Intermittent Hypoxia Hypercapnia Research Articles

Intermittent hypoxia and hypercapnia induces inhibitor of nuclear factor-κB kinase subunit β-dependent atherosclerosis in pulmonary arteries.

Clinical studies have shown that obstructive sleep apnea (OSA) increases atherosclerosis risk. The inflammation, especially mediated by the macrophages via nuclear factor-κB (NF-κB), has been speculated to contribute to atherogenicity in OSA patients. Inhibitor of NF-κB kinase-β (IKKβ) is an essential element of the NF-κB pathway and is linked to atherosclerosis. We previously reported that atherosclerosis was accelerated in pulmonary artery (PA) but not in aorta when low-density lipoprotein receptor knockout (Ldlr-/-) mice were exposed to intermittent hypoxia/hypercapnia (IHH), a surrogate for recurrent upper-airway obstruction. Therefore, we hypothesized that IKKβ-dependent NF-κB activation in monocytes and macrophages plays a role in IHH-induced PA atherosclerosis. To test this hypothesis, myeloid restricted IKKβ deletion (IkkβΔMye) or control (IkkβF/F) mice were crossed with Ldlr-/- mice to generate double-knockout mice. Then, the mice were exposed to IHH or room air (Air) on high-fat diet for 8 or 16 wk. Lesions of PA and aorta were examined in IkkβΔMye;Ldlr-/- and IkkβF/F;Ldlr-/- male mice under IHH vs. Air. The results revealed that IKKβ deletion abolished IHH-induced PA atherosclerosis after 8-wk exposure but not after 16-wk exposure (8 wk: IkkβF/F;Ldlr-/-, IHH 13.5 ± 1.4 vs. Air 5.7 ± 0.7%, P < 0.01; IkkβΔMye;Ldlr-/-, IHH 7.4 ± 1.9% vs. Air 4.6 ± 1.3%, P = 0.24). Both IKKβ deletion and IHH had no effects on atherosclerosis in the aorta. Our findings demonstrate that IKKβ-dependent NF-κB activity in myeloid-lineage cells plays a critical role in IHH-induced PA atherosclerosis at the early stage.

Transcriptional Response to Intermittent Hypoxia in the Heart of Germ‐Free Mice

Accumulating evidence has demonstrated that the gut microbiota plays a critical role in health and disease and regulates the organismal response to environmental stresses. For example, we have found that intermittent hypoxia/hypercapnia (IHH), a condition mimicking obstructive sleep apnea in humans, induced significant alterations in gut microbiome/metabolism and enhanced atherogenesis and progression in two atherosclerosis mouse models (i.e., the LDLR−/− and ApoE−/− mice). Indeed, with a joint microbiome and metabolome analysis, we have determined that IHH‐induced remarkable compositional changes in both microbial (&gt;10%, most remarkably in Clostridia) and metabolites (&gt;22%) in the gut of LDLR−/− mice, which are tightly correlated with a significantly enhanced atherosclerotic lesion formation in pulmonary arteries and aorta. In the current project, in order to further understand the role of the microbiome in the cardiovascular response to hypoxia and/or hypercapnia, we developed a set up that allows the treatment of experimental mice with different levels of O2 and/or CO2 in a germ‐free environment. Using this experimental set up, we studied the impact of the microbiome on transcriptional response to intermittent hypoxia (IH) in the left ventricle of mouse heart. We identified modest but significant changes in gene expression in both conventionally reared and germ‐free mice under IH condition. We found 52 significantly altered genes (19 up‐regulated and 33 down‐regulated) in the conventional mice and 76 significantly altered genes (24 upregulated and 52 down‐regulated) in the germ‐free mice. In addition, we found that these significantly altered genes play important roles in the development and function of cardiomyocytes and endothelial cells. Interestingly, there were only 15 genes (2 up‐regulated and 13 down‐regulated) that were commonly altered in both conventional and germ‐free mice, representing 28% changes in conventional mice and 20% changes in germ‐free mice, respectively. In summary, we discovered that IH induces distinct transcriptional alterations between conventional and germ‐free mice, which demonstrates that microbiome plays a critical role in the transcriptional response to IH in the left ventricle of mouse heart.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Electrical stimulation influences chronic intermittent hypoxia-hypercapnia induction of muscle fibre transformation by regulating the microRNA/Sox6 pathway.

