Impact of Hypoxia in Cystic Fibrosis Bronchial Epithelial Cells: Focus on CFTR and TRPA1 Channels

IntroductionCystic Fibrosis (CF) is an autosomal and recessive disease caused by the mutation of a gene located on the chromosome 7: CFTR (Cystic Fibrosis Transmembrane conductance Regulator). Its codes for CFTR, a protein which plays a role in mucus homeostasis by transporting both chloride ions and water. The most common mutation F508del-CFTR, leads to the absence and malfunction of CFTR at the surface of epithelial cells in various organs especially in lungs. It results in the loss of the mucus clearance properties in the airways, which will cause the obstruction of bronchi and alveola over time. The oxygen (O2) delivery, crucial for aerobic metabolism, is less effective especially for the lung's epithelial cells whose environment is gradually becoming hypoxic. The inability of lungs to realise haematosis, at tissue level is commonly named the respiratory failure.Our project aims to characterise the impact of hypoxia on ion channels, especially CFTR and TRPA1 (an oxygen sensible calcium channel).MethodsCystic Fibrosis Bronchial Epithelial (CFBE) cells-wt (WT) and CFBE F508del (DF) are cultivated in a controlled hypoxic atmosphere (1% O2). Protein expression and quantification have been realised by western blot. CFTR activity have been measured by automated patch-clamp (whole cell recording, WCR) and Ussing chamber while the activity of TRPA1 have been recorded using the Fluo4-AM probe. TRPA1 localisation has been studied by immunostaining.ResultsHere, we show that the change from normoxia (21% O2) to hypoxia (1% O2) is able to induce a fast cellular response with the accumulation of HIF-1α (Hypoxia Inducible Factor) in only 6hours in CFBE WT, CFBE-DF and CFBE-DF corrected by the tri-therapy Kaftrio® (Elexacaftor/Tezacaftor/Ivacaftor, ETI). We also observed, that HIF-1α accumulated is reduced in non-corrected CFBE-F508del. Regarding CFTR, our results shows that only F508del-CFTR is impacted by hypoxia at protein level and activity, despite the correction by ETI. Automated WCR patch-clamp and Ussing chamber recordings both show that Kaftrio® corrected F508del-CFTR activity decreases 24hours after hypoxia induction. F508del-CFTR currents are decreased by 43% in whole cell configuration while short-circuit current (Isc) CFTR dependent are diminished by around 48%. Concerning TRPA1, hypoxia does not impact the protein accumulation but instead decrease the channel activity by 49% in CFBE-wt, 40% in CFBE-F508del non corrected and 30% when corrected by ETI. Finally, it seems that hypoxia plays a role in TRPA1 location inducing its relocation close to the plasma membrane.ConclusionOur data show a reduced amount of CFTR protein accumulated in CFBE F508del corrected or not, which was not observed on the WT form. Electrophysiologic assays show a clear impact of hypoxia on F508del-CFTR activity at a cellular level and at a pseudo-epithelium level. Despite a lack of impact of hypoxia on TRPA1 protein accumulation, both activity (decreased by hypoxia) and his location (closer to the plasma membrane) are affected by a lower O2 concentration. It is also important to note that in hypoxic state, ETI is not as efficient as it could be in normoxia, which raises questions about the intake conditions of treatment for patients.