Constitutive expression of Hif2α confers acute O2 sensitivity to carotid body glomus cells

Depósito de investigación de la Universidad de Sevilla https://idus.us.es/ Esta es la versión aceptada del artículo publicado en: This is an accepted manuscript of a paper published in: Advances in experimental medicine and biology: June (2023) DOI: https://doi.org/10.1007/978-3-031-32371-3_17 Copyright: © 2021 Elsevier Ltd. All rights reserved El acceso a la versión publicada del artículo puede requerir la suscripción de la revista. Access to the published version may require subscription. “This is an Accepted Manuscript of an article published by Elsevier in Advances in experimental medicine and biology 2023, available at: https://doi.org/10.1007/978-3-031-32371-3_17”. Constitutive Expression of Hif2α Confers Acute O2 Sensitivity to Carotid Body Glomus Cells Olalla Colinas1,2,3, #, Alejandro Moreno-Domínguez1,2,3, #, Patricia Ortega- Sáenz1,2,3 and José López-Barneo1,2,3,* 1. Instituto de Biomedicina de Sevilla (IBiS), Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla, 41013 Seville, Spain 2. Departamento de Fisiología Médica y Biofísica, Facultad de Medicina, Universidad de Sevilla, 41009 Seville, Spain 3. Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), 28031 Madrid, Spain # These authors contributed equally to this work Running title: Role of HIF2α in carotid body O2 sensing * Corresponding author: Dr. José López-Barneo Phone: +34 955 923032 Email: [email protected] ! 2 Abstract Acute oxygen (O2) sensing and adaptation to hypoxia are essential for physiological homeostasis. The prototypical acute O2 sensing organ is the carotid body, which contains chemosensory glomus cells expressing O2-sensitive K+ channels. Inhibition of these channels during hypoxia leads to cell depolarization, transmitter release, and activation of afferent sensory fibers terminating in the brain stem respiratory and autonomic centers. Focusing on recent data, here we discuss the special sensitivity of glomus cell mitochondria to changes in O2 tension due to Hif2α-dependent expression of several atypical mitochondrial electron transport chain subunits and enzymes. These are responsible for an accelerated oxidative metabolism and the strict dependence of mitochondrial complex IV activity on O2 availability. We report that ablation of Epas1 (the gene coding Hif2α) causes a selective down-regulation of the atypical mitochondrial genes and a strong inhibition of glomus cell acute responsiveness to hypoxia. Our observations indicate that Hif2α expression is required for the characteristic metabolic profile of glomus cells and provide a mechanistic explanation for the acute O2 regulation of breathing. Introduction Oxygen (O2) is essential for life, with mammals being particularly susceptible to low O2 environments, even for brief periods. Decreases in blood O2 tension (PO2) induces acute adaptive responses, such as hyperventilation or increased cardiac output that within a few seconds augments O2 uptake by the lungs and its distribution to the most vulnerable organs such as the brain or heart (Weir et al., 2005; Teppema and Dahan, 2010). These systemic acute responses to O2 deficiency (hypoxia) are primarily mediated by the carotid body (CB), an arterial chemoreceptor and prototypical acute O2-sensing organ located in the carotid bifurcation. The CB contains neurosensory glomus cells (also called type I cells) with O2-sensitive K+ channels that are inhibited by hypoxia, thus leading to cell depolarization, Ca2+ influx, and the release of transmitters that activate afferent nerve fibers impinging upon the brainstem respiratory and autonomic centers (López-Barneo et al., 2001; Pérez-García et al., 2004; Buckler, 2015). The molecular mechanisms by which glomus cells acutely detect hypoxia have remained elusive (Prabhakar and Peers, 2014; Ortega-Sáenz and López-Barneo, 2020; Iturriaga et al., 2021). Although several O2-sensing mechanisms have been postulated (López-Barneo et al., 2016; Rakoczy and Wyatt, 2018), none have proven to be essential for CB function, because mice with ablation of genes coding for the relevant enzymes or receptors exhibit normal CB responses to hypoxia (Roy et al., 2000; He et al., 2002; Ortega- Sáenz et al., 2006; Mahmoud et al., 2016; Wang et al., 2017; Torres-Torrelo et al., 2018). 