Chronic Intermittent Hypoxia Exposure Research Articles

Involvement of overweight and lipid metabolism in the development of pulmonary hypertension under conditions of chronic intermittent hypoxia.

There is growing evidence that exposure to hypoxia, regardless of the source, elicits several metabolic responses in individuals. These responses are constitutive and are usually observed under hypoxia but vary according to the type of exposure. The aim of this review was to describe the involvement of obesity and lipid metabolism in the development of high-altitude pulmonary hypertension and in the development of acute mountain sickness under chronic intermittent hypoxia. Overweight or obesity, which are common in individuals with long-term chronic intermittent hypoxia exposure (high-altitude miners, shift workers, and soldiers), are thought to play a major role in the development of acute mountain sickness and high-altitude pulmonary hypertension. This association may be rooted in the interactions between obesity-related metabolic and physical alterations, such as increased waist circumference and neck circumference, among others, which lead to critical ventilation impairments; these impairments aggravate hypoxemia at high altitude, thereby triggering high-altitude diseases. Overweight and obesity are strongly associated with higher mean pulmonary artery pressure in the context of long-term chronic intermittent hypoxia. Remarkably, de novo synthesis of triglycerides by the sterol regulatory element-binding protein-1c pathway has been demonstrated, mainly due to the upregulation of stearoyl-CoA desaturase-1, which is also associated with the same outcomes. Therefore, overweight, obesity, and other metabolic conditions may hinder proper acclimatization. The involved mechanisms include respiratory impairment, alteration of the nitric oxide pathways, inflammatory status, reactive oxygen species imbalance, and other metabolic changes; however, further studies are required.

Adiponectin ameliorates lung injury induced by intermittent hypoxia through inhibition of ROS-associated pulmonary cell apoptosis.

Obstructive sleep apnea hypopnea syndrome has been reported to be associated with pulmonary hypertension (PH). Adiponectin (Ad) has many protective roles in the human body, including its function as an anti-inflammatory and an anti-oxidant, as well as its role in preventing insulin resistance and atherosclerosis. This study aimed to investigate the molecular mechanism of chronic intermittent hypoxia (CIH)-induced pulmonary injury and the protective role of Ad in experimental rats. Thirty male Sprague-Dawley rats were randomly divided into three groups with 10 rats in each group: normal control (NC) group, CIH group, and CIH + Ad group. Rats in the NC group were kept breathing room air for 12weeks. Rats in the CIH group were intermittently exposed to a hypoxic environment for 8h/day for 12weeks. Rats in the CIH + Ad group received 10μg Ad twice weekly via intravenous injection. After 12weeks of CIH exposure, we detected the pulmonary function, pulmonary artery pressure, lung histology, pulmonary cell apoptosis, pulmonary artery endothelial cell apoptosis, mitochondrial membrane potential (MMP), and reactive oxygen species (ROS) level. We also analyzed expression proteins involved in the mitochondria-, endoplasmic reticulum (ER) stress-, and Fas receptor-associated pulmonary apoptosis pathways, as well as the SIRT3/SOD2 pathway. CIH exposure for 12weeks did not lead to abnormal pulmonary function, PH, or pulmonary artery endothelial cell apoptosis. However, we observed a significant increase in the rate of pulmonary cell apoptosis, the expression of proteins involved in mitochondria-, ER stress-, and Fas receptor-associated pulmonary apoptosis pathways, and the generation of ROS in the CIH group compared with the NC group. In contrast, the MMP and protein expressions of SIRT3/SOD2 pathway were significantly decreased in the CIH group compared with the NC group. Ad supplementation in the CIH + Ad group partially improved these changes induced by CIH. Even though CIH did not cause abnormal pulmonary function or PH, early lung injury was detected at the molecular level in rats exposed to CIH. Treatment with Ad ameliorated the pulmonary injury by activating the SIRT3/SOD2 pathway, reducing ROS generation, and inhibiting ROS-associated lung cell apoptosis.

Cell-Autonomous Autophagy Protects Against Chronic Intermittent Hypoxia Induced Sensory Nerves and Endothelial Dysfunction of the Soft Palate.

BackgroundChronic intermittent hypoxia (CIH) is a key feature of obstructive sleep apnea (OSA) syndrome. The pathogenesis of CIH-induced soft palate lesion is not well understood. Understanding the mechanisms of CIH-induced soft palate damage could provide new strategies for clinical treatment.Material/MethodsTwenty male Sprague-Dawley rats were randomized into a control group (n=10) and experimental group (n=10). The experimental group were exposed to CIH for 28 days. The control experiments were run in parallel. Morphological changes of CIH-induced soft palate were examined by hematoxylin and eosin. Peripheral nerves and vascular associated markers were analyzed by western blot and immunohistochemical staining. LC3B expression and transmission electron microscopy analysis was detected to investigate the destiny of cells in CIH-induced soft palate.ResultsHistological studies demonstrated the thicken mucosal layer, muscular changes consistent with glands hyperplasia, and loose connective tissues of the soft palate in CIH induced rat models. CIH exposure significantly decreased the expression of annexin V but did not change argin level, suggesting that sensory nerves not motor nerves were damaged when exposed to intermittent hypoxia. Moreover, in response to CIH, the vascular vessel around the nerves and muscles became enlarged and caveolin-1 was overexpressed. Autophagy occurs in response to CIH-induced neuromuscular and vascular endothelial injury.ConclusionsSensory nerves and endothelial dysfunction contributed to the morphological damage of soft palate under intermittent hypoxia. Autophagy as a compensatory mechanism protects against CIH-induced injury. These findings have important implications for understanding mechanisms contributing to the increased soft palate lesion in patients with OSA.

