Patricia González-Rodríguez

Oxygen Sensing by Arterial Chemoreceptors Depends on Mitochondrial Complex I Signaling

O2 sensing is essential for mammalian homeostasis. Peripheral chemoreceptors such as the carotid body (CB) contain cells with O2-sensitive K(+) channels, which are inhibited by hypoxia to trigger fast adaptive cardiorespiratory reflexes. How variations of O2 tension (PO2) are detected and the mechanisms whereby these changes are conveyed to membrane ion channels have remained elusive. We have studied acute O2 sensing in conditional knockout mice lacking mitochondrial complex I (MCI) genes. We inactivated Ndufs2, which encodes a protein that participates in ubiquinone binding. Ndufs2-null mice lose the hyperventilatory response to hypoxia, although they respond to hypercapnia. Ndufs2-deficient CB cells have normal functions and ATP content but are insensitive to changes in PO2. Our data suggest that chemoreceptor cells have a specialized succinate-dependent metabolism that induces an MCI state during hypoxia, characterized by the production of reactive oxygen species and accumulation of reduced pyridine nucleotides, which signal neighboring K(+) channels.

Oxygen-sensing by arterial chemoreceptors: Mechanisms and medical translation

Acute O2 sensing is necessary for the activation of cardiorespiratory reflexes (hyperventilation and sympathetic activation), which permit the survival of individuals under hypoxic environments (e.g. high altitude) or medical conditions presenting with reduced capacity for gas exchange between the lung alveoli and the blood. Changes in blood O2 tension are detected by the arterial chemoreceptors, in particular the carotid body (CB), which act in concert with the adrenal medulla (AM) to facilitate rapid adaptations to hypoxia. The field of arterial chemoreception has undergone a considerable expansion in recent years, with many of the fundamental observations made at the molecular and cellular levels serving to improve our understanding of the pathogenesis of numerous medical disorders, and even to propose advances in the treatment strategies. In this review, after a short historical preface, we describe the current model of chemosensory transduction based on the modulation of membrane K(+) channels by O2 in specialized chemoreceptor cells. Recent progress in elucidating the molecular mechanisms underlying the modulation of ion channels by O2 tension, which involves mitochondrial complex I, is also discussed. The discovery in the last few years of a specific population of neural crest-derived stem cells in the CB explains the reversible growth of this organ, an intriguing and unusual property of this type of neuronal tissue that contributes to acclimatization under chronic hypoxia. The essential homeostatic role of the CB-AM axis is clearly evident in newly generated mouse models that reach adulthood, albeit with CB and AM atrophy. These animals exhibit a marked intolerance to even mild hypoxia. CB inhibition or over-activation can have important medical consequences. Respiratory depression by general anesthetics or by opioid use is a common clinical condition that frequently causes death in susceptible individuals. An exaggerated sympathetic outflow due to over-activation of the CB-AM axis may contribute to the pathogenesis of several highly prevalent medical conditions, such as chronic heart failure, obstructive sleep apnea, obesity, metabolic syndrome, and diabetes. A detailed understanding of the molecular mechanisms underlying acute O2 sensing may help in the design of more efficient therapeutic approaches to combat these disorders.

Oxygen sensing by the carotid body: mechanisms and role in adaptation to hypoxia

Oxygen (O2) is fundamental for cell and whole-body homeostasis. Our understanding of the adaptive processes that take place in response to a lack of O2(hypoxia) has progressed significantly in recent years. The carotid body (CB) is the main arterial chemoreceptor that mediates the acute cardiorespiratory reflexes (hyperventilation and sympathetic activation) triggered by hypoxia. The CB is composed of clusters of cells (glomeruli) in close contact with blood vessels and nerve fibers. Glomus cells, the O2-sensitive elements in the CB, are neuron-like cells that contain O2-sensitive K(+)channels, which are inhibited by hypoxia. This leads to cell depolarization, Ca(2+)entry, and the release of transmitters to activate sensory fibers terminating at the respiratory center. The mechanism whereby O2modulates K(+)channels has remained elusive, although several appealing hypotheses have been postulated. Recent data suggest that mitochondria complex I signaling to membrane K(+)channels plays a fundamental role in acute O2sensing. CB activation during exposure to low Po2is also necessary for acclimatization to chronic hypoxia. CB growth during sustained hypoxia depends on the activation of a resident population of stem cells, which are also activated by transmitters released from the O2-sensitive glomus cells. These advances should foster further studies on the role of CB dysfunction in the pathogenesis of highly prevalent human diseases.

