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

Rationale: Mitochondria in ductus arteriosus (DA) smooth muscle cells (DASMC) are oxygen sensors that trigger O2-induced vasoconstriction at birth; however, the molecular mechanisms of mitochondrial oxygen sensing are not fully understood. Many redox sensor proteins are conserved in the mammalian adult homeostatic oxygen sensing system, including NDUFS2 (NADH:Ubiquinone oxidoreductase core subunit S2), a component of mitochondrial Complex I that contributes to oxygen sensing in adult pulmonary arteries. Here we compared the role of NDUFS2 in DA oxygen sensing, to that of other Complex I subunits and putative O2-sensor subunits, including: NADH:Ubiquinone oxidoreductase core subunit S1 (NDUFS1), NADH:Ubiquionone oxidoreductase core subunit S7 (NDUFS7), Ubiquinol-cytochrome c reductase, Rieske iron-sulfur polypeptide 1 (UQCRFS1), and Cytochrome c oxidase subunit 4I2 (COX4I2). Methods: Human DASMC were grown in hypoxia (2.5% O2, pO2=41mmHg). Oxygen responsiveness of DASMC was assessed, measuring O2-induced changes in intracellular calcium, [Ca2+]i, cell length, and mitochondrial reactive oxygen species (mROS) production. DASMC were treated for 48-hours with silencing RNA (siRNA) targeting NDUFS2, NDUFS1, NDUFS7, UQCRFS1, or COX4I2; knockdown was confirmed using qPCR and immunoblot. Mitochondrial metabolic consequences were assessed with micropolarimetry and Complex I, III, and IV activity assays. 3'RNA sequencing was used to explore the impact of gene knockdown on the DASMC molecular signature. Results: The O2-induced increase in [Ca2+]i in siControl-treated cells (+18.6±2.3%) was reduced by siNDUFS2 (+5.5±1.5%, p<0.0001), but unchanged by other siRNAs. siNDUFS2 also uniquely depressed O2-induced DASMC shortening (from -18.4±1.1% to -8.9±0.77%, p<0.0001), and mROS generation (+24±4.9% untreated versus -6.6±5.4% siNDUFS2, p<0.0001). The mitochondrial antioxidant MitoTEMPO also inhibited mROS (+2.9±4.5%, p=0.001) and attenuated oxygen-induced cell shortening (8.43±0.91%, p=0.0003). Knockdown of NDUFS2 and other ETC subunits did not inhibit mitochondrial respiration or ETC activity. Transcriptomics revealed unique changes in mitochondrial pathways with siNDUFS2. Conclusions: NDUFS2 regulates mROS and acts as a mitochondrial oxygen sensor in human DASMC.