The molecular mechanisms of oxygen-sensing in human ductus arteriosus smooth muscle cells: A comprehensive transcriptome profile reveals a central role for mitochondria

• Published in: Genomics (San Diego, Calif.) Calif.), 2021-09, Vol.113 (5), p.3128-3140
• Main Authors: Bentley, Rachel E.T., Hindmarch, Charles C.T., Dunham-Snary, Kimberly J., Snetsinger, Brooke, Mewburn, Jeffrey D., Thébaud, Arthur, Lima, Patricia D.A., Thébaud, Bernard, Archer, Stephen L.
• Format: Article
• Language: English
• Subjects: CREBBP (Crebb binding protein); Ductus Arteriosus - metabolism; Ductus Arteriosus, Patent - etiology; Ductus Arteriosus, Patent - metabolism; Endothelial Cells - metabolism; EP300 (Histone Acetyltransferase P300); Humans; Infant, Newborn; Mitochondria - genetics; Myocytes, Smooth Muscle - metabolism; NADH-ubiquinone oxidoreductase (NDUF); Nucleoside biosynthesis; Oxygen - metabolism; Oxygen - pharmacology; Patent ductus arteriosus; Premature Birth - metabolism; Prostaglandin E synthase (PTGES); RNAsequencing; Transcriptome; Vasoconstriction - physiology
• ISSN: 0888-7543; 1089-8646
• DOI: 10.1016/j.ygeno.2021.07.006

The ductus arteriosus (DA) connects the fetal pulmonary artery and aorta, diverting placentally oxygenated blood from the developing lungs to the systemic circulation. The DA constricts in response to increases in oxygen (O2) with the first breaths, resulting in functional DA closure, with anatomic closure occurring within the first days of life. Failure of DA closure results in persistent patent ductus arteriosus (PDA), a common complication of extreme preterm birth. The DA's response to O2, though modulated by the endothelium, is intrinsic to the DA smooth muscle cells (DASMC). DA constriction is mediated by mitochondrial-derived reactive oxygen species, which increase in proportion to arterial partial pressure of oxygen (PaO2). The resulting redox changes inhibit voltage-gated potassium channels (Kv) leading to cell depolarization, calcium influx and DASMC constriction. To date, there has not been an unbiased assessment of the human DA O2-sensors using transcriptomics, nor are there known molecular mechanisms which characterize DA closure. DASMCs were isolated from DAs obtained from 10 term infants at the time of congenital heart surgery. Cells were purified by flow cytometry, negatively sorting using CD90 and CD31 to eliminate fibroblasts or endothelial cells, respectively. The purity of the DASMC population was confirmed by positive staining for α-smooth muscle actin, smoothelin B and caldesmon. Cells were grown for 96 h in hypoxia (2.5% O2) or normoxia (19% O2) and confocal imaging with Cal-520 was used to determine oxygen responsiveness. An oxygen-induced increase in intracellular calcium of 18.1% ± 4.4% and SMC constriction (−27% ± 1.5% shortening) occurred in all cell lines within five minutes. RNA sequencing of the cells grown in hypoxia and normoxia revealed significant regulation of 1344 genes (corrected p