Group 4 : KCNK3, NMDAR & ionic channel pathways
Fabrice Antigny and Sylvia Cohen-Kaminsky
The pathophysiology of PAH involve the remodelling the pulmonary arteries and the right heart. The functions of pulmonary vascular cells and right ventricular cells are regulated by many ion channels. We are studying different families of ion channels (K+, Ca2+ and Cl−), their regulatory mechanisms and their functions at the pulmonary vascular and cardiac levels, in order to understand their implication in the pathophysiology of PAH, and to explore their potential as new therapeutic targets for PAH.
Genetic mutations partly explain the abnormal phenotypes of pulmonary vascular cells in heritable and non-heritable PAH. Recently, mutations in genes coding for different ion channels have been identified in different cohorts of PAH patients, such as the KCNK3, ABCC8, ATP13A3, CRACRA2 genes. In addition, polymorphisms of the KCNA5gene (Kv1.5, Voltage-gated K+channels) and the TRPC6 (Transient Recepteur Potential Canonical 6, a cation channel)gene have been shown to predispose to the development of PAH. To date, 10 mutations in the KCNK3gene (Potassium Channel, Subfamily K, member 3) have been identified in PAH patients. The KCNK3gene codes for a potassium channel also called TASK-1 (TWIK-Related Acid-Sensitive K+Channel 1). Patch-clamp recordings show a loss of function for each identified mutation.
With the financial support of ANR JCJC KAPAH (https://anr.fr/Projet-ANR-18-CE14-0023), our group recently demonstrated that 1) the KCNK3 dysfunction contributes to the development of hereditary PAHs (BMPR2mutated patients) and also to idiopathic PAH, thus indicating that the loss of KCNK3-function is a hallmark of PAH; KCNK3 dysfunction contributes to the development of experimental pulmonary hypertension (PH) (Antigny F et al. Circulation 2016)2) that Kcnk3loss of function mutation in rats (by CRISPRCas9 approach) predisposes these animals to the development of experimental PH (Lambert M et al. Circ Research 2019)http://www.theses.fr/2019SACLS268, Mélanie Lambert PhD thesis) and 3) that the loss of function/expression of KCNK3 is also a characteristic of the RV hypertrophy/dysfunction in PH (Lambert M et al. Cardiovasc Res. 2018). We continue studying the consequences of KCNK3 dysfunction in PAH.
In 2018, 12 mutations in the ABCC8(ATP-binding cassette transporter sub-family C member 8) gene were identified in several cohorts of PAH patients. The protein encoded by thisABCC8gene, a member of the ABC transporter (ATP-binding cassette) superfamily, is known as SUR1 (Sulfonylurea Receptor 1), which is a regulatory subunit of K+channels-ATP-dependent (KATP), the Kir6.2 and Kir6.1 channels. Most of the identified mutations induce a loss of function of the SUR1/Kir6.2 channel. We continue studying the role of the ABCC8gene or SUR1 protein in pulmonary circulation and the involvementof this channel in the pathogenesis of PAH.
The voltage-dependent calcium channels are responsible for the ICaL and ICaT currents, the main source of calcium for excitable cells. Our group has shown that the Ca2+signaling generated by ICaT is altered in the PASMC from PAH patients, contributing to the “hyper-proliferation / resistance to apoptosis” phenotype of these cells (Sankhe S et al, Biochim Biophys Acta, 2017).
With the financial support of ANR NUTS (https://anr.fr/Projet-ANR-14-CE16-0016) and LabEx LERMIT (http://www.labex-lermit.fr/en/25-projets/projet-target/t1/30%20-t1-2), our group has recently identified the NMDAR receptor (N-methyl-D-aspartate receptor), another Ca2+ channel regulated by the resting membrane potential as a major player in PAH pulmonary vascular remodeling (Dumas S et al., Circulation 2018). We show that 1) NMDAR and its ligand, glutamate, are disregulated in pulmonary arteries. We highlighted that the NMDAR is over-expressed by the PAEC and PASMC isolated from PAH patients. 2) NMDAR is activated (by phosphorylation) via ET-1 and PDGF, two main pathways deregulated in PAH and the pulmonary vascular cell proliferation induced by PDGF or VEGF is inhibited by NMDAR antagonists. 3) mice deficient for nmdarin PASMC or PAEC are protected against pulmonary hypertension. Unexpectedly, we have shown that NMDAR is involved in angiogenesis (https://www.theses.fr/191147184, Sébastien Dumas PhD thesis), which is deregulated in PAH. 4) Finally, we have shown that the expression of the Glun2B (subunit of NMDARs) is reduced in PAH patients, and that the activation of GluN2B via Src kinase could confer proliferative and anti-migratory properties to PASMCs. The reduced levels of GluN2B in the pulmonary arteries of PAH patients may be responsible for the excessive proliferation of PASMC, thus contributing to medial hyperplasia and the development of PAH (Quatredeniers et al., AJP lung 2019).