Short Communication: Genetic Ablation of L-Type Ca2+ Channels Abolishes Depolarization-Induced Ca2+ Release in Arterial Smooth Muscle

Rationale: In arterial myocytes, membrane depolarization-induced Ca2+ release (DICR) from the sarcoplasmic reticulum (SR) occurs through a metabotropic pathway that leads to inositol trisphosphate synthesis independently of extracellular Ca2+ influx. Despite the fundamental functional relevance of DICR, its molecular bases are not well known. Objective: Biophysical and pharmacological data have suggested that L-type Ca2+ channels could be the sensors coupling membrane depolarization to SR Ca2+ release. This hypothesis was tested using smooth muscle–selective conditional Cav1.2 knockout mice. Methods and Results: In aortic myocytes, the decrease of Ca2+ channel density was paralleled by the disappearance of SR Ca2+ release induced by either depolarization or Ca2+ channel agonists. Cav1.2 channel deficiency resulted in almost abolition of arterial ring contraction evoked by DICR. Ca2+ channel–null cells showed unaltered caffeine-induced Ca2+ release and contraction. Conclusion: These data suggest that Cav1.2 channels are indeed voltage sensors coupled to the metabolic cascade, leading to SR Ca2+ release. These findings support a novel, ion-independent, functional role of L-type Ca2+ channels linked to intracellular signaling pathways in vascular myocytes.

Glucose sensing by carotid body glomus cells: potential implications in disease.

The carotid body (CB) is a key chemoreceptor organ in which glomus cells sense changes in blood O2, CO2, and pH levels. CB glomus cells have also been found to detect hypoglycemia in both non-primate mammals and humans. O2 and low-glucose responses share a common final pathway involving membrane depolarization, extracellular calcium influx, increase in cytosolic calcium concentration, and neurotransmitter secretion, which stimulates afferent sensory fibers to evoke sympathoadrenal activation. On the other hand, hypoxia and low glucose induce separate signal transduction pathways. Unlike O2 sensing, the response of the CB to low glucose is not altered by rotenone, with the low glucose-activated background cationic current unaffected by hypoxia. Responses of the CB to hypoglycemia and hypoxia can be potentiated by each other. The counter-regulatory response to hypoglycemia by the CB is essential for the brain, an organ that is particularly sensitive to low glucose. CB glucose sensing could be altered in diabetic patients, particularly those under insulin treatment, as well as in other medical conditions such as sleep apnea or obstructive pulmonary diseases, where chronic hypoxemia presents with plastic modifications in CB structure and function. The current review will focus on the following main aspects: (1) the CB as a low glucose sensor in both in vitro and in vivo models; (2) molecular and ionic mechanisms of low glucose sensing by glomus cells, (3) the interplay between low glucose and O2 sensing in CB, and (4) the role of CB low glucose sensing in the pathophysiology of cardiorespiratory and metabolic diseases, and how this may serve as a potential therapeutic target.

Redox signaling in acute oxygen sensing

Acute oxygen (O2) sensing is essential for individuals to survive under hypoxic conditions. The carotid body (CB) is the main peripheral chemoreceptor, which contains excitable and O2-sensitive glomus cells with O2-regulated ion channels. Upon exposure to acute hypoxia, inhibition of K+ channels is the signal that triggers cell depolarization, transmitter release and activation of sensory fibers that stimulate the brainstem respiratory center to produce hyperventilation. The molecular mechanisms underlying O2 sensing by glomus cells have, however, remained elusive. Here we discuss recent data demonstrating that ablation of mitochondrial Ndufs2 gene selectively abolishes sensitivity of glomus cells to hypoxia, maintaining responsiveness to hypercapnia or hypoglycemia. These data suggest that reactive oxygen species and NADH generated in mitochondrial complex I during hypoxia are signaling molecules that modulate membrane K+ channels. We propose that the structural substrates for acute O2 sensing in CB glomus cells are “O2-sensing microdomains” formed by mitochondria and neighboring K+ channels in the plasma membrane.

Hypoxic induction of T‐type Ca2+ channels in rat cardiac myocytes: role of HIF‐1α and RhoA/ROCK signalling

T‐type Ca2+ channels are expressed in the ventricular myocytes of the fetal and perinatal heart, but are downregulated as development progresses. However, these channels are re‐expressed in adult cardiomyocytes under pathological conditions. Hypoxia induces the upregulation of the T‐type Ca2+ channel Cav3.2 mRNA in cardiac myocytes, whereas Cav3.1 mRNA is not significantly altered. The effect of hypoxia on Cav3.2 mRNA requires hypoxia inducible factor‐1α (HIF‐1α) stabilization and involves the small monomeric G‐protein RhoA and its effector ROCKI. Our results suggest that the hypoxic regulation of the Cav3.2 channels may be involved in the increased probability of developing arrhythmias observed in ischemic situations, and in the pathogenesis of diseases associated with hypoxic Ca2+ overload.