Progesterone decreases apnoea and reduces oxidative stress induced by chronic intermittent hypoxia in ovariectomized female rats.

What is the central question of this study? Does progesterone reduce the effect of chronic intermittent hypoxia (CIH) on arterial blood pressure, respiratory control and oxidative stress in the central nervous system in ovariectomized rats? What is the main finding and its importance? Progesterone does not prevent the elevation of arterial blood pressure in rats exposed to CIH, but normalizes respiratory control, and reduces cerebral oxidative stress. This study draws focus to a potential role of progesterone and the consequences of sleep apnoea in menopausal women. We tested the hypothesis that progesterone (Prog) reduces the effect of chronic intermittent hypoxia (CIH) on arterial blood pressure, respiratory chemoreflexes and oxidative stress in the central nervous system. Ovariectomized female rats were implanted with osmotic pumps delivering vehicle (Veh) or Prog (4mgkg-1 day-1 ). Two weeks following the surgery, rats were exposed to room air (Air) or CIH (7days, 10% O2 , 10 cyclesh-1 , 8hday-1 ). We studied three groups: Veh-Air, Veh-CIH and Prog-CIH. After the CIH exposures, we measured the mean arterial pressure (MAP; tail cuff) and assessed the frequency of apnoeas at rest and ventilatory responses to hypoxia and hypercapnia (whole body plethysmography). The activities of the pro-oxidant enzyme NADPH oxidase (NOX) and antioxidant enzymes superoxide dismutase (SOD; in mitochondrial and cytosolic fractions) and glutathione peroxidase (GPx), as well as the concentration of malondialdehyde (MDA), a marker of lipid peroxidation, were measured in brain cortex and brainstem samples. CIH exposure increased the MAP, the frequency of apnoeas, and the respiratory frequency response to hypoxia and hypercapnia. Prog did not prevent the CIH-induced elevation in MAP, but it reduced the CIH-induced frequency of apnoeas and increased hypoxic and hypercapnic ventilatory responses. In the brain cortex, CIH increased NOX activity, and decreased the cytosolic and mitochondrial SOD activities. These effects were prevented by Prog. NOX activity was increased by CIH in the brainstem, and this was also blocked by Prog. The study draws focus to the links between ovarian hormones and the consequences of sleep apnoea in women.

Chronic Intermittent Hypoxia Triggers a Senescence-like Phenotype in Human White Preadipocytes

Obstructive sleep apnea (OSA) is a common sleep disorder associated with obesity. Emerging evidence suggest that OSA increases the risk of cardiovascular morbidity and mortality partly via accelerating the process of cellular aging. Thus, we sought to examine the effects of intermittent hypoxia (IH), a hallmark of OSA, on senescence in human white preadipocytes. We demonstrate that chronic IH is associated with an increased generation of mitochondrial reactive oxygen species along with increased prevalence of cells with nuclear localization of γH2AX & p16. A higher prevalence of cells positive for senescence-associated β-galactosidase activity was also evident with chronic IH exposure. Intervention with aspirin, atorvastatin or renin-angiotensin system (RAS) inhibitors effectively attenuated IH-mediated senescence-like phenotype. Importantly, the validity of in vitro findings was confirmed by examination of the subcutaneous abdominal adipose tissue which showed that OSA patients had a significantly higher percentage of cells with nuclear localization of γH2AX & p16 than non-OSA individuals (20.1 ± 10.8% vs. 10.3 ± 2.7%, Padjusted < 0.001). Furthermore, the frequency of dual positive γH2AX & p16 nuclei in adipose tissue of OSA patients receiving statin, aspirin, and/or RAS inhibitors was comparable to non-OSA individuals. This study identifies chronic IH as a trigger of senescence-like phenotype in preadipocytes. Together, our data suggest that OSA may be considered as a senescence-related disorder.

Astragaloside IV attenuates chronic intermittent hypoxia-induced myocardial injury by modulating Ca2+ homeostasis.