These works demonstrate that the activation of NMDAR at the pulmonary vascular level promotes arterial remodeling and PAH, and suggests that targeting NMDAR could be a new therapeutic strategy in PAH. The project is currently in its technological maturation phase at SATT Paris-Saclay (NUTS-MAT project) https://satt-paris-saclay.fr/vitrine-technologique/nuts-mat/), protected by3 patent familes (WO/2017/093354, WO/2017/017116, WO/2017/216159),and we have validated a drug candidate, targeting NMDAR without central effects (not passingthe blood-brain barrier). A start-up is being created to develop this new therapeutic approach.
We continue to decipher the mechanisms linked to the NMDAR/glutamate pathway in PAH, and we plan to develop therapeutic tools to directly target this pathway. We are developing a model of the involvement of NMDARs in the context of the known PAH pathways, using a systems biology approach (http://www.theses.fr/s142375, Marceau Quatredeniers PhD Thesis), and we have contributed to constructing a whole-atom dynamic molecular model of the NMDAR in order to design new NMDAR antagonists targeting the open channel (Palmai et al, Plos one, 2018).
Our team has recently proposed that a lung/gut axis inducing LPS translocation from the gut to the lungs which could contribute to pulmonary vascular remodeling of PAH (Ranchoux B et al AJRCM 2017). We hypothesized that whatever the mechanisms leading to a dysbiosis of the lung microbiota (disruption of pulmonary homeostasis, bacterial translocation, or migration of oropharyngeal bacteria), changes in the structure and the lung microbiota diversity could have direct effects on the pulmonary vascular remodelling leading to PAH. Thus, we will explore the lung microbiota as a new actor with a direct impact on vascular remodeling and the progression of PAH, via its metabolites and their potential actions on the different ion channels of the pulmonary vasculature (ANR LUMI, 2019, https://anr.fr/Projet-ANR-18-CE14-0043).
Other Ca2+channels activated independently of the resting membrane potential could contribute to the pathogenesis of PAH, the Store-Operated Ca2+channels (SOC). SOC participate to Ca2+homeostasis of many cell types. SOC are composed of three essential elements: the regulatory element, the family of stromal interaction molecules (STIM1-2) and two Ca2+ channel namely TRPC (Transient Receptor Potential Canonical 1-7) and Orai (Orai1-3). In some PAH patients, polymorphisms inducing gain of function of TRPC6 have been identified, which predisposes to the development of PAH. In addition, trpc1/c6deficient mice are resistant to the induction of PAH by chronic hypoxia. In collaboration with Dr Jessica Sabourin (INSERM U1180), our group recently demonstrated the involvement of the TRPC1, TRPC4 and Orai1 channels in the development of RV hypertrophy in an experimental model of PH in rats (Sabourin J et al., JMCC 2018). We continue deciphering the role of SOC in the PASMC and PAEC proliferation/apoptosis imbalance and in the regulation of pulmonary arterial tone in PAH.
To develop our projects, we developed a set of unique tools
- Transgenic animals: Kcnk3-deficient rats, Orai1-deficient rats in SMC, nmdar-deficient mice in SMC or nmdardeficient mice in EC.
- In vitro studies on isolated cells (PAEC, PASMC, pulmonary fibroblasts) isolated from PAH or controls pulmonary arteries,Calcium imaging, Patch-clamp, Molecular biology, Immunohistochemistry
- In situ studies with confocal imaging and correlative electron microscopy
- Ex vivo studies of the vascular reactivity on arteries or pulmonary veins isolated from samples from PAH patients and from control patients without pulmonary vascular disease and or isolated from our different animal models: myograph
- In vivo studies of cardiac function by echocardiography and cardiac catheterization of our different experimental PH models.
Publications (Best selected)
Antigny F, Mercier O, Humbert M, Sabourin J. Excitation-contraction coupling and relaxation alteration in right ventricular remodelling caused by pulmonary arterial hypertension.Arch Cardiovasc Dis. 2020 Jan 7. pii: S1875-2136(19)30224-4.
