The Role of Mitochondria in Oxygen Sensing of the Ductus Arteriosus and Fetal Pulmonary Arteries

Abstract

Rationale: Mitochondria underlie the oxygen responses of the ductus arteriosus (DA), adult pulmonary arteries (PA) and other oxygen sensing tissues. However, the fetal PA oxygen response has been little studied, the identity of the DA oxygen sensor remains elusive, and the basis for the opposing oxygen responses of the DA (vasoconstriction) and PA (vasodilation) are unknown. With the transition from fetal (hypoxic) to neonatal (normoxic) life, a failure of the DA to constrict or the PA to dilate in response to oxygen can precipitate patent ductus arteriosus and persistent pulmonary hypertension of the newborn, respectively. Objectives: 1) determine the molecular changes in human DA smooth muscle cells (DASMC) induced by exposure to postnatal oxygen concentrations, 2) examine the contribution of individual mitochondrial electron transport chain (ETC) subunits to DASMC oxygen response, and 3) explore the proteomic differences between fetal DA and PA that might underlie their opposing oxygen responses. General Methods: Cell lines were purified with fluorescence activated cell sorting and oxygen response measured with confocal calcium imaging. Human DASMC grown for 96-hours in hypoxia (2.5% oxygen) or normoxia (19.6% oxygen) were used for 3’RNA-sequencing. Silencing RNA was used to selectively knock down ETC subunits, with effects on oxygen responsiveness examined with calcium imaging. The bioenergetic effects of knockdown were examined with micropolarimetry and ETC activity assays. Liquid chromatography with tandem mass spectrometry was used to explore proteomic differences in whole DA versus PA and mitochondrially-enriched DASMC versus PASMC samples. General Results: 1) Transcriptomic analysis revealed mitochondrial pathway enrichment between hypoxic and normoxic DASMC. Among genes in ETC Complex I, NADH:Ubiquinone oxidoreductase core subunit S2 (NDUFS2) was uniquely downregulated by oxygen. 2) Knockdown of NDUFS2 reduced DASMC acute oxygen responsiveness without bioenergetic inhibition. 3) Rabbit DA and PA have unique proteomes, with differences in structural, proliferative, and metabolic proteins. Conclusions: This thesis furthers our understanding of the oxygen response of the human DA, demonstrating a key role for NDUFS2 and characterizing the molecular changes in gene transcription caused by oxygen. This first omic comparison between DA and PA identifies mitochondrial-metabolic protein differences that may explain their opposing response to oxygen.