormalities in respiratory control including sleep disordered breathing in former preterm infants who had received prior supplemental O2 and who might also be at increased risk of SIDS.

One and one is not always two: hypo- and hyper-additive effects of the chemoreflex during exercise.

One and one is not always two: hypo- and hyper-additive effects of the chemoreflex during exercise.

Desirability profiling to rank scalable mesenchymal stromal cell enhancement methods according to multimodal mechanisms

Background & Aim Mesenchymal stromal cells (MSCs) are widely investigated due to their anti-inflammatory, angiogenic, and anti-fibrotic properties. However, insufficient potency, MSC donor variability, and deficient knowledge on therapeutic mechanisms can limit MSC treatment efficacy. The Viswanathan lab has pioneered a novel, non-genetic proprietary strategy to culture MSCs in 3D aggregates under serum-free conditions to augment anti-inflammatory functions. Hypoxic culture has also been shown to enhance MSC paracrine functions. Our objective is to systematically characterize the anti-inflammatory, angiogenic, and anti-fibrotic properties of 3D and hypoxic MSCs, and to apply desirability profiling to empirically rank the potency of the MSCs according to these different properties. Methods, Results & Conclusion METHODS: Human adipose tissue (AT) or bone marrow (BM) MSCs were expanded with serum-free medium. MSCs were then cultured for 24-h under 3D (static culture or in 0.1 L vertical-wheel bioreactors) or 2D hypoxic (5% O2) conditions, with 2D normoxic MSCs as controls. Gene expression was measured by RT-qPCR and using a NanoString 50-gene panel, while microRNAs (miR) were examined by miR-sequencing. In ongoing work, desirability profiling will be performed on these data and on additional functional read-outs in MSC co-cultures with human monocytes, endothelial cells, or fibroblasts. RESULTS 3D MSC culture under static and bioreactor conditions resulted in similar high viable cell yields (>80%). 3D MSCs had increased expression of immunomodulatory genes (e.g. IDO, TSG6 with 4- to 10-fold increased expression vs. 2D normoxic control) while hypoxic MSCs had greater expression of angiogenic genes (e.g. VEGF with 2-fold greater expression vs. control). Preliminary investigation into miR expression revealed that AT-MSCs displayed a distinct miR profile relative to BM-MSCs, and that 3D enhanced AT-MSCs had a differential miR profile vs. controls. The miRs and their target pathways are being investigated to identify mechanisms underlying the enhanced MSC functionality. CONCLUSION MSCs act through multimodal mechanisms that are enhanced through 3D or hypoxic cultures. Statistical modeling is being used to systematically rank the potency attributes of enhanced MSCs in terms of their anti-inflammatory, angiogenic, and anti-fibrotic functionality in vitro and in inflammatory mouse models. Ultimately, this work will generate methods for improved MSC products with therapeutic profiles that are “fit-for-purpose.”

IL-11 and soluble VCAM-1 are important components of Hypoxia Conditioned Media and crucial for Mesenchymal Stromal Cells attraction.

Bone marrow stromal cells (BMSC) are highly attractive for tissue engineering due to their ability to differentiate into different cell types, to expand extensively in vitro and to release paracrine soluble factors with a high regenerative potential. They were observed to migrate towards the sites of injury in response to chemotactic signals in vivo. During the last years hypoxia has become a proven method to control proliferation, differentiation and multipotency of BMSC. Conditioned medium from hypoxia-treated BMSC (Hypoxia-conditioned Medium; HCM) has been shown to have various favorable properties on tissue regeneration - such as on cell recruitment, wound healing, angiogenesis and revascularization. Due to this regenerative potential many studies attempt to further characterize HCM and its main functional components. In this study we used HCM generated from umbilical cord mesenchymal stem cells (UC-MSC) instead of BMSC, because GMP-verified methods were used to isolate and cultivate the cells and ensure their constant quality. UC-MSC have a high regenerative potential and are still immunologically naive and therefore highly unlikely to cause an immune reaction. In our article we took the first steps to closer investigate the role of umbilical cord MSC-derived HCM components, namely stromal cell-derived factor 1 (SDF-1α), interleukin 11 (IL-11) and soluble vascular cell adhesion molecule 1 (sVCAM-1). Our results show previously unknown roles of IL-11 and sVCAM-1 in the attraction of BMSC. The synergistic effect of the investigated protein mixture consisting of IL-11, sVCAM-1 and SDF-1α as well as those recombinant proteins alone revealed a significantly higher chemoattractive capacity towards human BMSC compared to normoxic control medium. Both, the protein mixtures and proteins alone as well as UC-HCM showed an angiogenic effect by promoting the formation of significantly longer tubule structures and higher amounts of junctions and tubules compared to normoxic control medium. By showing the prominent upregulation of IL-11, sVCAM-1 and SDF-1α under hypoxic conditions compared to normoxic control and revealing their crucial role in migration of human BMSC we took a further step forward in characterization of the chemoattractive components of HCM.