Chronic obstructive pulmonary disease can cause muscle fibre transformation due to chronic intermittent hypoxia-hypercapnia (CIHH). Studies have shown that high expression of Sox6 in muscle could suppress type-I fibres through downregulating the PPARβ (peroxisome proliferator-activated receptor β)/ERRγ (oestrogen-related receptor γ)/microRNA pathway. However, whether this pathway is involved in CIHH-induced muscle fibre transformation is unknown. Electrical stimulation (ES) is an effective approach to ameliorate muscle dysfunction. Here, we explored the effects of ES on CIHH-induced muscle fibre transformation and the microRNA/Sox6 pathway. After CIHH exposure, both the soleus (SOL) and gastrocnemius (GC) muscles showed decreased type-I fibres. The PPARβ/ERRγ/mir-499&208b (PEM, for GC) and PPARβ/mir-499&208b (PM, for SOL) signalling cascades were suppressed, followed by elevated Sox6 expression. Low frequency electrical stimulation (LFES) activated the PEM/PM pathway and enhanced type-I fibre numbers through suppressing Sox6 in SOL and GC. High frequency electrical stimulation (HFES) promoted type-I fibre expression through activating the PEM pathway in GC. Although PPARβ expression and type-I fibres were suppressed in SOL after HFES, no significant change was found in mir-499&208b/Sox6 expression. These results suggest that the microRNA/Sox6 pathway is disturbed after CIHH. Both low and high frequency electrical stimulations induce muscle fibre transformation partly through regulating the microRNA/Sox6 pathway.

Electrical Stimulation Improves Rat Muscle Dysfunction Caused by Chronic Intermittent Hypoxia-Hypercapnia via Regulation of miRNA-Related Signaling Pathways

Skeletal muscle dysfunction in chronic obstructive pulmonary disease (COPD) patients is common. Neuromuscular Electrical Stimulation (NMES) is a powerful exercise training that may relieve muscle dysfunction in COPD. This study investigated whether electrical stimulation may have atypical adaptations via activation of miRNA related pathways in counteracting COPD muscle dysfunction. Forty-eight male Sprague-Dawley rats were randomly assigned to 3 groups. With the exception of the rats in the control group, the experimental rats were exposed to chronic intermittent hypoxia-hypercapnia (CIHH) (9∼11%O2,5.5∼6.5%CO2) for 2 or 4 weeks. Electrical stimulation was performed immediately after each CIHH session. Following assessment of the running capacity, biopsy samples were obtained from the gastrocnemius of the rats. The miR-1, miR-133a and miR-133b levels were measured, as well as their related proteins: phosphorylation of Akt (p-AKT), PGC-1alpha (PGC-1α), histone deacetylase 4 (HDAC4) and serum response factor (SRF). Myosin heavy chainⅡa (MHCⅡa) and myosin heavy chainⅡb (MHCⅡb) were also measured to assess fiber type changes. After 2 weeks, compared with the controls, only miR-1 and miR-133a were significantly increased (p<0.05) in the exposure group. After 4 weeks, the exposure group exhibited a decreased running distance (p = 0.054) and MHCⅡa-to-MHCⅡb shift (p<0.05). PGC-1α (p = 0.051), nuclear HDAC4 (p = 0.058), HDAC4, p-AKT, PGC-1α and SRF was also significantly decreased (p<0.05). In contrast, miR-1 and miR-133a were significantly increased (p<0.05). Four weeks of electrical stimulation can partly reversed those changes, and miR-133b exhibited a transient increase after 2 weeks electrical stimulation. Our study indicate miRNAs may have roles in the response of CIHH-impaired muscle to changes during electrical stimulation.

