P300/CBP Regulates HIF-1-Dependent Sympathetic Activation and Hypertension by Intermittent Hypoxia.

Obstructive sleep apnea (OSA), a widespread breathing disorder, leads to intermittent hypoxia (IH). Patients with OSA and IH-treated rodents exhibit heightened sympathetic nerve activity and hypertension. Previous studies reported transcriptional activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (Nox) by HIF-1 (hypoxia-inducible factor-1) contribute to autonomic dysfunction in IH-treated rodents. Lysine acetylation, regulated by KATs (lysine acetyltransferases) and KDACs (lysine deacetylases), activates gene transcription and plays an important role in several physiological and pathological processes. This study tested the hypothesis that acetylation of HIF-1α by p300/CBP (CREB-binding protein) (KAT) activates Nox transcription, leading to sympathetic activation and hypertension. Experiments were performed on pheochromocytoma-12 cells and rats treated with IH. IH increased KAT activity, p300/CBP protein, HIF-1α lysine acetylation, HIF-1 transcription, and HIF-1 binding to the Nox4 gene promoter in pheochromocytoma-12 cells, and these responses were blocked by CTK7A, a selective p300/CBP inhibitor. Plasma norepinephrine (index of sympathetic activation) and blood pressures were elevated in IH-treated rats. These responses were associated with elevated p300/CBP protein, HIF-1α stabilization, transcriptional activation of Nox2 and Nox4 genes, and reactive oxygen species, and all these responses were absent in CTK7A-treated IH rats. These findings suggest lysine acetylation of HIF-1α by p300/CBP is an important contributor to sympathetic excitation and hypertension by IH.

Carotid body hypersensitivity in intermittent hypoxia and obtructive sleep apnoea.

Carotid bodies are the principal sensory organs for detecting changes in arterial blood oxygen concentration, and the carotid body chemoreflex is a major regulator of the sympathetic tone, blood pressure and breathing. Intermittent hypoxia is a hallmark manifestation of obstructive sleep apnoea (OSA), which is a widespread respiratory disorder. In the first part of this review, we discuss the role of carotid bodies in heightened sympathetic tone and hypertension in rodents treated with intermittent hypoxia, and the underlying cellular, molecular and epigenetic mechanisms. We also present evidence for hitherto-uncharacterized role of carotid body afferents in triggering cellular and molecular changes induced by intermittent hypoxia. In the second part of the review, we present evidence for a contribution of a hypersensitive carotid body to OSA and potential therapeutic intervention to mitigate OSA in a murine model.

Protein phosphatase 1 regulates reactive oxygen species-dependent degradation of histone deacetylase 5 by intermittent hypoxia.

We recently reported pheochromocytoma 12 (PC12) cells and rats subjected to intermittent hypoxia (IH), a hallmark manifestation of obstructive sleep apnea (OSA), exhibit reduced histone deacetylase activity and HDAC5 protein. Our study further suggested that posttranslational modifications rather than transcriptional mechanism(s) mediate IH-induced HDAC5 degradation. These observations prompted our current study to investigate the mechanism(s) underlying HDAC5 degradation by IH in PC12 cell cultures. IH-induced HDAC5 degradation was blocked by an antioxidant, and reactive oxygen species (ROS) mimetics decreased HDAC5 protein, suggesting that ROS mediates HDAC5 degradation by IH. NADPH oxidases (NOX) 2 and 4 were identified as sources of ROS that mediate the effects of IH. HDAC5 degradation during IH was associated with dephosphorylation of HDAC5 at serine259, and this response was blocked by a NOX inhibitor, suggesting that ROS-dependent dephosphorylation mediates HDAC5 degradation. IH-induced dephosphorylation of HDCA5 was inhibited by calyculin A, an inhibitor of protein phosphatase (PP)-1 and -2, or by the overexpression of nuclear inhibitor of PP1 (NIPP1). HDAC5 dephosphorylation by IH lead to augmented hypoxia-inducible factor (HIF)-1α protein and an increase in its transcriptional activity. These data suggest that PP1-dependent dephosphorylation of S259 destabilizes HDAC5 protein in response to IH, resulting in HIF-1α stabilization and transcriptional activity. Our findings highlight hither to unexplored role of protein phosphatases, especially PP1 in regulating HDAC5 protein, which is an upstream activator of HIF-1 signaling by IH.