Variation in root growth responses of sweet potato to hypoxia and waterlogging

Climate change increase the probability of soil waterlogging due to severe rainfall. A low oxygen concentration in soil (hypoxia) is a major problem associated with waterlogging, and root development is an important indicator of resistance. However, little is known about the root development responses of sweet potato to hypoxia under waterlogging. In this study, the root hypoxia tolerance of four sweet potato cultivars was investigated using three environments (hydroponics, soil in pots, and upland field). In the hydroponic experiment, at 4 days after transplanting (DAT), the plants were transferred to either normoxia (control) or hypoxia and grown for 7 more days. In the soil pot experiment, seedlings at 7 DAT were grown for another 7 days under one of two treatments: drained (control) or waterlogging conditions. The root systems of individual plants were scanned with an image scanner using the WinRhizo software to measure root morphological traits. All the cultivars were tested for tuberous root performance under field waterlogging conditions imposed at the seedling stage. The obtained results showed that the root development responses to hypoxia were clearly associated and contributed to waterlogging tolerance in sweet potato plants. Of the four sweet potato varieties investigated, we identified Cuc Nhanh as the most waterlogging-tolerant variety and hence, it was noted as a promising and important genetic plant resource. The results of this study on root responses, hypoxia, and waterlogging relationships are fundamental towards exploring the mechanisms underlying hypoxia and waterlogging adaptation.

Microtubule Arrangement and FKBP51 Localization is Altered in BMPR2‐Deficient Pulmonary Artery Endothelial Cells Isolated from a Rat Model of PAH

Pulmonary arterial hypertension (PAH) is an incurable vasoconstrictive pathology of which there are limited palliative treatments. The hallmarks of PAH include vascular smooth muscle cell proliferation and endothelial dysfunction in the small pulmonary arteries. Endothelial cytoskeletal defects have been implicated in BMPR2‐associated PAH, and BMPR2‐silencing alters cytoskeletal arrangement in pulmonary artery endothelial cells (PAECs). Indeed, targeting cytoskeletal proteins, particularly microtubules, may be a novel approach to PAH treatment as microtubule disruption has been efficacious in the treatment of other inflammatory and proliferative diseases. FK506‐binding protein 51 (FKBP51), a peptidyl‐prolyl isomerase member of the immunophilin family, is a known regulator of the glucocorticoid receptor and its expression is upregulated upon glucocorticoid receptor activation. Furthermore, FKBP51 is a binding partner to Hsp90 and stabilizes microtubules in PAECs. Here, we show microtubule disruption and altered FKBP51 distribution in PAECs from a rat model of PAH. PAECs isolated from the Sugen/hypoxia rat model of PAH (PAH PAECs) or normoxic control cells were gifted from the University of South Alabama. The PAH PAECs have lower BMPR2 gene expression than normoxic controls, as shown by real‐time PCR. These cells also demonstrate decreased BMPR2 signaling compared to normoxic control PAECs, as demonstrated by lower ID‐1 expression levels upon BMP4 or BMP6 stimulation. Immunocytochemistry studies of α‐tubulin indicate microtubule disorganization in PAH PAECs, as compared to the normoxic control PAECs, which demonstrate normal perinuclear distribution of interphase microtubules. Immunocytochemical staining of FKBP51, using a novel antibody developed by our laboratory, reveals a microtubule‐like distribution of FKBP51 in normoxic control PAECs. Upon treatment with the synthetic glucocorticoid dexamethasone (DEX), FKBP51 is predominantly observed in the nucleus of normoxic control PAECs, with reduced cytosolic organization and distribution. PAH PAECs, however, showed diffuse cytoplasmic staining of FKBP51, with no change in distribution upon DEX treatment. Western blot analysis of PAEC nuclear fractions also demonstrated greater protein levels of FKBP51 upon DEX treatment, compared to DEX‐treated PAH PAECs. Taken together, these data indicate that microtubule rearrangement is evident in BMPR2‐deficient PAH PAECs, and FKBP51 dysregulation may play a role in cytoskeletal abnormalities as well as the disruption of metabolic homeostasis in these cells.Support or Funding InformationSupported by NIH1R15HL137135‐01A1

