Dissociation of UPR signaling and ER ultrastructure during 4-PBA therapy in shunt-driven pulmonary hypertension

Background Pulmonary arterial hypertension associated with congenital heart disease (PAH-CHD) arises from chronic left-to-right shunting and high-flow exposure, leading to pulmonary vascular remodelling and right-heart dysfunction. Whether high-flow shunting induces branch-selective unfolded protein response (UPR) activation and endoplasmic reticulum (ER) structural adaptation in pulmonary vascular smooth muscle remains incompletely defined. Methods Rats underwent right pulmonary artery ligation combined with left common carotid artery–external jugular vein shunting (RPAL/LCS), with or without 4-phenylbutyric acid (4-PBA) treatment. Hemodynamics, right-heart function, vascular remodeling, UPR signaling, transcriptomic profiles, apoptosis, and ER ultrastructure were assessed in vivo or in HPASMC gain- and loss-of-function experiments. Results RPAL/LCS increased RVSP from 22.46 ± 1.68 to 59.66 ± 7.06 mmHg, whereas 4-PBA reduced RVSP to 38.24 ± 1.71 mmHg. The wall area/total vessel area ratio increased from 46.88 ± 1.69% to 77.89 ± 3.08% after RPAL/LCS and decreased to 47.19 ± 2.00% after 4-PBA treatment. Lung immunoblotting showed increased p-IRE1α/IRE1α, whereas p-PERK/PERK and ATF6N/ATF6 did not differ significantly; these proximal UPR markers were not reduced by 4-PBA at the endpoint. In HPASMCs, ATF4 and ATF6N overexpression enriched apoptosis-related programs and induced ER swelling and fragmentation, whereas XBP-1s overexpression maintained organized ER cisternae and XBP-1s knockdown disrupted ER ultrastructure with increased Annexin V–positive apoptotic cells. Conclusions This study suggests that the IRE1α–XBP1s axis supports adaptive ER proteostasis and structural organization in shunt-driven pulmonary hypertension, whereas 4-PBA improves disease features without measurable suppression of canonical UPR branch activation markers.