Bartoli F, Bailey MA, Rode B, Mateo P, Antigny F, Bedouet K, Gerbaud P, Gosain R, Plante J, Norman K, Gomez S, Lefebvre F, Rucker-Martin C, Ainscough JFX, Kearney MT, Bruns AF, Shi J, Appleby HL, Young RS, Shawer HM, Debant M, Gomez AM, Beech DJ, Foster R, Benitah JP, Sabourin J. Orai1 Channel Inhibition Preserves Left Ventricular Systolic Function and Normal Ca2+ Handling After Pressure Overload.Circulation. 2020 Jan 21;141(3):199-216.
Bartoli F, Moradi Bachiller S, Antigny F, Bedouet K, Gerbaud P, Sabourin J, Benitah JP.Specific Upregulation of TRPC1 and TRPC5 Channels by Mineralocorticoid Pathway in Adult Rat Ventricular Cardiomyocytes.Cells. 2019 Dec 23;9(1). pii: E47.
Lambert M, Capuano V, Boet A, Tesson L, Bertero T, Nakhleh MK, Remy S, Anegon I, Pechoux C, Hautefort A, Rucker-Martin C, Manoury B, Domergue V, Mercier O, Girerd B, Montani D, Perros F, Humbert M, Antigny F. Characterization of Kcnk3-Mutated Rat, a Novel Model of Pulmonary Hypertension.Circ Res. 2019 Sep 13;125(7):678-695.
Antigny F. Potassium channels in vascular smooth muscle: a pathophysiological and pharmacological perspective.Fundam Clin Pharmacol. 2019 Oct;33(5):524-526.
Antigny F, Chaumais MC, Humbert M, Montani D. Pulmonary arterial hypertension in patient treated for multiple sclerosis with 4-aminopyridine.Fundam Clin Pharmacol. 2019 Aug;33(4):426-427.
Hautefort A, Mendes-Ferreira P, Sabourin J, Manaud G, Bertero T, Rucker-Martin C, Riou M, Adão R, Manoury B, Lambert M, Boet A, Lecerf F, Domergue V, Brás-Silva C, Gomez AM, Montani D, Girerd B, Humbert M, Antigny F*, Perros F*. Bmpr2 Mutant Rats Develop Pulmonary And Cardiac Characteristics Of Pulmonary Arterial Hypertension.Circulation. 2019 Feb 12;139(7):932-948. doi: 10.1161/CIRCULATIONAHA.118.033744. *co-last authors
Quatredeniers M, Nakhleh MK, Dumas SJ, Courboulin A, Vinhas MC, Antigny F, Phan C, Guignabert C, Bendifallah I, Vocelle M, Fadel E, Dorfmüller P, Humbert M, Cohen-Kaminsky S.Functional interaction between PDGFβand GluN2B-containing NMDA receptors in smooth muscle cell proliferation and migration in pulmonary arterial hypertension.Am J Physiol Lung Cell Mol Physiol. 2019 Mar 1;316(3):L445-L455
Lambert M, Capuano V, Olschewski A, Sabourin J, Nagaraj C, Girerd B, Weatherald J, Humbert M, Antigny F. Ion Channels in Pulmonary Hypertension: A Therapeutic Interest?Int J Mol Sci. 2018 Oct 14;19(10).
Sabourin J, Boët A, Rucker-Martin C, Lambert M, Gomez A-M, Benitah J-P, Perros F, Humbert M, Antigny F. Ca2+ handling remodeling and STIM1L/Orai1/TRPC1/TRPC4 upregulation in monocrotaline-induced right ventricular hypertrophy. J Mol Cell Cardiol. 2018 May;118:208-224. doi: 10.1016/j.yjmcc.2018.04.003. Epub 2018 Apr 7.
Dumas SJ, Bru-Mercier G, Courboulin A, Quatredeniers M, Rücker-Martin C, ANTIGNY F, Nakhleh MK, Ranchoux B, Gouadon E, Vinhas MC, Vocelle M, Raymond N, Dorfmüller P, Fadel E, Perros F, Humbert M, Cohen-Kaminsky S. NMDA-Type Glutamate Receptor Activation Promotes Vascular Remodeling and Pulmonary Arterial Hypertension. Circulation. 2018 Feb 14.
Vardhan Reddy Kh, Yen-Pon E, Cohen-Kaminsky S, Messaoudi S, Alami M. Convergent Strategy to Dizocilpine MK-801 and Derivatives. J Org Chem. 2018 83:4264-4269.
Palmai Z, Houenoussi K, Cohen-Kaminsky S, Tchertanov L. How does binding of agonist ligands control intrinsic molecular dynamics in human NMDA receptors? PLoS One. 2018 Aug 3;13(8):e0201234.