Intermittent Hypoxia Differentially Modulates Endogenous Tau Phosphorylation in Rats

Intermittent hypoxia, or periods of low oxygen, can have different effects depending on the dose of hypoxic episodes (duration, severity, number per day, etc). “Low dose” daily acute intermittent hypoxia (dAIH) elicits neuroprotection and neuroplasticity, whereas “high dose” chronic intermittent hypoxia (CIH) similar to that experienced during sleep apnea elicits neuropathology. Sleep apnea is comorbid with more than half of patients with Alzheimer’s Disease (AD)—a progressive, neurodegenerative disease consisting of brain amyloid plaque accumulation and tau pathology. Clinically, patients with sleep apnea present with higher levels of tau aggregation, consistent with levels observed in AD patients. However, it is not known if sleep apnea and the associated CIH accelerates the onset or progression of AD and related tauopathies. We hypothesized high dose intermittent hypoxia consistent with that experienced during mild or moderate sleep apnea would increase levels of soluble phosphorylated tau species in the hippocampus and frontal cortex. Thus, we measured Ser396/Ser404 (PHF‐1) and Ser202 (CP13)—sites associated with tau pathology—in adult male SD rats exposed to different intermittent hypoxia protocols for 7 or 28 consecutive days, including: 1) continuous normoxia; 2) dAIH (10, 5‐min 10.5% O2 episodes per day; 5‐min normoxic intervals); 3) mild chronic intermittent hypoxia (CIH5/5: 5‐min 10.5% O2 episodes, 5‐min intervals; 8 hrs/day); and 4) moderate chronic intermittent hypoxia (CIH2/2: 2‐min 10.5% O2 episodes, 2‐min intervals; 8 hrs/day). Rats were then anesthetized and hippocampal and prefrontal cortex tissues were freshly harvested for protein quantification by western blot or fixed in 4% paraformaldehyde for immunohistochemical analysis. PHF‐1 and CP13 protein was standardized to total tau and normalized to normoxic controls. After 7 days, there were significant increases in hippocampal PHF‐1 levels in CIH5/5 rats (p=0.039) and in PHF‐1 and CP13 levels of CIH2/2 rats (p=0.003 and p=0.004) vs. normoxic controls. Changes in phosphorylated tau were not detected in the prefrontal cortex. After 28 days, hippocampal PHF‐1 and CP13 were significantly elevated in rats exposed to CIH5/5 (p=0.018 and p=0.024) and CIH2/2 (p=0.012 and p=0.047). PHF‐1 and CP13 levels were also significantly elevated in the prefrontal cortex of rats exposed to CIH5/5 (p=0.007 and p&lt;0.001) and CIH2/2 (p=0.006 and p=0.003). 28 days of dAIH also significantly increased hippocampal PHF‐1 (p=0.047) and prefrontal cortex PHF‐1 (p=0.033) and CP13 (p=0.001), however these effects were smaller in magnitude versus corresponding tissues with either CIH5/5 or CIH2/2. Thus, intermittent hypoxia induces dose‐dependent, region‐specific tau phosphorylation. However, the functional significance of increased phosphorylation is not yet known in these neurotypical rats. Clarifying the differential effects of intermittent hypoxia on AD onset and progression has both biological and translational significance, potentially guiding new therapeutic strategies to prevent or manage AD or other tauopathies.Support or Funding InformationFlorida Department of Health, Ed &amp; Ethyl Moore Alzheimer’s Research Program; NIH HL148030 and McKnight Brain Institute.

