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Ndufs2, A Core Subunit Of Mitochondrial Complex I, Is Essential For Acute Oxygen-Sensing And Hypoxic Pulmonary Vasoconstriction

Kimberly J. Dunham-Snary, Danchen Wu, François Potus, Edward A. Sykes, Jeffrey D. Mewburn, Rebecca L. Charles, Philip Eaton, Richard A. Sultanian, Stephen L. Archer

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Rationale: Hypoxic pulmonary vasoconstriction (HPV) optimizes systemic oxygen delivery by matching ventilation to perfusion. HPV is intrinsic to pulmonary artery smooth muscle cells (PASMCs). Hypoxia dilates systemic arteries, including renal arteries. Hypoxia is sensed by changes in mitochondrial-derived reactive oxygen species, notably hydrogen peroxide (H 2 O 2 ) ([H 2 O 2 ] mito ). Decreases in [H 2 O 2 ] mito elevate pulmonary vascular tone by increasing intracellular calcium ([Ca 2+ ] i ) through reduction-oxidation regulation of ion channels. Although HPV is mimicked by the Complex I inhibitor, rotenone, the molecular identity of the O 2 sensor is unknown. Objective: To determine the role of Ndufs2 (NADH [nicotinamide adenine dinucleotide] dehydrogenase [ubiquinone] iron-sulfur protein 2), Complex I’s rotenone binding site, in pulmonary vascular oxygen-sensing. Methods and Results: Mitochondria-conditioned media from pulmonary and renal mitochondria isolated from normoxic and chronically hypoxic rats were infused into an isolated lung bioassay. Mitochondria-conditioned media from normoxic lungs contained more H 2 O 2 than mitochondria-conditioned media from chronic hypoxic lungs or kidneys and uniquely attenuated HPV via a catalase-dependent mechanism. In PASMC, acute hypoxia decreased H 2 O 2 within 112±7 seconds, followed, within 205±34 seconds, by increased intracellular calcium concentration, [Ca 2+ ] i . Hypoxia had no effects on [Ca 2+ ] i in renal artery SMC. Hypoxia decreases both cytosolic and mitochondrial H 2 O 2 in PASMC while increasing cytosolic H 2 O 2 in renal artery SMC. Ndufs2 expression was greater in PASMC versus renal artery SMC. Lung Ndufs2 cysteine residues became reduced during acute hypoxia and both hypoxia and reducing agents caused functional inhibition of Complex I. In PASMC, siNdufs2 (cells/tissue treated with Ndufs2 siRNA) decreased normoxic H 2 O 2 , prevented hypoxic increases in [Ca 2+ ] i , and mimicked aspects of chronic hypoxia, including decreasing Complex I activity, elevating the nicotinamide adenine dinucleotide (NADH/NAD + ) ratio and decreasing expression of the O 2 -sensitive ion channel, Kv1.5. Knocking down another Fe-S center within Complex I (Ndufs1, NADH [nicotinamide adenine dinucleotide] dehydrogenase [ubiquinone] iron-sulfur protein 1) or other mitochondrial subunits proposed as putative oxygen sensors (Complex III’s Rieske Fe-S center and COX4i2 [cytochrome c oxidase subunit 4 isoform 2] in Complex IV) had no effect on hypoxic increases in [Ca 2+ ] i . In vivo, siNdufs2 significantly decreased hypoxia- and rotenone-induced constriction while enhancing phenylephrine-induced constriction. Conclusions: Ndufs2 is essential for oxygen-sensing and HPV.