3 Mitochondria have been classically considered to be involved in CB O2 sensing due to the exquisite sensitivity of the CB cells to mitochondrial inhibitors (Mills and Jöbsis, 1972; Mulligan and Lahiri, 1982). Experiments performed on CB slices showed that similar to the response to hypoxia, the secretory response of single glomus cells to inhibitors of the mitochondrial electron transport chain (ETC), such as rotenone or cyanide, was dependent on extracellular Ca2+ influx. In addition, it was also shown that rotenone, a lipophilic membrane-permeant agent that inhibits mitochondrial complex (MC) I NADH (reduced form of nicotinamide adenine dinucleotide) dehydrogenase activity, is highly effective in occluding glomus cell responsiveness to hypoxia. These observations suggested that mitochondria signal the plasma membrane to regulate glomus cell excitability and that MCI could be involved in glomus cell O2 sensing (Ortega-Sáenz et al., 2003). For the last few years, a series of experiments performed on genetically modified mice have shown that CB glomus cells can repurpose their metabolism to survive after disruption of MCI. However, these cells are insensitive to hypoxia although they exhibit normal responses to hypercapnia, lactate, and hypoglycemia (Fernández- Agüera et al., 2015; Arias-Mayenco et al., 2018; Torres-Torrelo et al., 2021). These experimental findings have suggested a model of acute O2 sensing in which mitochondria, possibly located near the plasma membrane of glomus cells (Rakoczy et al., 2022), act as O2 sensors and effectors (Ortega-Sáenz and López-Barneo, 2020). In this model, acute hypoxia slows down the mitochondrial ETC, consequently producing an accumulation of NADH and reactive oxygen species (ROS), which after equilibration with the cytosol modulate ion channels to elicit membrane depolarization (Fernández-Agüera et al., 2015; Arias-Mayenco et al., 2018; Moreno-Domínguez et al., 2020). In parallel with the functional experiments, gene expression analyses have provided relevant clues for advancing in the understanding of the molecular bases of glomus cell acute O2 sensing (Zhou et al., 2016; Gao et al., 2017). These studies have described a gene expression “signature profile”, conferring acute O2 sensitivity upon glomus cells, which includes Hif2α, atypical mitochondrial ETC subunits, enzymes and receptors, as well as several types of ion channels. In this paper we focus on Hif2α and its role as the main responsible for the specialized acute O2 sensing properties of CB glomus cells. HIF2α-dependent gene expression profile in carotid body cells Hif2α is a hypoxia-inducible transcription factor that, similar to Hif1α, regulates the expression of numerous genes. However, the actions of Hif1α and Hif2α are not redundant, but rather complementary, as Hif2α expression is restricted to specific tissues to serve specialized adaptive functions (Ema et al., 1997). During development, Hif2α is constitutively expressed at high levels in catecholaminergic tissues (Tian et al., 1998), however in the adult high levels of Hif2α expression in normoxic conditions is restricted to the CB (Figure 1A) and to a lesser extent to 4 adrenal medulla (AM) cells (Gao et al., 2017). Overexpression of non-degradable Hif2α in embryonic catecholaminergic (TH positive) tissues produces an ~2.5- fold enlargement of the CB with increase in the number of TH positive cells (Macías et al., 2014). In contrast, embryonic deletion of the gene coding Hif2α (Epas1) in catecholaminergic cells results in viable animals although with a marked CB atrophy and abnormal cardiorespiratory and metabolic responses to hypoxia (Macías et al., 2018). Conditional generalized Hif2α ablation in adult mice causes anemia and cardiac hypertrophy (Gruber et al., 