Na ⁺ /H ⁺ exchanger isoform 1 (NHE1) modulates apoptotic pathways in pulmonary arterial smooth muscle cells (PASMC) via control of CHOP levels

Pulmonary hypertension (PH) is a severe condition that arises from vascular remodeling in the lung due to increased cell proliferation and resistance to apoptosis. Our lab showed pulmonary arterial smooth muscle cells (PASMCs) from the Su5416+hypoxia (SuHx) animal model are resistant to apoptosis. Our preliminary data found increased ER stress in SuHx PASMCs, but lower levels of apoptosis, suggesting an inability of SuHx cells to progress in the ER stress-induced apoptotic pathway. The extracellular signal-regulated kinase (ERK 1/2) is a MAPK kinase involved in the ER stress-induced apoptosis pathway. ERK1/2 upregulates CAAT/enhancer binding protein homologous transcription factor (CHOP), allowing induction of apoptosis. Na + /H + exchanger isoform 1 (NHE1) is a membrane protein that regulates intracellular pH (pH i ) and cell volume via export of H + and import of Na + . NHE1 also has a cytosolic tail region that is involved in non-canonical functions such scaffolding for cell signaling molecules, including ERK1/2. We previously found NHE activity was increased in PASMCs from the SuHx animal model, but it is unclear if increased NHE activity is involved in the inhibition of apoptotic pathways in SuHx PASMC. In this study, we tested the hypothesis that increased NHE activity suppresses apoptosis via facilitating increased phosphorylation of ERK1/2 and decreased CHOP expression. We used the rat SuHx model of PH. Wistar rats received a single subcutaneous injection of SU5416, a vascular endothelial growth factor receptor inhibitor, and were exposed to hypoxia (10% O 2 ) for 3 wks followed by 2 wks of normoxia after which PASMCs were isolated from distal pulmonary arteries. Control rats received a vehicle injection and were placed in normoxia for 5 wks. Apoptosis was assessed via Hoechst staining under baseline and stimulated conditions (250-500 uM H 2 O 2 ). In some experiments, cells were pretreated with the NHE inhibitor, 5-(N-ethyl-N-isopropyl)-amiloride (EIPA; 1 uM) or vehicle (DMSO) for 24 h. To deplete NHE1, SuHx PASMCs were transfected with small interfering RNA (siRNA) directed towards either the NHE1 protein (siNHE1) or a non-targeted sequence (siNT). Apoptosis in SuHx cells was similar to control cells at baseline and lower than controls following H 2 O 2 treatment. Treatment with EIPA restored apoptosis susceptibility in SuHx cells. Additionally, depleting NHE1 with siRNA increased H 2 O 2 -induced apoptosis in SuHx cells compared to siNT. Despite increased ER-stress levels, SuHx cells showed negligible CHOP levels at baseline. Following apoptotic stimulation, CHOP abundance increased in control, but not SuHx, PASMCs. However, EIPA treatment restored the ability of H 2 O 2 to induce CHOP in SuHx cells. To determine whether ERK1/2 could be a possible link between NHE1 and CHOP levels, wetreated PASMCs with the ERK1/2 activation inhibitor U0126(10 uM) and measured apoptosis. We found increased phosphorylated ERK1/2 (p-ERK) levels in SuHx cells at baseline, and a trend towards increased H 2 O 2 -induced apoptosis in the presence of U0126 in SuHx cells. However, EIPA had no effect on ERK1/2 phosphorylation. Our results indicate NHE1 modulates apoptosis susceptibility in PASMCs from SuHx rats. We also observed that NHE1 controls CHOP abundance; however, this appears to be through a mechanism independent of ERK1/2. These results suggest NHE1 and CHOP could be new targets in SuHx PASMCs to potentially restore normal cell apoptosis and reverse disease. AHA 23PRE1022720, HL159906, HL073859 This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.