Obstructive sleep apnea syndrome (OSAS) is an important consequence of chronic intermittent hypoxia (CIH). Astragaloside IV (AS-IV) exerts multiple protective effects in diverse diseases. However, whether AS-IV can attenuate CIH-induced myocardial injury is unclear. In this study, rats exposed to CIH were established and treated with AS-IV for 4 weeks. In vitro, H9C2 cardiomyocytes subjected to CIH exposure were treated with AS-IV for 48 hours. Then the cardiac function, morphology, fibrosis, apoptosis and Ca2+ homeostasis were determined to assess cardiac damage. Results showed that AS-IV attenuated cardiac dysfunction and histological lesions in CIH rats. The increased TUNEL-positive cells and activated apoptotic proteins in CIH rats were reduced by AS-IV. We also noticed that AS-IV reversed the accumulation of Ca2+ and altered expressions of Ca2+ handling proteins (decreases of SERCA2a and RYR2, and increases of p-CaMKII and NCX1) under CIH exposure. Furthermore, CIH-induced reduction of SERCA2a activity was increased by AS-IV in rats. Similar results were also observed in H9C2 cells. Altogether, these findings indicate that AS-IV modulates Ca2+ homeostasis to inhibit apoptosis, protecting against CIH-induced myocardial injury eventually, suggesting it may be a potential agent for cardiac damage of OSAS patients. SIGNIFICANCE OF THE STUDY: Chronic intermittent hypoxia (CIH) is a great contributor of OSAS, which is closely associated with cardiovascular diseases. It is necessary for developing a promising drug to attenuate CIH-induced myocardial injury. This work suggests that AS-IV can attenuate myocardial apoptosis and calcium disruption, thus protecting against CIH-induced myocardial injury. It may represent a novel therapeutic for cardiac damage of OSAS.

Potential Role of mRNAs and LncRNAs in Chronic Intermittent Hypoxia Exposure-Aggravated Atherosclerosis.

Atherosclerosis is the pathological basis of cardiovascular disease. Obstructive sleep apnea (OSA) aggravates atherosclerosis, and chronic intermittent hypoxia (CIH) as a prominent feature of OSA plays an important role during the process of atherosclerosis. The mechanisms of CIH in the development of atherosclerosis remain unclear. In the current study, we used microarray to investigate differentially expressed mRNAs and long non-coding RNAs (lncRNAs) in aorta from five groups of ApoE–/– mice fed with a high-fat diet and exposed to various conditions: normoxia for 8 weeks, CIH for 8 weeks, normoxia for 12 weeks, CIH for 12 weeks, or CIH for 8 weeks followed by normoxia for 4 weeks. Selected transcripts were validated in aorta tissues and RT-qPCR analysis showed correlation with the microarray data. Gene Ontology analysis and pathway enrichment analysis were performed to explore the mRNA function. Bioinformatic analysis indicated that short-term CIH induced up-regulated mRNAs involved in inflammatory response. Pathway enrichment analysis of lncRNA co-localized mRNAs and lncRNA co-expressed mRNAs were performed to explore lncRNA functions. The up-regulated mRNAs, lncRNA co-localized mRNAs and lncRNA co-expressed mRNAs were significantly associated with protein processing in endoplasmic reticulum pathway in atherosclerotic vascular tissue with long-term CIH exposure, suggesting that differentially expressed mRNAs and lncRNAs play important roles in this pathway. Moreover, a mRNA-lncRNA co-expression network with 380 lncRNAs, 508 mRNAs and 3238 relationships was constructed based on the correlation analysis between the differentially expressed mRNAs and lncRNAs. In summary, our study provided a systematic perspective on the potential function of mRNAs and lncRNAs in CIH-aggravated atherosclerosis, and may provide novel molecular candidates for future investigation on atherosclerosis exposed to CIH.

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.

Effects of 2‐APB, a Stim‐Activated TRPC‐ORAI Channels Blocker, on the Vascular Remodeling and Pulmonary Hypertension Induced by Chronic Intermittent Hypoxia

Obstructive sleep apnea (OSA), a breathing disorder characterized by episodes of chronic intermittent hypoxia (CIH), is an independent risk for systemic hypertension. Furthermore, 20–60% of OSA patients develop moderate pulmonary hypertension (PH). Rats exposed to CIH developed pulmonary vascular remodeling and PH, but the mechanisms underlaying these responses are not well known. Stim‐activated TRPC‐ORAI channel (STOC), cationic calciumpermeable channels, are overexpressed in the lung and plays a key role in the development of pulmonary vascular remodeling and PH in animals exposed to sustained hypoxia. We previously demonstrated that STOC are overexpressed in lung of rats exposed to CIH. However, whether these channels are involved in the pulmonary vascular remodeling and PH induced by CIH have not been addressed. Thus, we studied the effects of treatment of 2‐aminoethyldiphenylborinate (2‐APB), a STOC blocker, in rats exposed to CIH. Male Sprague‐Dawley rats (~200g) were exposed for 28 days to CIH (5% O2, 12 times/h, for 8h). At 14 days of CIH, rats under anesthesia were implanted with osmotic pumps containing 2‐APB (10 mg/kg/day) or its vehicle, and then rats were exposed to CIH for 14 more days. At the end of CIH exposure, the right ventricular systolic pressure (RVSP) was measure under anesthesia with urethane/α‐cloralose (800 mg/kg, i.p). Animals were then euthanized and lungs dissected to determine vascular remodeling in arteries of 200–300 μm, and the mRNA levels of the STOC forming subunits TRPC1, TRPC4, TRPC6, ORAI 1 and ORAI 2 by qPCR. 2‐APB infusion prevented the increase of the RVSP (36.5 ± 1.1 mmHg vs. 24.7 ± 1.6 mmHg, 28d‐CIH vs. 2‐APB respectively), and the pulmonary vascular remodeling (66.3 ± 9.4 % vs 48.2 ± 3.5 %, CIH, 28d‐CIH vs. 2‐APB, respectively). In addition, 2‐APB treatment reverted the increase in the mRNA levels of TRPC1, TRPC4 and TRPC6, but induced an overexpression of ORAI 1, while ORAI 2 remained unchanged. Present results suggest that STOC plays a key role in the developing of the vascular remodeling and pulmonary hypertension induced by CIH.Support or Funding InformationSUPPORTED BY FONDECYT 1150040 and 1180341