Highly Efficient Neural Conversion of Human Pluripotent Stem Cells in Adherent and Animal-Free Conditions

Neural differentiation of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) can produce a valuable and robust source of human neural cell subtypes, holding great promise for the study of neurogenesis and development, and for treating neurological diseases. However, current hESCs and hiPSCs neural differentiation protocols require either animal factors or embryoid body formation, which decreases efficiency and yield, and strongly limits medical applications. Here we develop a simple, animal‐free protocol for neural conversion of both hESCs and hiPSCs in adherent culture conditions. A simple medium formula including insulin induces the direct conversion of >98% of hESCs and hiPSCs into expandable, transplantable, and functional neural progenitors with neural rosette characteristics. Further differentiation of neural progenitors into dopaminergic and spinal motoneurons as well as astrocytes and oligodendrocytes indicates that these neural progenitors retain responsiveness to instructive cues revealing the robust applicability of the protocol in the treatment of different neurodegenerative diseases. The fact that this protocol includes animal‐free medium and human extracellular matrix components avoiding embryoid bodies makes this protocol suitable for the use in clinic. Stem Cells Translational Medicine 2017;6:1217–1226

Orai1 and TRPC1 Proteins Co-localize with CaV1.2 Channels to Form a Signal Complex in Vascular Smooth Muscle Cells

Voltage-dependent CaV1.2 L-type Ca2+ channels (LTCC) are the main route for calcium entry in vascular smooth muscle cells (VSMC). Several studies have also determined the relevant role of store-operated Ca2+ channels (SOCC) in vascular tone regulation. Nevertheless, the role of Orai1- and TRPC1-dependent SOCC in vascular tone regulation and their possible interaction with CaV1.2 are still unknown. The current study sought to characterize the co-activation of SOCC and LTCC upon stimulation by agonists, and to determine the possible crosstalk between Orai1, TRPC1, and CaV1.2. Aorta rings and isolated VSMC obtained from wild type or smooth muscle-selective conditional CaV1.2 knock-out (CaV1.2KO) mice were used to study vascular contractility, intracellular Ca2+ mobilization, and distribution of ion channels. We found that serotonin (5-HT) or store depletion with thapsigargin (TG) enhanced intracellular free Ca2+ concentration ([Ca2+]i) and stimulated aorta contraction. These responses were sensitive to LTCC and SOCC inhibitors. Also, 5-HT- and TG-induced responses were significantly attenuated in CaV1.2KO mice. Furthermore, hyperpolarization induced with cromakalim or valinomycin significantly reduced both 5-HT and TG responses, whereas these responses were enhanced with LTCC agonist Bay-K-8644. Interestingly, in situ proximity ligation assay revealed that CaV1.2 interacts with Orai1 and TRPC1 in untreated VSMC. These interactions enhanced significantly after stimulation of cells with 5-HT and TG. Therefore, these data indicate for the first time a functional interaction between Orai1, TRPC1, and CaV1.2 channels in VSMC, confirming that upon agonist stimulation, vessel contraction involves Ca2+ entry due to co-activation of Orai1- and TRPC1-dependent SOCC and LTCC.

Genetic Rescue of Mitochondrial and Skeletal Muscle Impairment in an Induced Pluripotent Stem Cells Model of Coenzyme Q10 Deficiency

Coenzyme Q10 (CoQ10) plays a crucial role in mitochondria as an electron carrier within the mitochondrial respiratory chain (MRC) and is an essential antioxidant. Mutations in genes responsible for CoQ10 biosynthesis (COQ genes) cause primary CoQ10 deficiency, a rare and heterogeneous mitochondrial disorder with no clear genotype–phenotype association, mainly affecting tissues with high‐energy demand including brain and skeletal muscle (SkM). Here, we report a four‐year‐old girl diagnosed with minor mental retardation and lethal rhabdomyolysis harboring a heterozygous mutation (c.483G > C (E161D)) in COQ4. The patients fibroblasts showed a decrease in [CoQ10], CoQ10 biosynthesis, MRC activity affecting complexes I/II + III, and respiration defects. Bona fide induced pluripotent stem cell (iPSCs) lines carrying the COQ4 mutation (CQ4‐iPSCs) were generated, characterized and genetically edited using the CRISPR‐Cas9 system (CQ4ed‐iPSCs). Extensive differentiation and metabolic assays of control‐iPSCs, CQ4‐iPSCs and CQ4ed‐iPSCs demonstrated a genotype association, reproducing the disease phenotype. The COQ4 mutation in iPSC was associated with CoQ10 deficiency, metabolic dysfunction, and respiration defects. iPSC differentiation into SkM was compromised, and the resulting SkM also displayed respiration defects. Remarkably, iPSC differentiation in dopaminergic or motor neurons was unaffected. This study offers an unprecedented iPSC model recapitulating CoQ10 deficiency‐associated functional and metabolic phenotypes caused by COQ4 mutation. Stem Cells 2017;35:1687–1703