Lysine demethylase KDM6B regulates HIF-1α-mediated systemic and cellular responses to intermittent hypoxia.

Intermittent hypoxia (IH) is a hallmark manifestation of obstructive sleep apnea (OSA). Rodents treated with IH exhibit hypertension. Hypoxia-inducible factor (HIF)-1-dependent transcriptional activation of NADPH oxidases (Nox) and the resulting increase in reactive oxygen species (ROS) levels is a major molecular mechanism underlying IH/OSA-induced hypertension. Jumanji C (JmjC)-containing histone lysine demethylases (JmjC-KDMs) are coactivators of HIF-1-dependent transcriptional activation. In the present study, we tested the hypothesis that JmjC-KDMs are required for IH-evoked HIF-1 transcriptional activation of Nox4 and the ensuing hypertension. Studies were performed on pheochromocytoma (PC)12 cells and rats. IH increased KDM6B protein and enzyme activity in PC12 cells in an HIF-1-independent manner as evidenced by unaltered KDM6B activation by IH in HIF-1α shRNA-treated cells. Cells treated with IH showed increased HIF-1-dependent Nox4 transcription as indicated by increased HIF-1α binding to hypoxia-responsive element (HRE) sequence of the Nox4 gene promoter demonstrated by chromatin immunoprecipitation (ChiP) assay. Pharmacological blockade of KDM6B with GSKJ4, a specific KDM6 inhibitor, or genetic silencing of KDM6B with shRNA abolished IH-induced Nox4 transcriptional activation by blocking HIF-1α binding to the promoter of the Nox4 gene. Treating IH-exposed rats with GSKJ4 showed: 1) absence of KDM6B activation and HIF-1-dependent Nox4 transcription in the adrenal medullae, and 2) absence of elevated plasma catecholamines and hypertension. Collectively, these findings indicate that KDM6B functions as a coactivator of HIF-1-mediated Nox4 transactivation and demonstrates a hitherto uncharacterized role for KDMs in IH-induced hypertension by HIF-1.

Role of olfactory receptor78 in carotid body-dependent sympathetic activation and hypertension in murine models of chronic intermittent hypoxia.

Chronic intermittent hypoxia (CIH) is a hallmark manifestation of obstructive sleep apnea (OSA), a widespread breathing disorder. CIH-treated rodents exhibit activation of the sympathetic nervous system and hypertension. Heightened carotid body (CB) activity has been implicated in CIH-induced hypertension. CB expresses high abundance of olfactory receptor (Olfr) 78, a G-protein coupled receptor. Olfr 78 null mice exhibit impaired CB sensory nerve response to acute hypoxia. Present study examined whether Olfr78 participates in CB-dependent activation of the sympathetic nervous system and hypertension in CIH-treated mice and in hemeoxygenase (HO)-2 null mice experiencing CIH as a consequence of naturally occurring OSA. CIH-treated wild-type (WT) mice showed hypertension, biomarkers of sympathetic nerve activation, and enhanced CB sensory nerve response to hypoxia and sensory long-term facilitation (sLTF), and these responses were absent in CIH-treated Olfr78 null mice. HO-2 null mice showed higher apnea index (AI) (58 ± 1.2 apneas/h) than WT mice (AI = 8 ± 0.8 apneas/h) and exhibited elevated blood pressure (BP), elevated plasma norepinephrine (NE) levels, and heightened CB sensory nerve response to hypoxia and sLTF. The magnitude of hypertension correlated with AI in HO-2 null mice. In contrast, HO-2/Olfr78 double null mice showed absence of elevated BP and plasma NE levels and augmented CB response to hypoxia and sLTF. These results demonstrate that Olfr78 participates in sympathetic nerve activation and hypertension and heightened CB activity in two murine models of CIH.NEW & NOTEWORTHY Carotid body (CB) sensory nerve activation is essential for sympathetic nerve excitation and hypertension in rodents treated with chronic intermittent hypoxia (CIH) simulating blood O2 profiles during obstructive sleep apnea (OSA). Here, we report that CIH-treated mice and hemeoxygenase (HO)-2-deficient mice, which show OSA phenotype, exhibit sympathetic excitation, hypertension, and CB activation. These effects are absent in Olfr78 null and Olfr78/HO-2 double null mice.

