Na+/H+ exchanger isoform 1 (NHE1) confers resistance to apoptosis in pulmonary arterial smooth muscle cells (PASMCs)

Abstract

Pulmonary hypertension (PH) is a condition caused by remodeling of the lung vasculature due to abnormal cell turnover involving increased cell proliferation and resistance to apoptosis. The mechanisms controlling abnormal cell growth and survival are incompletely understood. We previously found that Na+/H+ exchanger (NHE) activity is increased in PASMCs from PH patients and the Sugen Hypoxia (SuHx) animal model of PH. NHE isoform 1 (NHE1) is a membrane protein that regulates intracellular pH (pHi) and cell volume via export of H+ and import of Na+. This protein also has a cytosolic tail region shown to be involved in non-canonical functions such as cytoskeletal interactions and cell signaling via protein-protein interactions. NHE1 has been associated with increased PASMC proliferation; however, it is unclear if increased NHE activity, dysregulation of pHi or cytoskeletal interactions are involved in resistance to apoptosis. In this study, we tested the hypothesis that increased expression of NHE1 leads to apoptosis resistance in PASMCs via dysregulation of pHi. We first used the rat SuHx model of PH in which male Wistar rats receive a single subcutaneous injection of the vascular endothelial growth factor receptor inhibitor, SU5416, followed by 3 wks of exposure to hypoxia (10% O2) and 2 wks of normoxia. PASMCs were isolated from distal pulmonary arteries of control and SuHx Wistar rats and treated with the NHE inhibitor, 5-(N-ethyl-N-isopropyl)-amiloride (EIPA; 1 uM), or vehicle for 24 h before analyzing cell apoptosis via Hoechst staining under baseline and stimulated conditions (250 uM H2O2). Endoplasmic reticulum stress was measured using the fluorescent stain, Thioflavin T (ThT). In some experiments, cells from control rats were infected with adenovirus containing green fluorescent protein (AdGFP; control for infection), wild-type NHE1 (AdNHE1-WT) or NHE1 mutants: AdNHE1-E262I lacking ion translocation activity and AdNHE1-EM lacking the ezrin binding site which prevents cytoskeletal interactions. NHE1 protein expression and localization were assessed via immunoblotting (total and surface proteins). Effcacy of Na+/H+ translocation was assessed from Na+-dependent recovery following an ammonium pulse. At baseline, despite increased ThT levels in SuHx cells, apoptosis was not different compared to control cells, indicating SuHx cells have suppressed apoptotic response to increased ER stress. Following treatment with EIPA, apoptosis was increased in SuHx cells, possibly indicating they became sensitized to the high levels of ER stress and were able to proceed into the apoptotic pathway. Similarly, following treatment with H2O2, SuHx PASMCs showed resistance to apoptosis compared to control cells, which was alleviated with pre-treatment with EIPA. In control cells, wild-type NHE1 and NHE1 mutants were expressed in similar amounts which led to an expected increase in NHE activity for WT and NHE1-EM, but not for NHE1-E262I when compared to GFP. We found that enhanced expression of NHE1-WT was suffcient to inhibit apoptosis following treatment with H2O2. Interestingly, both NHE1 mutants also significantly decreased PASMC apoptosis following H2O2 stimulation. Our results indicate NHE1 is necessary for apoptosis resistance in PASMCs from animals with PH and that increased expression is suffcient to confer resistance to apoptosis in control PASMCs. Additionally, these results suggest neither pHi regulation nor interaction between NHE1 and ezrin are essential for NHE1-induced apoptosis resistance, indicating another mechanism through which NHE1 confers resistance to apoptosis in PH. Based on these findings, we speculate that development of NHE1-related therapeutic targets could potentially restore cell apoptosis and reverse disease. AHA 23PRE1022720, HL159906, HL073859. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.