Oxytocin neuron activation prevents hypertension that occurs with chronic intermittent hypoxia/hypercapnia in rats.

Hypertension is a common outcome associated with obstructive sleep apnea (OSA), a prevalent yet poorly treated cardiovascular disease. Recent studies showed oxytocin (OXT), released from hypothalamic paraventricular nucleus (PVN) neurons, activates cardiac vagal neurons in the dorsal motor nucleus of the vagus (DMNX) and may blunt cardiovascular responses to stress. This study tests whether the release of OXT from PVN fibers in the DMNX is diminished with chronic intermittent hypoxia-hypercapnia (CIH/H) exposure, an animal model of OSA, and whether activation of PVN OXT neurons restores OXT release in the DMNX and prevents the hypertension resulting from CIH/H. To assess OXT release from PVN fibers, Chinese hamster ovarian (CHO) cells were engineered to be highly sensitive to OXT by stable expression of the human recombinant OXT receptor and the calcium indicator R-GECO1. PVN fibers in the DMNX were selectively photoactivated in vitro by expression of channelrhodopsin. The release of OXT onto CHO cells in the DMNX was blunted in rats exposed to 21 days of CIH/H. Chronic activation of PVN OXT neurons in vivo, using designer receptors exclusively activated by designer drugs, restored the release of OXT onto CHO cells in the DMNX. Chronic PVN OXT neuron activation in vivo also prevented the hypertension that occurred in conscious unrestrained telemetry-equipped sham rats exposed to 3 wk of CIH/H. These results demonstrate that chronic activation of OXT neurons restores the release of OXT from PVN fibers in the DMNX and prevents the hypertension that occurs with 3 wk of CIH/H exposure.

Restoration of Oxytocin Neuron Activity Prevents Hypertension Caused by Chronic Intermittent Hypoxia/Hypercapnia

New promising work has indicated oxytocin (OXT) receptor activation is blunted in cardiovascular diseases (CVDs) and OXT receptor activation may be a novel target to increase parasympathetic activity to the heart and treat CVDs. This study tests the hypotheses that the release of OXT from hypothalamic paraventricular (PVN) neurons onto cardiac vagal neurons (CVNs) is diminished with chronic intermittent hypoxia‐hypercapnia (CIH/H), an animal model of OSA, and furthermore that selective activation of PVN OXT neurons can reduce adverse cardiovascular consequences that occur with CIH/H. Optogenetic stimulation of channelrhodopsin expressing PVN fibers evoked large transient increases in Ca2+ in CHO cells stably transfected to express the human recombinant OXT receptor and the red fluorescent calcium indicator, R‐GECO1, dispersed adjacent to CVNs. The release of OXT onto CVNs upon photoactivation of PVN fibers was blunted in animals exposed to 21 days of CIH/H. To examine if restoration of OXT neuron activity can prevent the adverse cardiovascular effects of CIH/H in vivo, we selectively expressed excitatory DREADDs in PVN OXT neurons, and implanted animals with telemetry devices to monitor blood pressure (BP) and EKG activity. Selective chronic activation of PVN OXT neurons decreased resting BP and HR, and perhaps more importantly chronic PVN OXT neuron activation prevented the elevations in BP and HR that occur with CIH/H. These results indicate excitation of parvocellular PVN fibers releases OXT at brainstem CVN targets, and selective activation of OXT neurons in the PVN decreases resting BP and HR, and prevents the elevations in BP and HR that occur after 21 days of CIH/H.

Protective effect of Dl-3n-butylphthalide on learning and memory impairment induced by chronic intermittent hypoxia-hypercapnia exposure.