NADPH oxidase 2 knockout prevents chronic intermittent hypoxia‐induced diaphragm muscle weakness but not increased propensity for apnoea in a mouse model of obstructive sleep apnoea syndrome

Obstructive sleep apnoea syndrome (OSAS) is characterized by exposure to chronic intermittent hypoxia (CIH), as a consequence of repetitive occlusions of the upper airway in patients during sleep. CIH evokes redox changes culminating in impaired diaphragm muscle function and aberrant respiratory plasticity which manifests as destabilised breathing during sleep. We sought to investigate the putative role of the superoxide‐generating NADPH oxidase 2 (NOX2) enzyme in CIH‐induced diaphragm muscle dysfunction and respiratory maladaptation.A mouse model of CIH was generated by the cycling of gas from normoxia (21% O2) for 210 seconds to hypoxia (5% O2 at the nadir) over 90 seconds for 8hr/day during light hours for 2 weeks. Adult male (C57BL/6J) mice were assigned to one of 5 groups: normoxic controls, CIH‐exposed and CIH+apocynin (NADPH oxidase 2 inhibitor, 2mM) given in the drinking water throughout the CIH exposure, NOX2 null (B6.129S‐Cybbtm1Din/J) assigned to a sham or CIH exposure. On day 15, whole body plethysmography was used to measure breathing parameters on a breath‐by‐breath basis in room air. An apnoea was defined as ≥ 2 missed breaths. Diaphragm muscle contractile function was examined ex vivo. Gene expression was examined by qRT‐PCR. Markers of oxidative stress were determined using spectrophotometric assays. Values are expressed as mean ± SD and data were statistically compared by unpaired Student t‐test.Exposure to CIH significantly decreased diaphragm muscle peak specific force by ~45% compared with sham exposure. Apocynin and NOX2 gene knockout completely prevented CIH‐induced diaphragm muscle weakness. CIH resulted in no overt oxidative stress in diaphragm evidenced by unaltered thiobarbituric acid reactive substances (TBARS) and citrate synthase activity. CIH increased mRNA expression of NRF2, MuRF1, BNIP3, LC3B, GABARAPL1, PARK2, Myostatin, Myogenin, MEF2C, MyoD. Apocynin and NOX2 gene knockout prevented these CIH‐induced increases. Basal minute ventilation was unchanged following 2 weeks of CIH exposure however the number of apnoeas per hour was significantly increased compared with sham exposed mice. Apocynin intervention significantly reduced the frequency of apnoeas compared with the CIH group. Apnoea index was increased in NOX2 null mice exposed to CIH compared with NOX2 null sham mice reminiscent of that observed in wild‐type mice.Mice show signs of profound diaphragm muscle dysfunction and increased mRNA expression of genes relating to muscle atrophy, mitophagy, autophagy and regeneration following 2 weeks of CIH. Studies in NOX2 null mice reveal that NOX2 is necessary for CIH‐induced diaphragm muscle weakness. CIH‐induced increase in the propensity for apnoea may be of biological relevance as it may underpin progression in the severity of OSAS pathology (i.e. mild to moderate OSA). The reduction in apnoea frequency following treatment with apocynin implicates ROS (that are not NOX2‐derived) in the manifestation of CIH‐induced respiratory disturbances. Our results have implications for human OSAS and point to antioxidant intervention as a potential therapeutic strategy.

Myristoylated Protein Kinase C Beta II Peptide Inhibitor Improves Cell Survival in Cultured Human Umbilical Vein Endothelial Cells Subjected to Hypoxia/Reoxygenation