Gaseous transmitter regulation of hypoxia-evoked catecholamine secretion from murine adrenal chromaffin cells.

Emerging evidence suggests that gaseous molecules, carbon monoxide (CO), and hydrogen sulfide (H2S) generated by heme oxygenase (HO)-2 and cystathionine γ-lyase (CSE), respectively, function as transmitters in the nervous system. Present study examined the roles of CO and H2S in hypoxia-induced catecholamine (CA) release from adrenal medullary chromaffin cells (AMCs). Studies were performed on AMCs from adult (≥6 wk of age) wild-type (WT), HO-2 null, CSE null, and HO-2/CSE double null mice of either gender. CA secretion was determined by carbon fiber amperometry and [Ca2+]i by microflurometry using Fura-2. HO-2- and CSE immunoreactivities were seen in WT AMC, which were absent in HO-2 and CSE null mice. Hypoxia (medium Po2 30-38 mmHg) evoked CA release and elevated [Ca2+]i. The magnitude of hypoxic response was greater in HO-2 null mice and in HO inhibitor-treated WT AMC compared with controls. H2S levels were elevated in HO-2 null AMC. Either pharmacological inhibition or genetic deletion of CSE prevented the augmented hypoxic responses of HO-2 null AMC and H2S donor rescued AMC responses to hypoxia in HO-2/CSE double null mice. CORM3, a CO donor, prevented the augmented hypoxic responses in WT and HO-2 null AMC. CO donor reduced H2S levels in WT AMC. The effects of CO donor were blocked by either ODQ or 8pCT, inhibitors of soluble guanylyl cyclase (SGC) or protein kinase G, respectively. These results suggest that HO-2-derived CO inhibits hypoxia-evoked CA secretion from adult murine AMC involving soluble guanylyl cyclase (SGC)-protein kinase G (PKG)-dependent regulation of CSE-derived H2S.NEW & NOTEWORTHY Catecholamine secretion from adrenal chromaffin cells is an important physiological mechanism for maintaining homeostasis during hypoxia. Here, we delineate carbon monoxide (CO)-sensitive hydrogen sulfide (H2S) signaling as an important mediator of hypoxia-induced catecholamine secretion from murine adrenal chromaffin cells. Heme oxygenase-2 derived CO is a physiological inhibitor of catcholamince secretion by hypoxia and the effects of CO involve inhibition of cystathionine γ-lyase-derived H2S production through soluble guanylyl cyclase-protein kinase G signaling cascade.

Hypoxia-inducible factor-1 mediates pancreatic ß-cell dysfunction by intermittent hypoxia.

The role of hypoxia-inducible factor (HIF)-1 in pancreatic ß-cell response to intermittent hypoxia (IH) was examined. Studies were performed on adult wild-type (WT), HIF-1α heterozygous (HET), ß-cell-specific HIF-1-/- mice and mouse insulinoma (MIN6) cells exposed to IH patterned after blood O2 profiles during obstructive sleep apnea. WT mice treated with IH showed insulin resistance, and pancreatic ß-cell dysfunction manifested as augmented basal insulin secretion, and impaired glucose-stimulated insulin secretion and these effects were absent in HIF-1α HET mice. IH increased HIF-1α expression and elevated reactive oxygen species (ROS) levels in ß-cells of WT mice. The elevated ROS levels were due to transcriptional upregulation of NADPH oxidase (NOX)-4 mRNA, protein and enzymatic activity, and these responses were absent in HIF-1α HET mice as well as in ß-HIF-1-/- mice. IH-evoked ß-cell responses were absent in adult WT mice treated with digoxin, an inhibitor of HIF-1α. MIN6 cells treated with in vitro IH showed enhanced basal insulin release and elevated HIF-1α protein expression, and these effects were abolished with genetic silencing of HIF-1α. IH increased NOX4 mRNA, protein, and enzyme activity in MIN6 cells and disruption of NOX4 function by siRNA or scavenging H2O2 with polyethylene glycol catalase blocked IH-evoked enhanced basal insulin secretion. These results demonstrate that HIF-1-mediated transcriptional activation of NOX4 and the ensuing increase in H2O2 contribute to IH-induced pancreatic ß-cell dysfunction.