Lambert M, Boet A, Rucker-Martin C, Mendes-Ferreira P, Capuano V, Hatem S, Adão R, Brás-Silva C, Hautefort A, Michel JB, Dorfmuller P, Fadel E, Kotsimbos T, Price L, Jourdon P, Montani D, Humbert M, Perros F, Antigny F. Loss of KCNK3 is a hallmark of RV hypertrophy/dysfunction associated with pulmonary hypertension. Cardiovasc Res. 2018 Jan 19.
Olschewski A, Veale EL, Nagy BM, Nagaraj C, Kwapiszewska G, Antigny F, Lambert M, Humbert M, Czirják G, Enyedi P, Mathie A. TASK-1 (KCNK3) channels in the lung: from cell biology to clinical implications. Eur Respir J. 2017 Nov 9;50(5). pii: 1700754. doi: 10.1183/13993003.00754-2017.
Sankhe S, Manousakidi S, Antigny F, Arthur Ataam J, Bentebbal S, Ruchon Y, Lecerf F, Sabourin J, Price L, Fadel E, Dorfmüller P, Eddahibi S, Humbert M, Perros F, Capuano V. T-type Ca2+ channels elicit pro-proliferative and anti-apoptotic responses through impaired PP2A/Akt1 signaling in PASMCs from patients with pulmonary arterial hypertension. Biochim Biophys Acta. 2017 Oct;1864(10):1631-1641.
Philippe R*, Antigny F*, Buscaglia P, Norez C, Huguet F, Castelbou C, Trouvé P, Becq F, Frieden M, Férec C, Mignen O. Calumenin contributes to ER-Ca2+ homeostasis in bronchial epithelial cells expressing WT and F508del mutated CFTR and to F508del-CFTR retention. Cell Calcium. 2017 Mar;62:47-59. doi: 10.1016/j.ceca.2017.01.011. Epub 2017 Feb 4. . *co-first authors
Antigny F, Sabourin J, Saüc S, Bernheim L, Koenig S, Frieden M. TRPC1 and TRPC4 channels functionally interact with STIM1L to promote myogenesis and maintain fast repetitive Ca2+ release in human myotubes. Biochim Biophys Acta. 2017 May;1864(5):806-813. doi: 10.1016/j.bbamcr.2017.02.003.
Sabourin J, Bartoli F, Antigny F, Gomez A-M, Benitah J-P. TRPCs/Orai1-dependent Store-Operated Ca2+ Channels, new targets of aldosterone in cardiomyocytes.J Biol Chem. 2016 Jun 17;291(25):13394-409.
Antigny F, Hautefort A, Meloche J, Belacel-Ouari M, Manoury B, Rucker-Martin C, Péchoux C, Potus F, Nadeau V, Tremblay E, Ruffenach G, Bourgeois A, Dorfmüller P, Breuils-Bonnet S, Fadel E, Ranchoux B, Jourdon P, Girerd B, Montani D, Provencher S, Bonnet S, Simonneau G, Humbert M, Perros F. Potassium Channel Subfamily K Member 3 (KCNK3) Contributes to the Development of Pulmonary Arterial Hypertension.Circulation2016 Apr 5;133(14):1371-85.
Boucherat O, Chabot S, ANTIGNY F, Perros F, Provencher S, Bonnet S. Potassium channels in pulmonary arterial hypertension. Eur Respir J. 2015 Oct;46(4):1167-77.
Bertrand J, Dannhoffer L, Antigny F, Vachel L, Jayle C, Vandebrouck C, Becq F, Norez C. A functional tandem between transient receptor potential canonical channels 6 and calcium-dependent chloride channels in human epithelial cells. Eur J Pharmacol. 2015Oct 15;765:337-45.
Saüc S, Bulla M, Nunes P, Orci L, Marchetti A, Antigny F, Bernheim L, Cosson P, Frieden M, Demaurex N. STIM1L traps and gates Orai1 channels without remodeling the cortical ER. J Cell Sci. 2015 Apr 15;128(8):1568-79.
Dumas SJ, Humbert M, COHEN-KAMINSKY S. The cancer paradigm in pulmonary arterial hypertension: towards anti-remodeling therapies targeting metabolic dysfunction?. Biol Aujourdhui. 2016; 210:171-189.
Philippe R*, Antigny F*, Buscaglia P, Norez C, Becq F, Frieden M, Mignen O.SERCA and PMCA pumps contribute to the deregulation of Ca2+homeostasis in human CF epithelial cells. *Co-first author. BBA Cell Molecular Research 2015 May;1853(5):892-903.
Girerd B, Perros F, Antigny F, Humbert M, Montani D. KCNK3: new gene target for pulmonary hypertension? Expert Rev Respir Med. 2014 Aug;8(4):385-7.