The Core Subunit NDUFS2 in Mitochondrial Complex I is Critical to Oxygen Sensing in Human Ductus Arteriosus Smooth Muscle Cells

Human DASMC were grown in hypoxia (pO2=41mmHg). Oxygen responsiveness was assessed, measuring oxygen-induced changes in intracellular calcium, [Ca2+]i, cell length, and mitochondrial reactive oxygen species (mROS) production. DASMC were treated for 48-hours with control or targeting.

Knockdown was confirmed using qPCR and immunoblot. 3’RNA sequencing assessed molecular changes following siRNA manipulation. Results: Oxygen increased mitochondrial fission, [Ca2+]I, and constricted DASMC. 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% 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 cell shortening (8.4±0.9%, p=0.0003). Transcriptomics revealed distinct changes in mitochondrial pathways 48-hours post siNDUFS2. Conclusions: NDUFS2 regulates mROS and is a mitochondrial oxygen sensor in human DASMC.