Oxygen-sensing in the Lung is Regulated by Mitochondrial Complex I Ndufs2: Role of Mitochondria-derived Hydrogen Peroxide in Hypoxic Pulmonary Vasoconstriction

Abstract

Resistance pulmonary arteries (PA) constrict in response to alveolar hypoxia; conversely the systemic vasculature dilates. Hypoxic pulmonary vasoconstriction (HPV) matches ventilation to perfusion, optimizing systemic oxygen delivery. The core mechanism of HPV is intrinsic to pulmonary artery smooth muscle cells (PASMC) and involves mitochondrial electron transport chain (ETC)-mediated redox regulation of oxygen-sensitive ion channels culminating in increased cytosolic calcium ([Ca2+]i). However, the molecular identity of the mitochondrial O2-sensor and the mechanism by which it regulates vascular tone remains unknown. We hypothesize that the O2-sensor resides in ETC complex I and initiates HPV by varying H2O2 in proportion to PO2. The primary objective was to identify the mitochondrial redox O2-sensor. A secondary goal was identify the ETC-derived reactive oxygen species (ROS) that initiates HPV. Mitochondria were isolated from lungs or kidneys of adult male Sprague-Dawley rats, housed under conditions of normoxia or chronic hypoxia (FiO2 0.1 for 2-3 weeks). Mitochondria-conditioned media (MCM) was then infused into an isolated, perfused lung to examine the effects on HPV. The effects of acute hypoxia on mitochondrial-derived H2O2 and [Ca2+]i was assessed in PASMC using with a dynamic, mitochondria-targeted H2O2 probe and FURA2-AM, respectively. Immunocaptured ETC complex I from pulmonary and renal arterial SMCs was assessed by gel electrophoresis to identify differentially expressed subunits. HPV was attenuated by MCM from normoxic lung (nLM), but not MCM from chronically hypoxic lung or kidney. Catalase attenuated the effects of nLM. Acute hypoxia (~40 mmHg) decreased H2O2 within 112±7 seconds, followed, within 205±34 seconds, by increased [Ca2+]i. The expression of a Complex I subunit, NADH dehydrogenase [ubiquinone] iron-sulfur protein 2 (Ndufs2), was increased in PA versus renal artery SMC. siRNA knockdown of Ndufs2 (siNdufs2) decreased normoxic H2O2 levels and prevented hypoxia-induced increases in [Ca2+]i. Right heart catheterization and intravital microscopy demonstrated that in vivo nebulization of siNdufs2 selectively attenuated HPV in rats and mice. Mitochondrial-derived H2O2 originating from ETC Complex I in PASMC acts as a normoxic vasodilator. Acute hypoxia decreases this vasodilator, leading to HPV. Ndufs2 is a requisite component of the PASMC’s mitochondrial O2-sensor, and acts through its dynamic ability to regulate H2O2 production.