2007; Moreno- Domínguez et al., 2020), as well as inhibition of the hypoxic ventilatory response (HVR) (Hodson et al., 2016; Moreno-Domínguez et al., 2020) and of CB cellular proliferation in chronic hypoxia (Hodson et al., 2016). Fig. 1. Changes in the expression of selected genes in CB cells from Epas1-null mice. (A) Epas1 expression determined by β-galactosidase staining in adult mice carotid body (CB), which is located at the bifurcation of the common carotid artery (CC) into the internal carotid (IC) and external carotid (EC) arteries. (Modified from Tian et al. 1998). (B-D) mRNA levels of CB signature metabolic profile genes from Epas1+/+ and Epas1f/−,Cre mice (n = 6 replicates per group, with each replicate consisting of five mice for each genotype). Data are presented as mean ± SEM. *P < 0.05. (Modified from Moreno-Domínguez et al., 2020). Knowledge of the impact of Hif2α on the molecular properties of CB chemoreceptor cells has come from studies performed in mice with ubiquitous conditional ablation of Epas1 in adulthood (Moreno-Domínguez et al., 2020). These mice were studied 2–3 months after initiation of tamoxifen treatment to allow for com- 5 plete disappearance of Hif2α and of Hif2α-dependent genes. The strict compliance with this experimental protocol was critical given the slow turnover of some proteins, in particular those expressed in mitochondria (Fornasiero et al., 2018). CBs of Epas1-null mice appeared normal without clear structural modifications and were removed to study the level of expression of specific genes by qPCR. In addition to Epas1 mRNA down-regulation, which was clearly seen in comparison with Hif α mRNA (Fig. 1B), we studied 19 selected genes with potential relevance in CB chemoreception (Zhou et al., 2016; Gao et al., 2017) (Fig. 1C-E). Ablation of Epas1 produced down-regulation of four ETC subunits (Fig. 1C) and altered the expression of three enzymes/receptors (Fig. 1D). mRNA encoding some ion channels believed to participate in modulation of glomus cell excitability in response to hypoxia, such as Task1 (Kcnk3), Task 3 (Kcnk9), T-type Ca2+ channels (Cacna1h) or cationic Trp5 channels, were not significantly altered in Hif2α- deficient CB cells (Fig. 1E) (Moreno-Domínguez et al., 2020). The four mitochondrial ETC subunits (Ndufa4l2, Cox4i2, Cox8b and Higd1C) down-regulated in Epas1-null (Epas1f/-,Cre) CB in comparison with wild-type (Epas1+/+) CB encode tissue-specific mitochondrial ETC subunit isoforms highly expressed in the CB (Zhou et al., 2016; Gao et al., 2017; Timón-Gómez et al., 2022). CB expression of Ndufa4l2, Cox4i2 and Cox8b was known to be regulated by Hif2α (Moreno-Domínguez et al., 2020). We now extend the action of Hif2α to Higd1C mRNA, whose level in Epas1 null cells decreased below 30% the values observed in wild type CBs (Fig. 1C). Cox4 and Cox8 are part of the 13 subunits forming the catalytic core of MCIV (Tsukihara et al., 1996; Kadenbach and Huttemann, 2015). These subunits have adjacent single transmembrane segments which run in parallel in the periphery of MCIV. Cox4i2 is an atypical isoform of the more broadly distributed Cox4i1 subunit. Besides the CB, Cox4i2 is expressed in organs (e.g., lungs, brain, and fish gills) involved in oxygen delivery or that are oxygen sensitive (Huttemann et al., 2001; Zhou et al., 2016). Cox4i2 induction by hypoxia has been observed in several cell types (Fakuda et al., 2007; Aras et al., 2013). Cox8b is a tissue-specific isoform of the ubiquitously expressed Cox8a subunit. Interestingly Cox8b promoter also contains Hif binding sites (Gao et al., 2017), although induction of Cox8b by hypoxia has not been reported. The functional roles of Cox4i2 and Cox8b are unknown. Cox4i2 lacks ATP regulatory sites present in Cox4i1 and its expression could accelerate the catalytic rate of cytochrome c oxidase (Napiwotzki et al.,1997; Napiwotzki and Kadenbach 1998; Pajuelo-Reguera et al., 2020). On the other hand, structural studies have suggested that the Cox8 subunit contributes to the formation of mitochondrial