Cognitive impairment is a common finding in patients with chronic obstructive pulmonary disease (COPD), but little attention has been focused on therapeutic intervention for this complication. Chronic intermittent hypoxia hypercapnia (CIHH) exposure is considered to be responsible for the pathogenesis of COPD. Dl-3n-Butylphthalide (NBP), extracted from Apium graveolens Linn, has displayed a broad spectrum of neuroprotective properties. Our study aimed to investigate the potential of NBP on CIHH-induced cognitive deficits. The cognitive function of rats after CIHH exposure was evaluated by the Morris water maze, which showed that the NBP treated group performed better in the navigation test. NBP activated BDNF and phosphorylated CREB, the both are responsible for neuroprotection. Additionally, NBP decreased CIHH induced apoptosis. Moreover, NBP further induced the expression of HIF-1α, accompanied by the up-regulation of the autophagy proteins Bnip3, Beclin-1 and LC3-II. Finally, NBP also reversed the decreased expression of SIRT1 and PGC-1α, but the expression of Tfam, Cox II and mtDNA remained unchanged. These results suggested that the neuroprotective effects of NBP under CIHH condition possibly occurred through the inhibition of apoptosis, promotion of hypoxia-induced autophagy, and activation of the SIRT1/PGC-1α signalling pathway, while stimulation of mitochondrial biogenesis may not be a characteristic response.

Chronic intermittent hypoxia-hypercapnia blunts heart rate responses and alters neurotransmission to cardiac vagal neurons.

Patients with obstructive sleep apnoea experience chronic intermittent hypoxia-hypercapnia (CIHH) during sleep that elicit sympathetic overactivity and diminished parasympathetic activity to the heart, leading to hypertension and depressed baroreflex sensitivity. The parasympathetic control of heart rate arises from pre-motor cardiac vagal neurons (CVNs) located in nucleus ambiguus (NA) and dorsal motor nucleus of the vagus (DMNX). The mechanisms underlying diminished vagal control of heart rate were investigated by studying the changes in blood pressure, heart rate, and neurotransmission to CVNs evoked by acute hypoxia-hypercapnia (H-H) and CIHH. In vivo telemetry recordings of blood pressure and heart rate were obtained in adult rats during 4 weeks of CIHH exposure. Retrogradely labelled CVNs were identified in an in vitro brainstem slice preparation obtained from adult rats exposed either to air or CIHH for 4 weeks. Postsynaptic inhibitory or excitatory currents were recorded using whole cell voltage clamp techniques. Rats exposed to CIHH had increases in blood pressure, leading to hypertension, and blunted heart rate responses to acute H-H. CIHH induced an increase in GABAergic and glycinergic neurotransmission to CVNs in NA and DMNX, respectively; and a reduction in glutamatergic neurotransmission to CVNs in both nuclei. CIHH blunted the bradycardia evoked by acute H-H and abolished the acute H-H evoked inhibition of GABAergic transmission while enhancing glycinergic neurotransmission to CVNs in NA. These changes with CIHH inhibit CVNs and vagal outflow to the heart, both in acute and chronic exposures to H-H, resulting in diminished levels of cardioprotective parasympathetic activity to the heart as seen in OSA patients.

Efficacy of lovastatin on learning and memory deficits caused by chronic intermittent hypoxia-hypercapnia: through regulation of NR2B-containing NMDA receptor-ERK pathway.

