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bioRxiv · 10.1101/2025.07.08.663799

NDUFS2 (NADH:Ubiquinone oxidoreductase core subunit S2) in Mitochondrial Electron Transport Chain Complex I is Critical to Oxygen Responsiveness of Human Ductus Arteriosus Smooth Muscle Cells

Bentley, R. E. T.·Dunham-Snary, K. J.·Martin, A. Y.·Mewburn, J.·Ott, B. P.·Thebaud, B.·Friedberg, M. K.·Hindmarch, C. C.·Archer, S. L.
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Abstract

BackgroundMitochondria in ductus arteriosus smooth muscle cells (DASMCs) are oxygen sensors triggering vasoconstriction at birth; however, the oxygen sensing mechanisms are incompletely understood. Given the conserved role of mitochondrial Complex I subunit NDUFS2 in other oxygen-sensing tissues, we examined its role in DASMC oxygen sensing, comparing it to other Complex I subunits (NDUFS1 and NDUFS7) and putative O2-sensor subunits (UQCRFS1 and COX4I2). MethodsHuman DASMCs were grown in hypoxia (pO2=41mmHg). Oxygen responsiveness was assessed, measuring changes in intracellular calcium, [Ca2+]i, cell length, and mitochondrial reactive oxygen species (mROS). DASMCs were treated for 48-hours with control siRNA versus siRNA targeting NDUFS2, NDUFS1, NDUFS7, UQCRFS1, or COX4I2. qPCR and immunoblotting confirmed knockdown. 3RNA sequencing assessed transcriptional changes following siRNA. ResultsOxygen increased mitochondrial fission, [Ca2+]i, and constricted DASMCs. 48-hours post-treatment, siNDUFS2 selectively depressed oxygen-induced increase in [Ca2+]i (siControl +18.6{+/-}2.3%, siNDUFS2 +5.5{+/-}1.5%, p<0.0001), DASMC shortening (from 18.4{+/-}1.1% to 8.9{+/-}0.8%, p<0.0001), and mROS (+24{+/-}4.9% untreated, -6.6{+/-}5.4% post-siNDUFS2, p<0.0001), without altering the KCl response or depressing respiration. The mitochondrial antioxidant MitoTEMPO reduced mROS (2.9{+/-}4.5%, p=0.001) and attenuated oxygen-induced DASMC shortening (8.4{+/-}0.9%, p=0.0003). Transcriptomics revealed unique changes in mitochondrial pathways post siNDUFS2. ConclusionsNDUFS2 regulates mROS and is a mitochondrial oxygen sensor in human DASMCs. ImpactO_LIWe demonstrated a unique role of Complex I subunit NDUFS2 (NADH:Ubiquinone Oxidoreductase Core Subunit S2), amongst putative oxygen-sensing electron transport chain subunits, in the responsiveness of human ductus arteriosus (DA) smooth muscle cells (DASMCs) to oxygen. C_LIO_LINDUFS2 knockdown inhibited oxygen-induced DASMC constriction and generation of mitochondrial reactive oxygen species, at a timepoint prior to inhibition of mitochondrial respiration and without inhibition of KCl-induced constriction. C_LIO_LIWhile mitochondria are known DA oxygen sensors, this work identifies NDUFS2 as a molecular mediator of human DA oxygen sensing within the mitochondria, enhancing our understanding of a vital physiologic phenomenon and providing a novel potential therapeutic target to modulate ductal patency. C_LI

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Bentley, R. E. T., Dunham-Snary, K. J., Martin, A. Y., Mewburn, J., Ott, B. P., Thebaud, B., Friedberg, M. K., Hindmarch, C. C., Archer, S. L.. 2025-07-14. NDUFS2 (NADH:Ubiquinone oxidoreductase core subunit S2) in Mitochondrial Electron Transport Chain Complex I is Critical to Oxygen Responsiveness of Human Ductus Arteriosus Smooth Muscle Cells. https://doi.org/10.1101/2025.07.08.663799

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