Endothelial dysfunction as a result of ischemia/reperfusion (I/R) injury is known to contribute to damage in myocardial infarction and organ transplant patients. In vascular cells, NADPH oxidase (NOX) and the mitochondrial electron transport chain are primary sources of reactive oxygen species (ROS) during I/R injury resulting in reduced nitric oxide bioavailability. Protein kinase C beta II (PKCβII) is an attractive therapeutic target due to its regulation of these downstream mediators of ROS production. PKCβII phosphorylates p66Shc to enhance mitochondrial‐derived ROS production and p47 phox to promote ROS release from NOX. We have previously shown that myristoylated PKCβII peptide inhibitor (N‐myr‐SLNPEWNET; myr‐PKCβII‐) improved post‐reperfusion cardiac function and reduced infarct size in rat myocardial I/R injury compared to non‐treated controls. The decrease in myocardial I/R injury with myr‐PKCβII‐treatment may be attributed to improved vascular endothelial function. The myristoylated peptide sequence (cargo sequence) targets a highly conserved peptide sequence among mammalian species and inhibits PKCβII translocation to protein substrates (e.g. NOX and p66Shc) after second messenger activation. We hypothesize that myr‐PKCβII‐ will confer protection by directly inhibiting ROS production from NOX and mitochondria in human umbilical vein endothelial cells (HUVECs) subjected to hypoxia/reoxygenation (H/R) injury. HUVECs were cultured in gelatin‐coated 96‐well plates and subjected to 24h hypoxia and 24h reoxygenation in Billups‐Rothenburg hypoxia chamber with 1% O2, 5% CO2, and balanced nitrogen. Myr‐PKCβII‐ (20 mM) treatment was administered at the beginning of the 24h reoxygenation period. Cell viability was assessed using tetrazolium‐salt (WST‐8) colorimetric assay with a microplate reader (450 nm) and normalized against the normoxia control group. Data were analyzed using Student‐Newman‐Keuls post‐hoc analysis. At the end of the 24h reoxygenation period, cell viability (%) was significantly reduced to 78±2% (p&lt;0.05; n=5) in the non‐treated H/R group compared to normoxia controls (n=5). Myr‐PKCβII‐significantly improved HUVEC survival (95±4%; p&lt;0.01; n=5) compared to non‐treated H/R controls and was not significantly changed compared to normoxia controls. The data suggests that PKCβII inhibition promotes cell survival subjected to H/R possibly due to directly attenuating NOX and mitochondrial derived ROS. Further studies are needed to control for the myristoylated acid conjugation of the peptide cargo.Support or Funding InformationThis research was supported by the Division of Research, Department of Biomedical Sciences, and the Center for Chronic Disorders of Aging at the Philadelphia College of Osteopathic Medicine. Current license is supported by Young Therapeutics, LLC.

Oxygen‐Directed Laboratory Evolution and Conserved Mechanisms Underlying Hypoxia Tolerance

Oxygen is essential for metazoan life on earth. In this work, we have taken advantage of Drosophila melanogaster as a genetic model system to dissect mechanisms of genomic adaptation to extreme oxygen conditions. We conducted a ~15‐year laboratory evolution experiment over &gt;290 generations by chronically exposing multiple fly populations to normal, decreasing, or increasing O2 levels to determine the effect of selection pressure on their genomes, and to understand the mechanisms of adaptation to O2 imbalance. We have obtained Drosophila melanogaster populations and strains that can tolerate and perpetually survive environments containing extremely low (&lt;4%) or high (&gt;90%) level of O2, which are lethal for naïve controls. A time series of whole genome sequencing (WGS) analysis of multiple generations of the Low (L‐O2) and high (H‐O2) populations and normoxia control (N‐populations) demonstrated clear genomic changes along evolution between environments and generations. Furthermore, four genomic regions in the H‐O2 populations and five genomic regions in the L‐O2 populations were identified, which contain selection sweeps generated by selections with low or high oxygen condition. A total of 433 candidate genes and 215 candidate genes were identified in the selection sweeps in the L‐O2 populations and H‐O2 populations, respectively. This suggested a number of evolutionarily conserved mechanisms (e.g., the Notch signaling pathway) that may play a role in regulating adaptation to the extreme oxygen conditions. We believe that these mechanisms have a strong potential to be translated into humans and serve as novel targets for developing therapeutic strategies to prevent and treat hypoxia‐related diseases and oxidative stress‐induced injuries.

Activation of a non‐neuronal cholinergic system in rat cardiomyocytes by cardioprotective humoral factors from pigs with remote ischemic conditioning