MiRNA‐Mediated Suppression of a Cardioprotective Cardiokine as a Novel Mechanism Exacerbating Post‐MI Remodeling by Sleep Breathing Disorders

BackgroundObstructive sleep apnea (OSA), a sleep breathing disorder in which chronic intermittent hypoxia (CIH) is the primary pathology, is associated with multiple cardiovascular diseases. However, whether and how CIH may affect cardiac remodeling following myocardial infarction (MI) remains unknown.Methods and ResultsAdult male mice were subjected to MI (4 weeks) with and without CIH (4 or 8 weeks). CIH prior to MI (CIH+MI) had no significant effect upon post‐MI remodeling. However, double CIH exposure (CIH+MI+CIH) or CIH only during the MI period (MI+CIH) significantly exacerbated pathologic remodeling and reduced survival rate. Mechanistically, CIH activated TGF‐b/Smad signaling and enhanced cardiac epithelial to mesenchymal transition, markedly increasing post‐MI cardiac fibrosis. Transcriptome analysis revealed that, among 15 genes significantly downregulated (MI+CIH versus MI), Ctrp9 (a novel cardioprotective cardiokine) was one of the most significantly inhibited genes. Rt‐PCR/Western analysis confirmed that cardiomyocyte Ctrp9 expression was significantly reduced in MI+CIH mice. RNA‐seq, Rt‐PCR, and dual‐luciferase reporter assays identified that miR‐214‐3p is a novel Ctrp9 targeting miRNA. Its upregulation is responsible for Ctrp9 gene suppression in MI+CIH. Finally, AAV9‐mediated cardiac‐specific Ctrp9 overexpression or rCTRP9 administration inhibited TGF‐b/Smad and Wnt/β‐catenin pathways, attenuated interstitial fibrosis, improved cardiac function, and enhanced survival rate in MI+CIH animals.ConclusionsThis study provides the first evidence that MI+CIH upregulates miR‐214‐3p, suppresses cardiac CTRP9 expression, and exacerbates cardiac remodeling, suggesting that CTRP9 may be a novel therapeutic target against pathologic remodeling in MI patients with OSA.

Huperzine A, reduces brain iron overload and alleviates cognitive deficit in mice exposed to chronic intermittent hypoxia

Chronic intermittent hypoxia (CIH) is a consequence of obstructive sleep apnea (OSA), which increases reactive oxygen species (ROS) generation, resulting in oxidative damage and neurocognitive impairment. This study was designed to determine whether abnormal iron metabolism occurs in the brain under conditions of CIH and whether Huperzine A (HuA) could improve abnormal iron metabolism and neurological damage. The mouse model of CIH was established by reducing the percentage of inspired O2 (FiO2) from 21% to 9% 20 times/h for 8 h/day, and Huperzine A (HuA, 0.1 mg/kg, i.p.) was administered during CIH exposure for 21 days. HuA significantly improved cognitive impairment and neuronal damage in the hippocampus of CIH mice via increasing the ratio of Bcl-2/Bax and inhibiting caspase-3 cleavage. HuA considerably decreased ROS levels by downregulating the high levels of NADPH oxidase (NOX 2, NOX 4) mediated by CIH. There was an overload of iron, which was characterized by high levels of ferritin (FTL and FTH) and transferrin receptor 1 (TfR1) and low levels of ferroportin 1 (FPN1) in the hippocampus of CIH mice. Decreased levels of TfR1 and FTL proteins observed in HuA treated CIH group, could reduce iron overload in hippocampus. HuA increased PSD 95 protein expression, CREB activation and BDNF protein expression to protect against synaptic plasticity impairment induced by CIH. HuA acts as an effective iron chelator to attenuate apoptosis, oxidative stress and synaptic plasticity mediated by CIH.

