Na + /H + Exchanger Isoform 1 Regulates Apoptosis Susceptibility in Pulmonary Arterial Smooth Muscle from the Sugen/Hypoxia model of Pulmonary Hypertension

Pulmonary hypertension (PH) is characterized by vascular remodeling driven in part by apoptosis-resistant pulmonary arterial smooth muscle cells (PASMCs). Na + /H + exchanger isoform 1 (NHE1) regulates intracellular pH and plasma membrane cytoskeleton anchoring, but the role of NHE1 in PASMC apoptosis remains unclear. PASMCs were isolated from distal pulmonary arteries from control and Sugen/Hypoxia (SuHx) rats, a model of PH. NHE activity was measured via the ammonium pulse technique, protein via immunoblot, and apoptosis via Hoechst staining, Caspase-Glo ® 3/7 Assay System and Annexin V/ propidium iodide immunofluorescence. NHE activity and NHE1 surface expression were increased in SuHx PASMCs compared to controls. Despite increased endoplasmic reticulum stress at baseline, SuHx PASMCs were resistant to apoptosis following hydrogen peroxide (H 2 O 2 ) challenge. Inhibition of NHE activity with ethyl-isopropyl amiloride (EIPA) and NHE1 silencing with siRNA restored apoptosis in SuHx PASMCs. Conversely, NHE1 overexpression in control PASMCs conferred apoptosis resistance. Expression of mutant NHE1 constructs lacking ion translocation or ezrin binding also reduced H 2 O 2 -induced apoptosis. Mechanistically, H 2 O 2 increased p38 phosphorylation (p-p38) in PASMCs from control, but not SuHx, rats, indicating impaired activation of this pro-apoptotic pathway. NHE1 suppression via EIPA or siRNA restored p-p38 in SuHx PASMCs, while overexpression of NHE1 (wild-type or mutants) suppressed p-p38 following apoptotic stimulation. Inhibition of p-p38 with SB203580 prevented the pro-apoptotic effect of EIPA, validating a role for p-p38 signaling in NHE1-mediated apoptosis resistance in SuHx PASMCs. These findings identify NHE1 as necessary and sufficient for PASMC apoptosis resistance in PH, by a mechanism involving suppression of p-p38.