Validation of a human cell model to investigate the role of Ca2+-activated Cl- channels encoded by Anoctamin-1 in human pulmonary arterial smooth muscle cells
Pulmonary arterial hypertension (PAH) is a rare chronic disease in humans. While new genetic markers have been identified and new therapeutic agents developed within the last two decades, survival rate and quality of care have only seen incremental improvement. For this fact, investigation into the mechanisms behind pulmonary arterial hypertension (PAH) is merited. Animal models of pulmonary hypertension demonstrate increased activity and expression of Ca2+ -activated Cl- channels (CaCC) encoded by Anocatmin-1 (ANO1). It is known that CaCCs activity and ANO1 expression are in increased in animal models of PAH, which is thought to promote smooth muscle cell depolarization, enhanced Ca2+ entry and tone (Leblanc et al., 2015). The goal of this study is to create a human cell model of pulmonary arterial smooth muscle cells (hPASMCs) to later investigate the role of CaCCs in PAH. This study measured intracellular Ca2+ concentration in hPASMCs with the calcium indicator Fluo-4/AM. The Fluo-4 fluorescence (F/F0) was recorded using a Photo Detection System and Total Internal Reflection Fluorescence (TIRF) Microscopy whereby the constricting agonists Phenylephrine and Serotonin, and the TRPV4 agonist GSK 1016790A, were perfused on hPASMCs. Phenylephrine and Serotonin did not consistently elicit Ca2+ responses in hPASMCs, however, the TRPV4 agonist GSK exhibited a more consistent effect on Fluo-4 fluorescence in hPASMCs. In order to gather more conclusive results, the number of experiments using GSK should be increased substantially and more cell lines need to be made available including samples of hPASMCs from patients of all ages and sexes, and PAH patients. Once these additional resources and results are met, this will allow for a more comprehensive validation of hPASMCs as a model to investigate the role of CaCCs and ANO1 in PAH