Effects of Xiaotan Huayu Liqiao Formula on cognitive impairment in mice exposed to chronic intermittent hypoxia

Objective: To investigate the improvement effects of Xiaotan Huayu Liqiao Formula on cognitive impairment in mice exposed to chronic intermittent hypoxia (CIH), and to explore the related mechanisms. Methods: Forty-eight male C57 BL/6 mice were randomly divided into four groups as Normoxia, CIH, Formula+CIH and Formula group. Mice were exposed to normoxia in the normoxia and formula group, or intermittent hypoxia in CIH or Formula+CIH group (in the chambers, mice were filled with 100% N2 to produce FiO2 of 9% for 1. 5 min. The FiO2 gradually returned to 21% over the remainder of each cycle. The exposure cycle was repeated every 3 min, 8 h/day for 35 days). Mice were treated with Xiaotan Huayu Liqiao Formula at the dose of 26. 8 g/kg by intragastric administration before CIH exposure. Meanwhile, mice in CIH and normoxia group were given the same volume of normal saline. When the experiment lasts for 26-35 d, Morris water maze was used to detect cognitive dysfunction in mice. At the end of 35 days, Y-maze was performed in each group. After anesthesia, hippocampus was isolated for morphological observation and Western blot ananlysis. Nissl staining and electron microscopy were adopted to assess the neuronal damage in hippocampus, and Western blot was used to detect the levels of PSD-95 and synapsin expression. Results: Compared with normoxia group,the performance of CIH mice was significantly reduced in Morris water maze and Y-maze(P＜0. 01,P＜0. 01). Both the number of Nissl staining positive cells and the thickness of the postsynaptic density in hippocampus were significantly reduced. And, the levels of PSD-95 expression in hippocampus was also decreased in the CIH group(P＜0. 01), however, no significant change of synapsin expression was observed. Compared to CIH group, administration of Xiaotan Huayu Liqiao Formula markedly improved performance of mice in Morris water maze and Y-maze (P＜0. 01), increased Nissl staining positive cells and the thickness of the postsynaptic density and PSD-95 expression in hippocampus (P＜0. 01). Conclusion: Xiaotan Huayu Liqiao Formula could alleviate the structural and functional impairment of the postsynaptic dense area, and improved CIH-induced cognitive dysfunction.

Resveratrol Attenuates Intermittent Hypoxia-Induced Lung Injury by Activating the Nrf2/ARE Pathway.

Obstructive sleep apnea (OSA) is associated with lung injury. As a novel pathophysiological hallmark of OSA, chronic intermittent hypoxia (CIH) enhances apoptosis. The present study aims to evaluate the effect of resveratrol (Res) on CIH-induced lung apoptosis and inflammation in a rat model of CIH. Rats were randomly allocated to normoxia (control), CIH, and CIH + Res groups (n = 10 in each group). The CIH exposure duration was 12weeks. Rats in the CIH + Res group were additionally administered Res (50mgkg-1d-1). Inflammatory cytokine levels were detected by enzyme-linked immunosorbent assays (ELISAs). A terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay was conducted to evaluate the apoptosis rate. Bax, cleaved caspase-3, Nrf2 and HO-1 protein levels were detected by western blotting. The IL-6 and TNF-α levels in the serum and alveolar lavage fluid in the CIH group were markedly higher than those in the control group. The percentage of apoptotic cells in the CIH group was higher than that in the control group. Bax and cleaved caspase-3 protein levels were increased in the CIH group compared with those in the control group. Nrf2 and HO-1 protein levels were decreased in the CIH group compared with those in the control group (p < 0.05). Compared with the CIH group, rats in the CIH + Res group had lower percentages of apoptotic cells, lower IL-6, TNF-α, Bax and cleaved caspase-3 protein levels, and higher Nrf2 and HO-1 protein levels (p < 0.05). Res attenuates CIH-related inflammatory reactions and apoptosis in lung tissue by activating the Nrf2/ARE pathway.

MiRNA-Mediated Suppression of a Cardioprotective Cardiokine as a Novel Mechanism Exacerbating Post-MI Remodeling by Sleep Breathing Disorders.