Olfactory receptor 78 participates in carotid body response to a wide range of low O2 levels but not severe hypoxia.

The role of olfactory receptor 78 (Olfr78) in carotid body (CB) response to hypoxia was examined. BL6 mice with global deletion of Olfr78 manifested an impaired hypoxic ventilatory response (HVR), a hallmark of the CB chemosensory reflex, CB sensory nerve activity, and reduced intracellular Ca2+ concentration ([Ca2+]i) response of glomus cells to hypoxia (Po2 ~ 40 mmHg). In contrast, severe hypoxia (Po2 ~ 10 mmHg) depressed breathing and produced a very weak CB sensory nerve excitation but robust elevation of [Ca2+]i in Olfr78 null glomus cells. CB sensory nerve excitation evoked by Olfr78 ligands, lactate, propionate, acetate, and butyrate were unaffected in mutant mice and were smaller than that evoked by hypoxia (Po2 ~ 40mmHg). Similar results were obtained in Olfr78 null mice on a JAX genetic background. These results demonstrate a role for Olfr78 in CB responses to a wide range of hypoxia, but not severe hypoxia, and do not require either lactate or any other short-chain fatty acids.NEW & NOTEWORTHY The current study demonstrates that olfactory receptor 78 (Olfr78), a G protein-coupled receptor, is an integral component of the hypoxic sensing mechanism of the carotid body to a wide range of low oxygen levels, but not severe hypoxia, and that Olfr78 participation does not require either lactate or any other short-chain fatty acids, proposed ligands of Olfr78.

Role of the carotid chemoreceptors in insulin-mediated sympathoexcitation in humans.

We examined the contribution of the carotid chemoreceptors to insulin-mediated increases in muscle sympathetic nerve activity (MSNA) in healthy humans. We hypothesized that reductions in carotid chemoreceptor activity would attenuate the sympathoexcitatory response to hyperinsulinemia. Young, healthy adults (9 male/9 female, 28 ± 1 yr, 24 ± 1 kg/m2) completed a 30-min euglycemic baseline followed by a 90-min hyperinsulinemic (1 mU·kg fat-free mass-1·min-1), euglycemic infusion. MSNA (microneurography of the peroneal nerve) was continuously measured. The role of the carotid chemoreceptors was assessed at baseline and during hyperinsulinemia via 1) acute hyperoxia, 2) low-dose dopamine (1-4 µg·kg-1·min-1), and 3) acute hyperoxia + low-dose dopamine. MSNA burst frequency increased from baseline during hyperinsulinemia (P < 0.01). Acute hyperoxia had no effect on MSNA burst frequency at rest (P = 0.74) or during hyperinsulinemia (P = 0.83). The insulin-mediated increase in MSNA burst frequency (P = 0.02) was unaffected by low-dose dopamine (P = 0.60). When combined with low-dose dopamine, acute hyperoxia had no effect on MSNA burst frequency at rest (P = 0.17) or during hyperinsulinemia (P = 0.85). Carotid chemoreceptor desensitization in young, healthy men and women does not attenuate the sympathoexcitatory response to hyperinsulinemia. Our data suggest that the carotid chemoreceptors do not contribute to acute insulin-mediated increases in MSNA in young, healthy adults.

Complementary roles of gasotransmitters CO and H2S in sleep apnea.

Sleep apnea, which is the periodic cessation of breathing during sleep, is a major health problem affecting over 10 million people in the United States and is associated with several sequelae, including hypertension and stroke. Clinical studies suggest that abnormal carotid body (CB) activity may be a driver of sleep apnea. Because gaseous molecules are important determinants of CB activity, aberrations in their signaling could lead to sleep apnea. Here, we report that mice deficient in heme oxygenase-2 (HO-2), which generates the gaseous molecule carbon monoxide (CO), exhibit sleep apnea characterized by high apnea and hypopnea indices during rapid eye movement (REM) sleep. Similar high apnea and hypopnea indices were also noted in prehypertensive spontaneously hypertensive (SH) rats, which are known to exhibit CB hyperactivity. We identified the gaseous molecule hydrogen sulfide (H2S) as the major effector molecule driving apneas. Genetic ablation of the H2S-synthesizing enzyme cystathionine-γ-lyase (CSE) normalized breathing in HO-2-/- mice. Pharmacologic inhibition of CSE with l-propargyl glycine prevented apneas in both HO-2-/- mice and SH rats. These observations demonstrate that dysregulated CO and H2S signaling in the CB leads to apneas and suggest that CSE inhibition may be a useful therapeutic intervention for preventing CB-driven sleep apnea.