supercomplexes (Wu et al., 2016; Rieger et al., 2017). Although Cox4i2 and Cox8b are located relatively far from the catalytic site (heme a3/CuB), they could induce subtle structural changes in MCIV and decrease the affinity for O2, thus explaining the modulation of MCIV activity in glomus cells within physiological levels of PO2. It could be also that Cox4i2 and Cox8b decrease the accessibility of O2 to the catalytic site and therefore make cytochrome c oxidase activity highly sensitive to changes in O2 tension (Moreno-Domínguez et al., 2020). Ndufa4l2, one of the most abundant mRNA species in CB glomus cells (Zhou et al., 2016; Gao et al., 2017), encodes an isoform of the most widely expressed Ndufa4 subunit, which 6 appears to be associated to MCIV rather than to MCI (Carroll et al., 2006; Balsa et al., 2012). Ndufa4l2 is highly expressed in lung and brain pericytes and some tumor cells (Lucarelli et al., 2018) but its function is poorly known. Expression of Ndufa4l2, which is induced by hypoxia in a HIF-dependent manner, attenuates oxygen consumption and decreases ROS production in mitochondria (Tello et al., 2011). Higd1C is a member of the hypoxia inducible gene domain (HIGD) family that is highly expressed in the CB. Higd1C is a MCIV-associated protein that may interact with Cox4i2 and contribute to the O2 sensitivity of mitochondria in CB cells (Timón-Gómez et al., 2022). However, the location and function of this protein is not yet well known. ! Fig. 2. Electrophysiological properties of CB glomus cells from Epas1+/+ and Epas1-null mice. (A) Representative recordings of outward K+ currents in whole-cell patch clamped glomus cells from Epas1+/+ and Epas1f/-,Cre mice. Depolarizing (50 ms) voltage steps between -30 and +50 mV were applied from a holding potential of -70 mV. (B) Average peak K+ current density versus membrane potential relationship obtained from Epas1+/+ (back) and Epas1f/-,Cre (red) glomus cells. (C) Representative macroscopic Ca2+ currents recorded in glomus cells from Epas1+/+ and Epas1f/-,Cre mice. Depolarizing (10 ms) voltage steps (to 10 mV) were applied from a holding potential of -70 mV. (D) Average peak Ca2+ current density versus membrane potential relationship measured in glomus cells obtained from Epas1+/+ (black) and Epas1f/-,Cre (red) mice. (Modified from Moreno-Domínguez et al., 2020). In addition to the changes in the expression of mitochondrial ETC subunits, CBs from Epas1-null mice also show alterations in the mRNA expression of two enzymes, phosphoglycerate kinase 1 (Pgk1) and pyruvate carboxylase (Pcx) and one olfactory receptor (Olfr78) (Moreno-Domínguez et al., 2020) (Fig. 1D). Pgk1 is a typical Hif1α-dependent gene encoding a glycolytic enzyme, whose expression 7 also increases in liver (Rankin et al., 2007) and lung (Elorza et al., 2012) after Epas1 ablation. Hif2α probably exerts a dominant negative action on Pgk1 expression, which is relieved in Epas1-deficient cells. Pyruvate carboxylase (Pcx) is an enzyme highly expressed in CB cells in comparison with other peripheral catecholaminergic tissues (Gao et al., 2017). Pcx expression is compatible with the longstanding view that CB glomus cells contain highly active mitochondria with an elevated O2 consumption. Pcx accelerates oxidative phosphorylation by the generation of oxaloacetate from pyruvate, thereby replenishing the pool of TCA cycle intermediates. In this anaplerotic reaction (Owen et al., 2002), Pcx utilizes biotin, a cofactor which is stored in high quantities in CB glomus cells and in lesser amounts in AM chromaffin cells (Ortega-Sáenz et al., 2016). Olfr78 is an atypical olfactory receptor that is expressed outside the nasal mucosa and particularly in CB glomus cells. CB Olfr78 expression is strongly dependent on Hif2α (Fig. 1D) however its relevance in glomus cell function is unknown (see below). Selective inhibition of acute responsiveness to hypoxia in Hif2α- deficient glomus cells The alteration in gene expression profile induced by Epas1 deficiency does not have obvious consequences on the normal