Obstructive sleep apnea-hypopnea syndrome, a sleep breathing disorder in which chronic intermittent hypoxia (CIH) is the primary pathology, is associated with multiple cardiovascular diseases. However, whether and how CIH may affect cardiac remodeling following myocardial infarction (MI) remains unknown. To determine whether CIH exposure at different periods of MI may exacerbate post-MI heart failure and to identify the mechanisms underlying CIH-exacerbated post-MI remodeling. Adult male mice were subjected to MI (4 weeks) with and without CIH (4 or 8 weeks). CIH before MI (CIH+MI) had no significant effect on post-MI remodeling. However, double CIH exposure (CIH+MI+CIH) or CIH only during the MI period (MI+CIH) significantly exacerbated pathological remodeling and reduced survival rate. Mechanistically, CIH activated TGF-β (tumor growth factor-β)/Smad (homologs of both the Drosophila protein MAD and the C. elegans protein SMA) signaling and enhanced cardiac epithelial to mesenchymal transition, markedly increasing post-MI cardiac fibrosis. Transcriptome analysis revealed that, among 15 genes significantly downregulated (MI+CIH versus MI), Ctrp9 (a novel cardioprotective cardiokine) was one of the most significantly inhibited genes. Real-time polymerase chain reaction/Western analysis confirmed that cardiomyocyte CTRP9 expression was significantly reduced in MI+CIH mice. RNA-sequencing, real-time polymerase chain reaction, and dual-luciferase reporter assays identified that microRNA-214-3p is a novel Ctrp9 targeting miRNA. Its upregulation is responsible for Ctrp9 gene suppression in MI+CIH. Finally, AAV9 (adeno-associated virus 9)-mediated cardiac-specific CTRP9 overexpression or rCTRP9 (recombinated CTRP9) administration inhibited TGF-β/Smad and Wnt/β-catenin pathways, attenuated interstitial fibrosis, improved cardiac function, and enhanced survival rate in MI+CIH animals. This study provides the first evidence that MI+CIH upregulates miR-214-3p, suppresses cardiac CTRP9 (C1q tumor necrosis factor-related protein-9) expression, and exacerbates cardiac remodeling, suggesting that CTRP9 may be a novel therapeutic target against pathological remodeling in MI patients with obstructive sleep apnea-hypopnea syndrome.

Potential Contribution of Carotid Body-Induced Sympathetic and Renin-Angiotensin System Overflow to Pulmonary Hypertension in Intermittent Hypoxia.

Obstructive sleep apnea (OSA), featured by chronic intermittent hypoxia (CIH), is an independent risk for systemic hypertension (HTN) and is associated with pulmonary hypertension (PH). The precise mechanisms underlying pulmonary vascular remodeling and PH in OSA are not fully understood. However, it has been suggested that lung tissue hypoxia, oxidative stress, and pro-inflammatory mediators following CIH exposure may contribute to PH. New evidences obtained in preclinical OSA models support that an enhanced carotid body (CB) chemosensory reactiveness to oxygen elicits sympathetic and renin-angiotensin system (RAS) overflow, which contributes to HTN. Moreover, the ablation of the CBs abolished the sympathetic hyperactivity and HTN in rodents exposed to CIH. Accordingly, it is plausible that the enhanced CB chemosensory reactivity may contribute to the pulmonary vascular remodeling and PH through the overactivation of the sympathetic-RAS axis. This hypothesis is supported by the facts that (i) CB stimulation increases pulmonary arterial pressure, (ii) denervation of sympathetic fibers in pulmonary arteries reduces pulmonary remodeling and pulmonary arterial hypertension (PAH) in humans, and (iii) administration of angiotensin-converting enzyme (ACE) or blockers of Ang II type 1 receptor (ATR1) ameliorates pulmonary remodeling and PH in animal models. In this review, we will discuss the supporting evidence for a plausible contribution of the CB-induced sympathetic-RAS axis overflow on pulmonary vascular remodeling and PH induced by CIH, the main characteristic of OSA.

Effect of age on the cardiovascular remodelling induced by chronic intermittent hypoxia as a murine model of sleep apnoea.

Chronic intermittent hypoxia (CIH) is a major determinant of the cardiovascular morbidity associated with obstructive sleep apnoea (OSA), and the magnitude of CIH impact may be influenced by ageing. Here, we assessed the role of ageing in the early cardiovascular structural remodelling induced by severe CIH in a murine model of OSA. Cardiovascular remodelling was assessed in young (2 months old, n = 20) and aged (18 months old, n = 20) C57BL/6 female mice exposed to CIH (20% O2 for 40 s, 5% O2 for 20 s) or normoxia (room air) for 8 weeks (6 h/day). Early vascular remodelling was observed in young mice exposed to CIH as illustrated by intima-media thickening (mean change: 4.6 ± 2.6 μm; P = 0.02), elastin fibre disorganization (mean change: 9.2 ± 4.5%; P = 0.02) and fragmentation (mean change: 2.5 ± 0.8%; P = 0.03), and collagen (mean change: 3.2 ± 0.6%; P = 0.001) and mucopolysaccharide accumulation (mean change: 2.4 ± 0.8%; P = 0.01). In contrast, vascular remodelling was not apparent in aged mice exposed to CIH. Furthermore, left ventricular perivascular fibrosis (mean change: 0.71 ± 0.1; P < 0.001) and hypertrophy (mean change: 0.17 ± 0.1; P = 0.038) were increased by CIH exposure in young mice, but not in aged mice. Principal component analysis identified similar cardiovascular alterations among the young mice exposed to CIH and both older mouse groups, suggesting that CIH induces premature cardiovascular senescence. Cardiovascular remodelling induced by severe CIH is affected by the age at which CIH onset occurs, suggesting that the deleterious cardiovascular effects associated with CIH may be more pronounced in younger populations, and such changes resemble chronological age-related declines in cardiovascular structural integrity.