BackgroundChronic intermittent hypoxia-hypercapnia (CIHH) exposure leads to learnning and memory deficits in rats. Overactivation of N-methyl-D-aspartate receptors(NMDARs) can lead to the death of neurons through a process termed excitotoxicity, which is involved in CIHH-induced cognitive deficits. Excessively activated NR2B (GluN2B)-containing NMDARs was reported as the main cause of excitotoxicity. The ERK1/2 (extracellular signal-regulated kinase 1/2) signaling cascade acts as a key component in NMDARs-dependent neuronal plasticity and survival. Ca2+/calmodulin-dependent protein kinase II (CaMKII), synapse-associated protein 102 (SAP102) and Ras GTPase-activating protein (SynGAP) have been shown to be involved in the regulation of NMDAR-ERK signalling cascade. Recent studies revealed statins (the HMG-CoA reductase inhibitor) have effect on the expression of NMDARs. The present study intends to explore the potential effect of lovastatin on CIHH-induced cognitive deficits and the NR2B-ERK signaling pathway.Methods and FindingsEighty male Sprague Dawley rats were randomly divided into five groups. Except for those in the control group, the rats were exposed to chronic intermittent hypoxia-hypercapnia (CIHH) (9∼11%O2, 5.5∼6.5%CO2) for 4 weeks. After lovastatin administration, the rats performed better in the Morris water maze test. Electron microscopy showed alleviated hippocampal neuronal synaptic damage. Further observation suggested that either lovastatin or ifenprodil (a selective NR2B antagonist) administration similarly downregulated NR2B subunit expression leading to a suppression of CaMKII/SAP102/SynGAP signaling cascade, which in turn enhanced the phosphorylation of ERK1/2. The phosphorylated ERK1/2 induced signaling cascade involving cAMP-response element-binding protein (CREB) phosphorylation and brain-derived neurotrophic factor (BDNF) activation, which is responsible for neuroprotection.ConclusionsThese findings suggest that the ameliorative cognitive deficits caused by lovastatin are due to the downregulation of excessive NR2B expression accompanied by increased expression of ERK signaling cascade. The effect of NR2B in upregulating pERK1/2 maybe due, at least in part, to inactivation of CaMKII/SAP102/SynGAP signaling cascade.

Intermittent hypoxia and hypercapnia induce pulmonary artery atherosclerosis and ventricular dysfunction in low density lipoprotein receptor deficient mice

Patients with obstructive sleep apnea, who experience episodic hypoxia and hypercapnia during sleep, often demonstrate increased inflammation, oxidative stress, and dyslipidemia. We hypothesized that sleep apnea patients would be predisposed to the development of atherosclerosis. To dissect the mechanisms involved, we developed an animal model in mice whereby we expose mice to intermittent hypoxia/hypercapnia (IHH) in normobaric environments. Two- to three-month-old low-density lipoprotein receptor deficient (Ldlr(-/-)) mice were fed a high-fat diet for 8 or 16 wk while being exposed to IHH for either 10 h/day or 24 h/day. Plasma lipid levels, pulmonary artery and aortic atherosclerotic lesions, and cardiac function were then assayed. Surprisingly, atherosclerosis in the aorta of IHH mice was similar compared with controls. However, in IHH mice, atherosclerosis was markedly increased in the trunk and proximal branches of the pulmonary artery of exposed mice; even though plasma cholesterol and triglycerides were lower than in controls. Hemodynamic analysis revealed that right ventricular maximum pressure and isovolumic relaxation constant were significantly increased in IHH exposed mice and left ventricular % fractional shortening was reduced. In conclusion, 1) Intermittent hypoxia/hypercapnia remarkably accelerated atherosclerotic lesions in the pulmonary artery of Ldlr(-/-) mice and 2) increased lesion formation in the pulmonary artery was associated with right and left ventricular dysfunction. These findings raise the possibility that patients with obstructive sleep apnea may be susceptible to atherosclerotic disease in the pulmonary vasculature, an observation that has not been previously recognized.

Intermittent hypoxia/hypercapnia alters the transcriptome of mouse coronary artery endothelial cells

Intermittent hypoxia/hypercapnia (IHH) occurs in a variety of diseases, especially in sleep‐disordered breathing conditions (e.g. obstructive sleep apnea–OSA). IHH may contribute to the development of hypertension and atherosclerosis. To study the impact of IHH on global transcriptional remodeling, we exposed neonatal (NN) and adult C57BL/6 mice to IHH (8% O2; 8% CO2; 4 min cycles) for 4 wks. A recovery group of IHH‐exposed adult mice were returned to room air for 4 wks. Global mRNA levels of isolated coronary artery endothelial cells (ECs) were determined with microarrays. Clustering of differentially expressed genes revealed very little overlap of altered genes in adult and NN ECs (e.g. Notch 1 and Notch 4 were downregulated in NN ECs while Notch 3 was upregulated in adult ECs). In adult ECs three patterns of gene regulation were observed: 1) genes altered by IHH that returned to basal levels with reoxygenation (e.g. leukocyte migration and blood vessel development); 2) genes with sustained changes in IHH and reoxygenation (e.g. autophagy and antigen binding); 3) genes altered only by reoxygenation (e.g. immune response and DNA repair). In conclusion, IHH differentially affects the transcriptome of NN and adult ECs. IHH mediates distinct signaling pathways related to immune and cell stress responses in adult ECs. These changes may contribute to EC dysfunction and the pathophysiology of OSA.