IntroductionBrief cycles of ischemia/reperfusion (I/R) in a tissue/organ remote from the heart (remote ischemic conditioning, RIC) reduce myocardial I/R injury. Although operative in all species tested so far, including humans, the translation of RIC into clinical practice has been largely disappointing. Such failure in translation has in part been attributed to incomplete understanding of RIC’s signal transduction. RIC’s cardioprotection can be transferred with plasma between different individuals, even across species. Prior studies have suggested that the intrinsic nervous system is causally involved in RIC’s protection, since the ganglionic blocker hexamethonium attenuated the infarct size reduction in isolated rat hearts in response to plasma taken from different rats after RIC. However, recently a non‐neuronal cardiomyocyte‐based cholinergic system (NNCS) has been identified, and its involvement in the signal transduction of RIC is entirely unclear.We therefor aimed to study whether the NNCS is activated by humoral factor(s) released by RIC.MethodsAnesthetized open‐chest minipigs were subjected to 60/180 min occlusion/reperfusion of the left anterior descending coronary artery (n=7, placebo, PLA). RIC was induced by 4×5/5 min hindlimb I/R (n=8) 90 min before coronary occlusion. Arterial blood was sampled after PLA/RIC and plasma separated. Rat ventricular cardiomyocytes were isolated and incubated with PLA or RIC plasma‐dialysate (1:10; cut‐off &lt;12–14 kDa) for 30 min ± hexamethonium (1 μmol/L) or ± atropine (100 nmol/L), respectively, subjected to 30/5 min hypoxia/reoxygenation (H/R) and compared to cardiomyocytes exposed to normoxic buffer (time control, TC) for 35 min. Cardiomyocyte viability was quantified before and after H/R or TC, respectively, as the percent fraction of rod‐shaped, unstained (trypan blue) cells over all cells. Diluted (1:10) PLA or RIC plasma was also infused into isolated perfused rat hearts for 8 min before 30/120 min global ischemia/reperfusion (GI/R) ± hexamethonium (50 μmol/L) or ± atropine (100 nmol/L), respectively. Infarct size (IS) was demarcated by triphenyltetrazolium chloride and calculated as percent of ventricular mass.ResultsIncubation with RIC plasma‐dialysate preserved cardiomyocyte viability after H/R in comparison to PLA plasma‐dialysate. This protection was attenuated by hexamethonium and by atropine. Hexamethonium or atropine had no impact on cardiomyocyte viability per se (Figure A). In isolated hearts, IS was less with infusion of plasma from pigs with RIC than with that from pigs with PLA. With infusion of PLA plasma, hexamethonium or atropine did not impact on IS per se. The protective effect of RIC plasma, however, was abrogated by hexamethonium and by atropine (Figure B).ConclusionRIC’s humoral transfer of cardioprotection is mediated via an activation of cholinergic receptors on cardiomyocytes and attenuated to the same extent by hexamethonium or by atropine. Whether or not hexamethonium also and additionally attenuates the cardioprotection of RIC at the ganglionic level therefore remains unclear.Support or Funding InformationSupported by the German Research Foundation (SFB 1116 B8)Figure legend: A) Viability of rat ventricular cardiomyocytes incubated with placebo (PLA)/remote ischemic conditioning (RIC) plasma‐dialysate after hypoxia (H)/reoxygenation(R), or after normoxic time control (TC). Cardiomyocytes with plasma‐dialysate were incubated with saline, ± hexamethonium (hexa), or ± atropine. Data are means ± standard deviations. †p&lt;0.05 vs. PLA plasma‐dialysate; ‡p&lt;0.05 vs. saline RIC plasma‐dialysate; two‐way ANOVA with Fisher’s least significant differences post‐hoc tests. B) Infarct size in isolated perfused rat hearts (± hexa or ± atropine) with infusion of PLA or RIC plasma and after global ischemia/reperfusion. Data are means ± standard deviations. #p&lt;0.05 vs. PLA plasma; *p&lt;0.05 vs. saline RIC plasma; two‐way ANOVA with Fisher’s least significant differences post‐hoc tests.Figure 1

Increased fatty acid oxidation promotes mitochondrial ROS (mtROS) production and Ca2+ entry via TRPV4 in microvascular endothelial cells in PAH