Role of NADPH oxidase in chronic intermittent hypoxia‐induced respiratory dysfunction: Insights from pharmacological and transgenic approaches

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 upper airway and diaphragm muscle function. Excessive ROS are also associated with impaired respiratory control, which manifests as destabilised breathing during sleep. There is a paucity of information regarding the molecular mechanisms underlying these effects. We sought to investigate the putative role of the superoxide‐generating NADPH oxidase 2 (NOX2) enzyme in CIH‐induced respiratory 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 for 2 weeks. Adult male (C57BL/6J) mice were assigned to one of 5 groups: normoxic controls (sham), CIH‐exposed, CIH+apocynin (NOX2 inhibitor, 2mM) given in the drinking water throughout the CIH and NOX2 null (B6.129S‐Cybbtm1Din/J) sham or CIH exposed. On day 15, whole body plethysmography was used to measure breathing parameters in unrestrained, unaesthetized mice in room air. Flow signals recorded were analysed offline and an apnoea was defined as ≥ 2 missed breaths. Sternohyoid and diaphragm muscle contractile function was examined ex vivo. Gene expression was examined by qRT‐PCR. Western blot was used to measure protein expression. NOX enzyme activity was determined using a spectrophotometric assay.CIH decreased sternohyoid and diaphragm muscle peak specific force by ~45% compared with sham exposure. CIH increased NOX enzyme activity in the sternohyoid, with no alterations in gene or protein expression of NOX subunits compared with sham. CIH decreased the expression of p22phox and rac genes and increased protein expression of NOX4 in the diaphragm, while NOX activity remained unchanged compared with sham. Administration of apocynin and NOX2 gene knockout completely prevented CIH‐induced sternohyoid and diaphragm muscle weakness. Basal minute ventilation remained unchanged following CIH exposure however the number of apnoeas per hour was increased compared with sham. Apocynin intervention 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 in wild‐type mice.Mice show signs of profound respiratory muscle dysfunction following exposure to 2 weeks of CIH. The putative NOX inhibitor, apocynin, prevents CIH‐induced respiratory muscle weakness. Studies in NOX2 null mice reveal that NOX2 is necessary for this CIH‐induced respiratory 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 OSAS). The reduction in apnoea frequency following treatment with apocynin implicates ROS in the manifestation of CIH‐induced respiratory disturbances in a non‐NOX2 dependant manner. Our results have implications for human OSAS and point to antioxidant intervention as a potential therapeutic strategy.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Role of Alpha‐1 Adrenergic Receptors (α1AR) and Norepinephrine Transporter in Parvocellular Neurons of the Hypothalamic Paraventricular Nucleus (PVN) After Chronic Intermittent Hypoxia Exposure

The Paraventricular nucleus (PVN) of the hypothalamus is critical for autonomic homeostasis and cardiorespiratory reflex responses to a variety of stimuli and stressors. This is accomplished, in part, via dense catecholaminergic and norepinephrine (NE) projections from nucleus tractus solitarii (nTS) and ventrolateral medulla (VLM). NE signaling is balanced by the uptake of NE by transporters (NET's) and the activation of one or more adrenergic receptor (AR). Within the PVN, stimulation of aAR's increases the frequency of spontaneous excitatory postsynaptic currents (sEPSCs), decreases the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) and depolarizes resting membrane potential (RMP) which elevates blood pressure (BP) and sympathetic outflow. Chronic intermittent hypoxia (CIH) is a model for obstructive sleep apnea (OSA) that elevates respiration, BP, sympathoexcitation and circulating NE levels. CIH activates not only PVN neurons but also the catecholaminergic neurons in the VLM and nTS. However, the contribution of aAR and NET in PVN neurons after CIH exposure remains elusive. In the present work, we determined the electrophysiological properties of PVN parvocellular neurons in response to a1AR activation and NET inhibition after normoxia (Norm) and CIH. Male Sprague‐Dawley rats (110–150g) were exposed to either 10 days normoxia (Norm, FiO2 = 21%) or CIH (alternating FiO2 = 21% and 6%, 8 hr/day). PVN slices (~280 μm) were generated and cell capacitance (Cm), initial input resistance (Rin), and sEPSCs were examined by whole cell patch clamp. sEPSCs were recorded under aCSF baseline or in the presence of one or more of the following: NE (10–100 μM, general AR agonist), phenylephrine (Phe, a1AR agonist, 100 μM), prazosin (a1AR antagonist, 10 μM), or NET inhibitor (desipramine, 100 μM). The Cm of Norm neurons was smaller than CIH, but CIH decreased Rin. Between Norm and CIH, baseline sEPSC frequency and amplitude were similar. Phe and NE significantly increased sEPSC frequency in Norm but not in CIH. Block of the a1AR with prazosin alone did not alter sEPSC frequency or amplitude in Norm (n=10) or CIH (n=6). NE in the presence of prazosin (n=7) prevented the increase in sEPSC frequency in Norm. In Norm (n=5) and CIH (n=6), NET block with desipramine increased sEPSC frequency but did not alter amplitude. However, the magnitude of increase in sEPSC frequency was comparable. These results demonstrate that a1AR activation participate of neuronal responses in Norm, but are minimized in CIH. NET function may be elevated after CIH exposure. The latter may contribute to reduced neuronal responses evoked by Phe after CIH, as well as compensatory mechanism caused by over sympathetic activity.Support or Funding InformationHL 098602 and HL128454This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Chronic Intermittent Hypoxia and Respiratory Motor Plasticity after Cervical Spinal Cord Contusion