structure and function of resting glomus cells. Glomus cell capacitance (proportional to cell membrane surface) and input resistance (determined by the density of background ion channels in the plasma membrane) measured with the whole cell configuration of the patch clamp technique are unchanged in Epas1 null cells in comparison with controls (Moreno- Domínguez et al., 2020). In addition, the time course and density of voltage-gated K+ and Ca2+ currents, which influence cell excitability, are also remarkably similar in control and Epas1-null glomus cells (Fig. 2A-D). However, Epas1-deficient glomus cells loaded with Fura-2 exhibit a clear inhibition of the hypoxia-induced increase in cytosolic [Ca2+] without changes in the responses to CO2 or high K+ (Fig. 3A-E). In Epas1-null CBs there is a drastic decrease in the number of cells that respond to hypoxia (Fig. 3C) and in the amplitude of the Ca2+ signals (Fig. 3D). In agreement with these results, the responsiveness of glomus cells to hypoxia, as determined by amperometric measurement of exocytotic dopamine release with a carbon fiber electrode (Fernández-Agüera et al., 2015; Moreno-Domínguez et al., 2020), is markedly inhibited in Epas1f/-,Cre mice in comparison with wild type mice (Fig. 4 A-B). The percentage of cells that respond to hypoxia is decreased in Epas1-null mice, whereas responsiveness to hypercapnia and high K+ is unchanged (Fig. 4C and D). In addition, Epas1-deficient cells that exhibit some secretory activity in hypoxia also have a decreased secretion rate in comparison with controls (Fig. 4D). These data indicate that although Epas1-deficient glomus cells have normal electrical parameters and excitability, as well as some chemosensory functions, they are practically unresponsive to hypoxia. 8 Fig. 3. Cytosolic [Ca2+] signals elicited by hypoxia, hypercapnia, and high K+ in dispersed glomus cells from Epas1+/+ and Epas1-null mice. (A and B) Representative recordings of the increases in cytosolic [Ca2+] elicited by hypoxia (Hx; ~10 to 15 mmHg), hypercapnia (CO2; 20% CO2) and high K+ (40K; 40 mM KCl) in Fura2-loaded glomus cells from from Epas1+/+ and Epas1-null mice. (C) Percentage of responding cells (exhibiting an increase in cytosolic [Ca2+] in response to hypoxia or hypercapnia) relative to high K+ responding cells from Epas1+/+ and Epas1f/−,Cre mice. (D and E) Quantification of the increase in cytosolic [Ca2+] elicited by hypoxia or hypercapnia (D) and high K+ (E) in dispersed cells from Epas1+/+ and Epas1-null mice. Data are presented as mean ± SEM. *P < 0.05. (From Moreno-Domínguez et al., 2020). The absolute requirement for Hif2α to maintain acute sensitivity to hypoxia in glomus cells depends on the gene expression profile described above. Conditional deletion of Cox4i2 in catecholaminergic tissues result in a phenotype similar to the one described in Epas1-deficient glomus cells (Moreno-Domínguez et al., 2020). In addition, deletion of Higd1C also inhibits the sensitivity to hypoxia in glomus cells (Timón-Gómez et al., 2022). The role of other Hif2α-dependent transcripts in acute O2 sensing is as yet unknown. Deletion of Ndufa4l2 does not seem to elicit any clear alteration in systemic HVR or glomus cell response to hypoxia (Moreno-Domínguez et al., 2020). Therefore, the precise role of Ndufa4l2 in the context of acute O2 sensing remains to be determined. On the other hand, Olfr78, which is a G protein-coupled receptor highly expressed in glomus cells (Zhou et al., 2016; Gao et al., 2017), was proposed to be a lactate receptor essential for the hypoxic ventilatory response (Chang et al., 2015). However, subsequent studies have shown that CB responsiveness to hypoxia and the HVR are maintained in Olfr78 - null mice (Torres-Torrelo et al., 2018). Moreover, glomus cell activation by lactate is independent on Olfr78 expression (Torres-Torrelo et al., 2018, 2021). Although Olfr78 is dispensable for lactate or acute O2 sensing, it may have a relevant role in glomus cell homeostasis. Decreased sensitivity to mild hypoxia of Olfr78-