Adaptive and maladaptive cardiorespiratory responses to continuous and intermittent hypoxia mediated by hypoxia-inducible factors 1 and 2.

Hypoxia is a fundamental stimulus that impacts cells, tissues, organs, and physiological systems. The discovery of hypoxia-inducible factor-1 (HIF-1) and subsequent identification of other members of the HIF family of transcriptional activators has provided insight into the molecular underpinnings of oxygen homeostasis. This review focuses on the mechanisms of HIF activation and their roles in physiological and pathophysiological responses to hypoxia, with an emphasis on the cardiorespiratory systems. HIFs are heterodimers comprised of an O(2)-regulated HIF-1α or HIF-2α subunit and a constitutively expressed HIF-1ß subunit. Induction of HIF activity under conditions of reduced O(2) availability requires stabilization of HIF-1α and HIF-2α due to reduced prolyl hydroxylation, dimerization with HIF-1ß, and interaction with coactivators due to decreased asparaginyl hydroxylation. Stimuli other than hypoxia, such as nitric oxide and reactive oxygen species, can also activate HIFs. HIF-1 and HIF-2 are essential for acute O(2) sensing by the carotid body, and their coordinated transcriptional activation is critical for physiological adaptations to chronic hypoxia including erythropoiesis, vascularization, metabolic reprogramming, and ventilatory acclimatization. In contrast, intermittent hypoxia, which occurs in association with sleep-disordered breathing, results in an imbalance between HIF-1α and HIF-2α that causes oxidative stress, leading to cardiorespiratory pathology.

Long-term facilitation of catecholamine secretion from adrenal chromaffin cells of neonatal rats by chronic intermittent hypoxia.

In neonates, catecholamine (CA) secretion from adrenal medullary chromaffin cells (AMC) is an important mechanism for maintaining homeostasis during hypoxia. Nearly 90% of premature infants experience chronic intermittent hypoxia (IH) because of high incidence of apnea of prematurity, which is characterized by periodic stoppage of breathing. The present study examined the effects of repetitive hypoxia, designed to mimic apnea of prematurity, on CA release from AMC of neonatal rats. Neonatal rats were exposed to either control conditions or chronic intermittent hypoxia (IH) from ages postnatal days 0-5 (P0-P5), and CA release from adrenal medullary slices was measured after challenge with repetitive hypoxia (5 episodes of 30-s hypoxia, Po2 ~35 mmHg). In response to repetitive hypoxia, chronic IH-treated AMC exhibited sustained CA release, and this phenotype was not seen in control AMC. The sustained CA release was associated with long-lasting elevation of intracellular Ca2+ concentration ([Ca2+]i), which was due to store-operated Ca2+ entry (SOCE). 2-Aminoethoxydiphenyl borate, an inhibitor of SOCE, prevented the long-lasting [Ca2+]i elevation and CA release. Repetitive hypoxia increased H2O2 abundance, and polyethylene glycol (PEG)-catalase, a scavenger of H2O2 blocked this effect. PEG-catalase also prevented repetitive hypoxia-induced SOCE activation, sustained [Ca2+]i elevation, and CA release. These results demonstrate that repetitive hypoxia induces long-term facilitation of CA release in chronic IH-treated neonatal rat AMC through sustained Ca2+ influx mediated by SOCE.NEW & NOTEWORTHY Apnea of prematurity and the resulting chronic intermittent hypoxia are major clinical problems in neonates born preterm. Catecholamine release from adrenal medullary chromaffin cells maintains homeostasis during hypoxia in neonates. Our results demonstrate that chronic intermittent hypoxia induces a hitherto uncharacterized long-term facilitation of catecholamine secretion from neonatal rat chromaffin cells in response to repetitive hypoxia, simulating hypoxic episodes encountered during apnea of prematurity. The sustained catecholamine secretion might contribute to cardiovascular morbidities in infants with apnea of prematurity.