Augmented Endothelin Vasoconstriction in Intermittent Hypoxia-Induced Hypertension

We reported previously that simulating sleep apnea in rats by exposing them 7 hours per day to intermittent hypoxia/hypercapnia (IH) elevates plasma endothelin-1 and causes hypertension, which is reversed by an endothelin-1 antagonist. We hypothesized that in this model of sleep apnea-induced hypertension, vascular sensitivity to endothelin-1 is increased in combination with the elevated plasma endothelin-1 to cause the endothelin-1-dependent hypertension. In small mesenteric arteries with endothelial function disabled by passing air through the lumen, diameter and vessel wall [Ca2+] were recorded simultaneously. IH arteries demonstrated increased constrictor sensitivity to endothelin-1 (percentage max constriction 100+/-0% IH versus 80+/-10% Sham; P<0.05). This was accompanied by increased calcium sensitivity of IH arteries. In contrast, constrictor sensitivity and increases in vessel wall [Ca2+] to KCl and phenylephrine were not different between IH and Sham arteries. We have shown previously that endothelin-1 constriction in mesenteric arteries is mediated by endothelin A receptors. In the current study, the selective increase in endothelin-1 constriction in IH resistance arteries was accompanied by increased expression of endothelin A receptor expression (densitometry units 271+/-23 IH versus 158+/-25 Sham; P<0.05). Thus, IH hypertension appears to cause alterations in signaling components unique to endothelin-1 at the receptor level and in postreceptor signaling that increases calcium sensitivity during endothelin A activation. Future studies will determine the specific changes in vascular smooth muscle signaling in IH hypertension causing this augmented contractile phenotype.

Role of endothelin in intermittent hypoxia-induced hypertension.

Clinical studies suggest that sleep apnea causes systemic hypertension. In addition, patients with sleep apnea have elevated plasma levels of endothelin-1 (ET-1). We hypothesized that the intermittent hypoxia/hypercapnia (IH) associated with sleep apnea causes hypertension by increasing ET-1 production. To test this hypothesis, rats with arterial and venous catheters were placed in Plexiglas chambers. IH rat chambers were flushed with an N(2)-CO(2) mixture for 90 seconds to achieve hypoxia/hypercapnia (5% O(2)-5% CO(2)) followed by 90 seconds of compressed air to achieve normoxia (21% O(2)-0% CO(2)). Control rat chambers were flushed with 90 seconds of air-air cycles. Cycles for both groups were repeated 8 hours per day for 11 days. Resting mean arterial pressure (MAP) and heart rate were recorded daily before the start of exposure. After 11 days, MAP was significantly elevated in IH rats compared with initial MAP (109+/-5 mm Hg initial, 139+/-11 mm Hg day 11) and compared with air-air rats (110+/-4 mm Hg). On day 11, cumulative doses of PD145065 (a nonselective ET-receptor antagonist) were administered intravenously to the rats breathing room air. PD145065 caused a dose-dependent decrease in MAP in IH rats but did not alter MAP in air-air rats. Plasma ET-1 measured by radioimmunoassay was significantly increased on days 5 and 11 in the IH rats compared with day 1 and compared with air-air rats. There was no significant change in plasma ET-1 over time in air-air rats. We conclude that IH exposure increases both MAP and plasma ET-1 and that the increased ET-1 may contribute to the hypertension.