B28-13 Mercaptopyruvate Sulfurtransferaseloss Promotes Mitochondrial Dysfunction and Impairs Airway Epithelial Differentiation in a Neonatal Patient-derived 3D Model

Abstract Rationale Supraphysiological oxygen (hyperoxia) is lifesaving for preterm infants, however it damages the developing airway epithelium, leading to aberrant remodeling and long-term respiratory complications such as wheezing and early-onset COPD. The cellular mechanisms that underlie this remodeling remain poorly defined. Using a 3D organotypic model of neonatal patient-derived tracheal airway epithelial cells (nTAECs), we found that hyperoxia downregulates mitochondrial Complex I, disrupts bioenergetics, and impairs terminal differentiation into ciliated cells. MPST (3-mercaptopyruvate sulfurtransferase), a mitochondrial redox enzyme, functions as an upstream checkpoint that protects Complex I from oxidative injury and determines whether oxidant stress propagates or resolves. Our data show that hyperoxia suppresses MPST expression in nTAECs. This study will test whether MPST loss drives mitochondrial dysfunction and impaired epithelial differentiation in the developing airway epithelium. Methods nTAECs were isolated from tracheal aspirates of intubated neonates, were seeded in a 3D air-liquid interface (ALI) Fig. 1A system and differentiated over 7 days (ALI day 0 to 7). nTAECs were treated with siRNA (or scrambled (Scr) control) targeting MPST at the time of cell seeding, achieving 70% knockdown. To capture early transcriptomic changes following MPST loss, bulk RNA sequencing was performed on ALI day 3. On ALI day 7, immunofluorescence was used to assess differentiation into club and ciliated lineages and immunoblotting was utilized to evaluate NDUFS1 expression, a key Complex I subunit critical for mitochondrial bioenergetics. Cellular bioenergetic function was measured by Seahorse metabolic flux analysis. Results Bulk RNAseq (ALI day 3) following MPST knockdown in nTAECs showed SCGB1A1, a club cell marker, among top downregulated genes Fig 1B, indicating early disruption in differentiation trajectory. On ALI day 7, immunofluorescence revealed reduced terminal differentiation into ciliated cells Fig 1C, recapitulating hyperoxia-induced differentiation defects. MPST knockdown also decreased NDUFS1 expression Fig 1D. Metabolic flux analysis demonstrated reduced baseline and maximal oxygen consumption rate (OCR) [Fig 1E, consistent with mitochondrial bioenergetic dysfunction. Conclusion MPST knockdown impairs mitochondrial bioenergetics and disrupts airway epithelial differentiation in neonatal airway cells, mimicking hyperoxia-induced epithelial injury. These findings implicate MPST as a key regulator of mitochondrial function and epithelial maturation in the developing airway. MPST-targeted therapies could mitigate hyperoxia-induced long-term airway remodeling in neonates. This abstract is funded by: PHF, OSCTR