The role of hypoxia-inducible factors in carotid body (patho) physiology.

Hypoxia-inducible factors mediate adaptive responses to reduced O2 availability. In patients with obstructive sleep apnoea, repeated episodes of hypoxaemia and reoxygenation (intermittent hypoxia) are sensed by the carotid body (CB). The ensuing CB chemosensory reflex activates the sympathetic nervous system and increased secretion of catecholamines by the adrenal medulla, resulting in hypertension and breathing abnormalities. In the CB, intermittent hypoxia induces the formation of reactive oxygen species (ROS) and increased intracellular Ca2+ levels, which drive increased expression of hypoxia-inducible factor (HIF) 1α and a decrease in the levels of HIF-2α.  Intermittent hypoxia increases HIF-1α-dependent expression of Nox2, encoding the pro-oxidant enzyme NADPH oxidase 2, and decreased HIF-2α-dependent expression of Sod2, encoding the anti-oxidant enzyme superoxide dismutase 2. These changes in gene expression drive persistently elevated ROS levels in the CB, brainstem, and adrenal medulla that are required for the development of hypertension and breathing abnormalities. The ROS generated by dysregulated HIF activity in the CB results in oxidation and inhibition of haem oxygenase 2, and the resulting reduction in the levels of carbon monoxide leads to increased hydrogen sulfide production, triggering glomus cell depolarization. Thus, the pathophysiology of obstructive sleep apnoea involves the dysregulation of O2 -regulated transcription factors, gasotransmitters, and sympathetic outflow that affects blood pressure and breathing.

DNA methylation in the central and efferent limbs of the chemoreflex requires carotid body neural activity.

KEY POINTS: The mechanisms underlying long-term (30 days) intermittent hypoxia (LT-IH)-evoked DNA methylation of anti-oxidant enzyme (AOE) gene repression in the carotid body (CB) reflex pathway were examined. LT-IH-treated rats showed increased reactive oxygen species (ROS) levels in the CB reflex pathway. Administration of a ROS scavenger or CB ablation blocked LT-IH-evoked DNA methylation and AOE gene repression in the central and efferent limbs of the CB reflex. LT-IH increased DNA methyltransferase (Dnmt) activity through upregulation of Dnmt1 and 3b proteins by ROS-dependent inactivation of glycogen synthase kinase 3ß (GSK3ß) by Akt. A pan-Akt inhibitor prevented LT-IH-induced GSK3ß inactivation, elevated Dnmt protein expression and activity, AOE gene methylation, sympathetic activation and hypertension. ABSTRACT: Long-term exposure to intermittent hypoxia (LT-IH; 30 days), simulating blood O2 profiles during sleep apnoea, has been shown to repress anti-oxidant enzyme (AOE) gene expression by DNA methylation in the carotid body (CB) reflex pathway, resulting in persistent elevation of plasma catecholamine levels and blood pressure. The present study examined the mechanisms by which LT-IH induces DNA methylation. Adult rats exposed to LT-IH showed elevated reactive oxygen species (ROS) in the CB, nucleus tractus solitarius (nTS) and rostroventrolateral medulla (RVLM) and adrenal medulla (AM), which represent the central and efferent limbs of the CB reflex, respectively. ROS scavenger treatment during the first ten days of IH exposure prevented ROS accumulation, blocked DNA methylation, and normalized AOE gene expression, suggesting that ROS generated during the early stages of IH activate DNA methylation. CB ablation prevented the ROS accumulation, normalized AOE gene expression in the nTS, RVLM, and AM and blocked DNA methylation, suggesting that LT-IH-induced DNA methylation in the central and efferent limbs of the CB reflex is indirect and requires CB neural activity. LT-IH increased DNA methyl transferase (Dnmt) activity through upregulation of Dnmt1 and 3b protein expression due to ROS-dependent inactivation of glycogen synthase kinase 3ß (GSK3ß) by protein kinase B (Akt). Treating rats with the pan-Akt inhibitor GSK690693 blocked the induction of Dnmt activity, Dnmt protein expression, and DNA methylation, leading to normalization of AOE gene expression as well as plasma catecholamine levels and blood pressure.

