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

Abstract

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 < 0.05). We examined these genes using Gene Ontology (GO). This unbiased assessment of altered gene expression indicated significant enrichment of the following GOterms: mitochondria, cellular respiration and transcription. The top regulated biologic process was generation of precursor metabolites and energy. The top regulated cellular component was mitochondrial matrix. The top regulated molecular function was transcription coactivator activity. Multiple members of the NADH-ubiquinone oxidoreductase (NDUF) family are upregulated in human DASMC (hDASMC) following normoxia. Several of our differentially regulated transcripts are encoded by genes that have been associated with genetic syndromes that have an increased incidence of PDA (Crebb binding protein and Histone Acetyltransferase P300). This first examination of the effects of O₂ on human DA transcriptomics supports a putative role for mitochondria as oxygen sensors.

Keywords: CREBBP (Crebb binding protein); EP300 (Histone Acetyltransferase P300); NADH-ubiquinone oxidoreductase (NDUF); Nucleoside biosynthesis; Patent ductus arteriosus; Prostaglandin E synthase (PTGES); RNAsequencing.

Fig. 1.

Human Ductus Arteriosus cell lines were enriched for smooth muscle cells. A. Two female patients, six male patients and two patients with undisclosed sex* were used in this study. B. Unstained sample used for gating CD31 (endothelial cells) and CD90 (fibroblasts) double negative population. C. hDASMCs were negatively sorted using antibodies for CD31 and CD90. Representative plot shown. D. All hDASMC cell lines used for transcriptomics experiments were highly enriched SMCs when sorted using SMA antibody. E. Five randomly selected hDASMC cell lines were confirmed to be proliferative, with a significant increase in cell number between day 1 and day 4, both when grown for 96 h in hypoxia (p = 0.040) and when grown for 96 h in normoxia (p = 0.037). There was no significant difference in cell index between hypoxia and normoxia at any time point. VSD = ventricular septal defect, CHF/PH = Congestive heart failure/pulmonary hypertension, HLHS = hypoplastic left heart syndrome, D-TGA = dextro-transposition of the great arteries, PFO = patent foramen ovale, TGA = transposition of the great arteries. *sex of these patients were determined to be male, a consequence of Xist gene expression from the RNAseq data (Supplementary File 1).

Fig. 2.

Ductus arteriosus smooth muscle cells (DASMC) are functionally responsive to oxygen. A. Change in cytosolic calcium in response to oxygen was measured using confocal imaging with the dynamic Cal-520 AM dye, reflected in an increased fluorescent signal following oxygen exposure within the indicated region of interest (ROI). B. Normoxia increased Cal-520 fluorescent signal within each cell line, relative to hypoxia. Each patient DASMC cell line was responsive to oxygen, as determined by a significant increase in cytosolic calcium. *indicates p < 0.001C. Normoxia caused an 18% increase in cytosolic calcium signal versus the hypoxic baseline level. There was no significant difference in the percent change in cytosolic calcium between cell lines. D. Each DASMC cell line showed a significant decrease in cell length upon exposure to normoxia. *indicates p < 0.001, #indicates p < 0.005, $indicates p < 0.05. E. Normoxia caused a 27% ± 1.5% decrease in cell length relative to the hypoxic baseline.

Fig. 3.

RNAsequencing reveals normoxia-induced differential gene expression in human DASMC. Ductus arteriosus smooth muscle cell (DASMC) lines were studied after culture in hypoxia or normoxia for 96 h using RNAsequencing. 1344 genes were differentially regulated by 96-h of oxygen exposure (corrected p-value < 0.05).

Fig. 4.

Functional annotation of RNAseq data reveals enriched pathways in human DASMC after oxygen exposure. The differentially regulated genes were placed into biological context through analysis of enriched Gene Ontology terms (GOterms). GOterms are resolved into Biological Process (BP), Cellular Component (CC) and Molecular Function (MF) in an unbiased manner. Data are coloured according to corrected p-value (p < 0.05), and the symbol sized according to GeneRatio (the number of genes within our data as a ratio of the total number of genes within that GOterm). The GOterm is then ranked by the number of genes involved in each GOterm, such that the most regulated pathways have the highest count on the X-axis. Note that most regulated pathways relate to mitochondria and metabolism. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 5.

Network of genes in each GO pathway within each Domain. Nodes for genes are coloured according to fold-change. Edges are coloured according to GOterm category. A. Differentially regulated genes involved in overlapping most enriched Gene Ontology terms (GOterms) within the Cellular Component Domain. There is a high clustering of genes involved in GOterms involved with mitochondrial function and the mitochondria’s electron transport chain. B. Differentially regulated genes involved in overlapping most enriched Gene Ontology terms (GOterms) within the Molecular Function Domain. There is a high clustering of genes involved in GOterms involved with ribonuclease activity and transcription. C. Differentially regulated genes involved in overlapping most enriched Gene Ontology terms (GOterms) within the Biological Process Domain. There is a high clustering of genes involved in GOterms involved with cellular respiration, energy production and nucleoside metabolism.