Chronic intermittent hypoxia (CIH), characterized by &gt; 100 hypoxic episodes per day, is a hallmark of moderate sleep apnea, a condition highly prevalent in individuals with spinal cord injury. Both 1 day (Huxtable et al., 2013) and 7 days (Gonzalez‐Rothi, in preparation) of CIH (15 episodes/hour; 8 hours/day) abolish phrenic motor plasticity in intact rats due to neural inflammation. Although a milder CIH protocol (40 episodes/day) enhances phrenic activity in acutely injured rats (Fuller et al., 2003), the effects of CIH protocols that more closely approximate moderate sleep apnea have not been explored in animal models of chronic spinal injury. Thus, we are evaluating the impact of moderate CIH (2 min hypoxic episodes, 2 min intervals; 8 hrs; single day) on phrenic nerve activity and the capacity to elicit phrenic motor plasticity in rats with chronic C3/4 midline contusion injury (12 wks). Neurophysiology experiments conducted one day post‐CIH are ongoing and include assessments of: 1) phrenic nerve activity during baseline and maximal chemoreflex activation; and 2) moderate AIH‐induced phrenic long‐term facilitation (pLTF). Preliminary analysis suggests reduced pLTF in contused rats exposed to CIH. These experiments are the first to explore the effects of CIH on respiratory outcomes following chronic spinal contusion. Further experiments are required to explore the impact of: 1) prolonged CIH exposures on breathing ability after chronic cervical contusion; and 2) whether reducing neural inflammation will enhance phrenic plasticity and translate into improved functional recovery following chronic spinal cord injury.Support or Funding InformationThe Department of Defense: SCI160123 (EGR); and the National Institutes of Health: K12 HDO55929 (EGR)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Enhanced Carotid Body Chemosensory Discharge is Essential for the Hypertension Induced by Chronic Intermittent Hypoxia

Chronic intermittent hypoxia (CIH), the main feature of obstructive sleep apnea, enhances carotid body (CB) discharges, leading to sympathetic overflow and hypertension. CIH produces oxidative stress (ROS) in the CB, which contributes to enhance the CB drive to the brainstem and hypothalamic nuclei related to the cardiorespiratory control, such as the nucleus of the tractus solitary (NTS), the rostral ventrolateral medulla (RVLM) and the paraventricular nucleus (PVN). We hypothesized whether the enhanced CB chemosensory drive may increase ROS levels in central cardiovascular nuclei during CIH‐induced hypertension. Accordingly, we studied the effects of CB ablation on ROS formation in the NTS, PVN and RVLM of hypertensive CIH‐rats. Male Sprague‐Dawley rats (200 g) were exposed to CIH (5–6% inspired O2 for 20s, followed by room air for 280s, 12 times/h, 8 h/day), for 28 days. Arterial blood pressure (BP) was measured by radio‐telemetry (DSI, USA). Following 21 days of CIH exposure, rats were anesthetized with isofluorane in 2% O2, and the CBs were cryogenically destroyed bilaterally using a fine‐tipped forcep cooled in liquid N2. Then, rats were maintained in CIH conditions for one more week. Another group of rats were maintained in CIH or Sham conditions for 28 days. At the end of the experiments, all animals were euthanized (pentobarbital 120mg/kg i.p.) and brains were removed and frozen at −20°C. Coronal sections of the NTS, RVLM and PVN were cut and incubated for 30 min in the dark with the ROS sensitive fluorescent probe dihydroethidium (DHE 10μM, Invitrogen USA). DHE fluorescence was measured using epifluorescence microscopy (Olympus Optical, Japan) at an excitation wavelength of 543 nm. Data were analyzed by one‐way ANOVA followed by Bonferroni's test. Compared to Sham rats, CIH‐rats showed a significant increase in mean BP (□ 10 mmHg, p&lt;0.05). The CIH‐induced hypertension was abolished by the ablation of the CBs (CBA). ROS formation was markedly reduced by CBA in the NTS, RVLM and PVN of CIH‐exposed rats (CIH vs. CIH‐CBA: NTS: 6.2±1.5 vs. 1.5±02 au, RVLM: 4.9± 1.5 vs. 2.1±1.6 au, PVN: 5.4±1.8 vs. 1.6±05 au, p &lt;0.05, n= 5–6 rats). Results support the evidence for an essential role of the CBs in the development and maintenance of the CIH‐induced hypertension and oxidative stress present in the NTS, RVLM and PVNSupport or Funding InformationFONDECYT 1150040This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.