Reactive oxygen radicals and gaseous transmitters in carotid body activation by intermittent hypoxia.

Sleep apnea is a prevalent respiratory disease characterized by periodic cessation of breathing during sleep causing intermittent hypoxia (IH). Sleep apnea patients and rodents exposed to IH exhibit elevated sympathetic nerve activity and hypertension. A heightened carotid body (CB) chemoreflex has been implicated in causing autonomic abnormalities in IH-treated rodents and in sleep apnea patients. The purpose of this article is to review the emerging evidence showing that interactions between reactive oxygen species (ROS) and gaseous transmitters as a mechanism cause hyperactive CB by IH. Rodents treated with IH exhibit markedly elevated ROS in the CB, which is due to transcriptional upregulation of pro-oxidant enzymes by hypoxia-inducible factor (HIF)-1 and insufficient transcriptional regulation of anti-oxidant enzymes by HIF-2. ROS, in turn, increases cystathionine γ-lyase (CSE)-dependent H2S production in the CB. Blockade of H2S synthesis prevents IH-evoked CB activation. However, the effects of ROS on H2S production are not due to direct effects on CSE enzyme activity but rather due to inactivation of heme oxygenase-2 (HO-2), a carbon monoxide (CO) producing enzyme. CO inhibits H2S production through inactivation of CSE by PKG-dependent phosphorylation. During IH, reduced CO production resulting from inactivation of HO-2 by ROS releases the inhibition of CO on CSE thereby increasing H2S. Inhibiting H2S synthesis prevented IH-evoked sympathetic activation and hypertension.

Therapeutic Targeting of the Carotid Body for Treating Sleep Apnea in a Pre-clinical Mouse Model.

Sleep apnea with periodic cessation of breathing during sleep is a highly prevalent respiratory disorder affecting an estimated 10% of adults. Patients with sleep apnea exhibit several co-morbidities including hypertension, stroke, disrupted sleep, and neurocognitive and metabolic complications. Emerging evidence suggests that a hyperactive carotid body (CB) chemo reflex is an important driver of apneas in sleep apnea patients. Gasotransmitters carbon monoxide (CO) and hydrogen sulfide (H2S) play important roles in oxygen sensing by the CB. We tested the hypothesis that an augmented CB chemo reflex stemming from disrupted CO-H2S signaling may lead to sleep apnea. This possibility was tested in mice deficient in hemeoxygenase-2 (HO-2), an enzyme involved in CO synthesis, which were shown to exhibit hyperactive CB activity due to high H2S levels. We found that HO-2-/- mice exhibit a high incidence of apneas during sleep compared to wild type mice. Blocking the CB hyperactivity with L-propargylglycine, an inhibitor of cystathionine-γ-lyase (CSE), which catalyzes H2S synthesis, prevented apneas in HO-2-/- mice. These findings suggest that targeting CB with inhibitors of CSE might be a novel therapeutic strategy for preventing sleep apnea.

Epigenetic regulation of redox state mediates persistent cardiorespiratory abnormalities after long-term intermittent hypoxia.