RationaleIn pulmonary arterial hypertension (PAH), highly proliferative microvascular endothelial cells (MVECs) are thought to contribute to vaso‐occlusive lesion formation and increased pulmonary artery pressures. Using MVECs isolated from the Sugen/Hypoxia (SuHx) model of PAH, we recently showed that increased mtROS production activates the transient receptor potential vanilloid‐4 (TRPV4) channel and contributes to increased MVEC proliferation in vitro. However, the mechanistic basis for sustained mtROS production in MVECs isolated from SuHx rats (SuHx‐MVECs) was unclear. Along with increased mtROS production, we observed evidence of significant mitochondrial dysfunction, including increased fragmentation and glycolytic shift. Since mtROS production can be regulated by shifts in TCA fuel substrates, we hypothesized that generation of TCA metabolites using non‐glucose sources (i.e. anaplerosis) could be the source of mitochondrial dysfunction and mtROS production in SuHx‐MVECs.MethodsCa2+ was measured using Fura‐2AM loaded MVEC in a flow chamber perfused with Krebs buffer. Mitochondrial morphology was measured computationally using previously validated algorithms applied to confocal images of mitochondria in N‐ and SuHx‐MVEC treated with MitoTracker. ROS measurements were made using the ratiometric ROS sensor roGFP. Lipid droplets were assessed using BODIPY staining in live MVECs, and quantified using CellProfiler. Targeted metabolomics were conducted using a standardized LC/MS approach and MS‐grade reagent standards.ResultsDespite evidence of glycolytic shift (decreased oxygen consumption rate, increased extracellular acidification rate, increased extracellular lactate levels), levels of TCA metabolites (measuring using targeted metabolomics) were not significantly lower in SuHx‐MVECs compared to normoxic (N‐MVEC) controls, suggesting presence of significant anaplerosis. Serum free fatty acids (FFA) were increased in SuHx rats at 5 weeks. Intracellular lipid droplet number was increased in SuHx‐MVECs, while droplet size was decreased, suggesting increased FA utilization. Untargeted metabolomics performed in N‐MVEC and SuHx‐MVEC cell extracts revealed evidence of metabolite shunting towards nucleotide and amino acid synthesis as well as increased levels of fatty acid oxidation byproducts. In cultured SuHx‐MVECs, inhibition of mitochondrial fatty acid import (with Etoximir) attenuated basal Ca2+ and mitochondrial fragmentation to a similar extent as mtROS quenching and TRPV4 inhibition.ConclusionIncreased utilization of fatty acid oxidation for anaplerotic carbons is associated with mitochondrial fragmentation and mtROS production, which in turn increases [Ca2+]I via a TRPV4‐dependent mechanism. These data suggest a novel link between EC metabolism, mitochondrial dysfunction and TRPV4 activation in the SuHx model of PAH.Support or Funding InformationThis work was supported by by the National Heart, Lung and Blood Institute Grants F32HL124930 and K08HL132055 (KS), R01HL073859, R25HL084762 and R01HL126514 (LAS), F32HL124727, and K08HL133475 (JH) and T32HL007534 (KS and JH).

Unfolding the Mysteries of Oxygen Sensing ‐ A Comprehensive Analysis of the Hypoxic Response in Zebrafish Gills One Cell at a Time via Single Cell RNA Sequencing

All vertebrates “sense” changes in O2 through specialized cells, called chemoreceptors, which interact with the peripheral and central nervous systems to maintain O2 homeostasis. Aquatic vertebrates are especially prone to fluctuations in environmental O2 availability. Therefore, it is vital for them to readily detect changes in PO2 and make appropriate respiratory and cardiovascular adjustments. Teleost fish, such as zebrafish (Danio rerio), have O2 chemoreceptors, called neuroepithelial cells (NECs), present in the gill epithelium and undergo membrane depolarization and vesicular recycling when exposed to acute hypoxia. NECs have also been shown to increase in number and size following chronic hypoxia. Here we present a comprehensive analysis of the effects of hypoxia on gill cells including NECs through a transcriptomic study. Using the transgenic line ETvmat2:GFP, zebrafish were acclimated to normoxia (PO2 = 160 mmHg) or severe hypoxia (PO2 = 35 mmHg ) and maintained for 14 days. Isolated cells from the distal ends of gill filaments, areas rich in NECs, were analyzed using single cell RNA sequencing (scRNA‐seq) analysis. Four normoxia (control) and four hypoxia‐treated biological samples were individually labelled before scRNA‐seq library construction using the 10X Genomics chromium technology. A total of 45,000 gill cells were consequently sequenced on a NextSeq500 (Illumina) sequencer with a read depth of 17,000 per cell. Our preliminary results separated 21 cell clusters and identified one cluster to be NECs based on gene expression. Highly differentially expressed genes in NECs included tph1a encoding tryptophan hydroxylase 1, the rate limiting enzyme in 5‐HT biosynthesis, and sv2 encoding synaptic vesicle 2 protein. Other differentially expressed genes in NECs are involved in mitochondrial oxidative metabolism and ATP synthesis. Moreover, differential expression analysis showed a clear shift in the transcriptome of NECs following 14 days of chronic hypoxia. NECs in the hypoxia group showed a high expression of genes involved in stress‐regulated polyubiquitination in contrast to the control group. The transcriptomic change implies a change in dynamics of the NEC population. We are the first group to conduct a single cell transcriptomic study in zebrafish gill cells. Our study is expected to provide a complete atlas for the hypoxic response in single gill cells and serve as a platform for future work on peripheral oxygen sensing.Support or Funding InformationThis research project is funded by Natural Sciences and Engineering Research Council of Canada (NSERC).