KEY POINTS: The effects of short-term (ST; 10 days) and long-term (LT; 30 days) intermittent hypoxia (IH) on blood pressure (BP), breathing and carotid body (CB) chemosensory reflex were examined in adult rats. ST- and LT-IH treated rats exhibited hypertension, irregular breathing with apnoea and augmented the CB chemosensory reflex, with all these responses becoming normalized during recovery from ST- but not from LT-IH. The persistent cardiorespiratory responses to LT-IH were associated with elevated reactive oxygen species (ROS) levels in the CB and adrenal medulla, which were a result of DNA methylation-dependent suppression of genes encoding anti-oxidant enzymes (AOEs). Treating rats with decitabine either during LT-IH or during recovery from LT-IH prevented DNA methylation of AOE genes, normalized the expression of AOE genes and ROS levels, reversed the heightened CB chemosensory reflex and hypertension, and also stabilized breathing. ABSTRACT: Rodents exposed to chronic intermittent hypoxia (IH), simulating blood O2 saturation profiles during obstructive sleep apnoea (OSA), have been shown to exhibit a heightened carotid body (CB) chemosensory reflex and hypertension. CB chemosensory reflex activation also results in unstable breathing with apnoeas. However, the effect of chronic IH on breathing is not known. In the present study, we examined the effects of chronic IH on breathing along with blood pressure (BP) and assessed whether the autonomic responses are normalized after recovery from chronic IH. Studies were performed on adult, male, Sprague-Dawley rats exposed to either short-term (ST; 10 days) or long-term (LT, 30 days) IH. Rats exposed to either ST- or LT-IH exhibited hypertension, irregular breathing with apnoeas, an augmented CB chemosensory reflex as indicated by elevated CB neural activity and plasma catecholamine levels, and elevated reactive oxygen species (ROS) levels in the CB and adrenal medulla (AM). All these effects were normalized after recovery from ST-IH but not from LT-IH. Analysis of the molecular mechanisms underlying the persistent effects of LT-IH revealed increased DNA methylation of genes encoding anti-oxidant enzymes (AOEs). Treatment with decitabine, a DNA methylation inhibitor, either during LT-IH or during recovery from LT-IH, prevented DNA methylation, normalized the expression of AOE genes, ROS levels, CB chemosensory reflex and BP, and also stabilized breathing. These results suggest that persistent cardiorespiratory abnormalities caused by LT-IH are mediated by epigenetic re-programming of the redox state in the CB chemosensory reflex pathway.

Epigenetic changes by DNA methylation in chronic and intermittent hypoxia.

DNA methylation of cytosine residues is a well-studied epigenetic change, which regulates gene transcription by altering accessibility for transcription factors. Hypoxia is a pervasive stimulus that affects many physiological processes. The circulatory and respiratory systems adapt to chronic sustained hypoxia, such as that encountered during a high-altitude sojourn. Many people living at sea level experience chronic intermittent hypoxia (IH) due to sleep apnea, which leads to cardiovascular and respiratory maladaptation. This article presents a brief update on emerging evidence suggesting that changes in DNA methylation contribute to pathologies caused by chronic IH and potentially mediate adaptations to chronic sustained hypoxia by affecting the hypoxia-inducible factor (HIF) signaling pathway.

Oxygen Sensing by the Carotid Body: Past and Present.

It is now well established that carotid bodies are sensory organs for monitoring arterial blood oxygen levels and trigger reflexes that are critical for maintaining homeostasis during hypoxemia. This review article provides a brief account of the early studies leading to the discovery of the carotid body as a sensory receptor and addresses current views of O2 sensing mechanism(s) in the carotid body and their physiological importance.

Inherent variations in CO-H2S-mediated carotid body O2 sensing mediate hypertension and pulmonary edema.

Oxygen (O2) sensing by the carotid body and its chemosensory reflex is critical for homeostatic regulation of breathing and blood pressure. Humans and animals exhibit substantial interindividual variation in this chemosensory reflex response, with profound effects on cardiorespiratory functions. However, the underlying mechanisms are not known. Here, we report that inherent variations in carotid body O2 sensing by carbon monoxide (CO)-sensitive hydrogen sulfide (H2S) signaling contribute to reflex variation in three genetically distinct rat strains. Compared with Sprague-Dawley (SD) rats, Brown-Norway (BN) rats exhibit impaired carotid body O2 sensing and develop pulmonary edema as a consequence of poor ventilatory adaptation to hypobaric hypoxia. Spontaneous Hypertensive (SH) rat carotid bodies display inherent hypersensitivity to hypoxia and develop hypertension. BN rat carotid bodies have naturally higher CO and lower H2S levels than SD rat, whereas SH carotid bodies have reduced CO and greater H2S generation. Higher CO levels in BN rats were associated with higher substrate affinity of the enzyme heme oxygenase 2, whereas SH rats present lower substrate affinity and, thus, reduced CO generation. Reducing CO levels in BN rat carotid bodies increased H2S generation, restoring O2 sensing and preventing hypoxia-induced pulmonary edema. Increasing CO levels in SH carotid bodies reduced H2S generation, preventing hypersensitivity to hypoxia and controlling hypertension in SH rats.