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

Highlights

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Ductus arteriosus smooth muscle cells (DASMC) were isolated from DAs obtained from 10 term infants at the time of congenital heart surgery.

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Highly purified DASMC were demonstrated to be >99% SMC and retained their contractile phenotype in response to oxygen.

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Cells were grown in either hypoxia mimicking the in-utero environment, or normoxia (mimicking the neonatal environment

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RNAsequencing revealed that 1,344 unique genes were differentially regulated in response to normoxia.

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Functional analysis of these genes revealed a central role for mitochondria in the response of hDASMC to oxygen.

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These data have relevance for understanding normal DA closure and patent ductus arteriosus

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.

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Keywords

Nucleoside biosynthesisPatent ductus arteriosusNADH-ubiquinone oxidoreductase (NDUF)Prostaglandin E synthase (PTGES)CREBBP (Crebb binding protein)EP300 (Histone Acetyltransferase P300)RNAsequencing

1. Introduction

In utero the ductus arteriosus (DA) connects the aorta to the pulmonary artery, ensuring that placentally oxygenated blood is directed away from the developing lungs to the aorta [1]. High pulmonary vascular resistance and relatively low systemic vascular resistance facilitates the fetal shunting of blood from the pulmonary to the systemic circulation through the widely patent DA [2,3]. With a newborn's first breath, the DA rapidly constricts in response to increased arterial partial pressure of oxygen (PaO2) as the circulatory system quickly transitions from placental oxygenation to oxygenation by neonatal respiration. Initial DA constriction in response to oxygen elicits functional closure, beginning within minutes of a rise in PaO2 [4]. Subsequent to functional closure, the DA undergoes remodelling leading to anatomical closure [5] eventually creating a fibrous structure, the ligamentum arteriosum. Failure of DA closure creates a persistent patent DA (PDA). PDA is one of the most common complications of preterm birth, accounting for approximately 5–10% of all congenital heart disease [6]. Although the incidence of PDA is fairly low among full-term infants (57 per 100,000), preterm infants are particularly susceptible, with more than 55% of infants with a birth weight < 1000 g being affected [4,7]. PDA is associated with serious adverse outcomes if not treated, including heart failure, necrotizing enterocolitis, intraventricular hemorrhage, renal dysfunction and chronic lung disease [7]. Therefore, understanding the physiology of DA closure becomes crucial in the development of therapeutic interventions in addition to nonsteroidal inflammatory drugs (NSAIDs), percutaneous DA closure devices, or surgery.

Currently, treatments for PDA include invasive surgical intervention and percutaneous interventions [8] in infants who fail a trial of NSAIDs (neonates <32 week's gestational age) [[9], [10], [11]]. Intervention to close the patent DA includes risks of complications like recurrent laryngeal nerve damage whilst the use of NSAIDs may cause gastrointestinal, renal and cerebral side-effects [[12], [13], [14], [15]]. Therefore, the development of alternative PDA treatments is necessary; and the understanding of the physio-molecular pathways in the functional and anatomical closure of DA is a useful, early step that should accelerate the discovery of molecules relevant to the pathogenesis and therapy of PDA.

Functional and anatomical closure of the DA is primarily mediated by exposure to oxygen [16]. With the transition from hypoxia in the fetal environment (PaO2 < 40 mmHg) to normoxia in neonatal life (PaO2 80-100 mmHg), multiple pathways act to permit or enhance DA constriction. Withdrawal of endogenous vasodilatory prostanoids, like prostaglandin E, promotes DA constriction [17]. There is also increased endothelial-mediated, oxygen-induced production of the vasoconstrictor endothelin-1, which induces release of intracellular calcium stores resulting in sustained constriction [18]. While the endothelium plays an important role in DA constriction and is the target of common therapies to close the DA (prostaglandin synthesis inhibitors) or to maintain DA patency (intravenous prostaglandin E), the DA constricts in response to increased oxygen tension in the absence of endothelium [19]. Moreover, oxygen-induced DA constriction in human DAs persists in the presence of prostaglandin synthesis inhibitors, endothelin converting enzyme inhibitors and endothelin receptor antagonists [20]. This suggests the DA oxygen-sensing pathway is intrinsic to the DASMC. Our current understanding of the DASMC's intrinsic oxygen response is that oxygen induces mitochondrial reactive oxygen species (ROS) production by increasing mitochondrial fission [21]. These mitochondrial derived ROS inhibit voltage-gated potassium channels (Kv channels) [20], including Kv1.5 and Kv2.1 [22]. Kv channel inhibition depolarizes the cell, causing voltage dependant L-type calcium channels to open, inducing calcium influx followed by SMC constriction [19]. The L-type calcium channel is also directly oxygen sensitive in the DASMC and developmental impairment of this mechanism contributes to patency of the preterm DA [23]. While the physiological mechanisms of oxygen-induced DA constriction are well described, the molecular pathways underlying these mechanisms remain unclear. Moreover, there has never been an unbiased look at mechanisms of DA oxygen sensing using multi-omics, specifically transcriptomics (i.e. the measurement of RNA produced by expressed genes).

Transcriptomics can provide a powerful and unbiased insight into the differentially regulated pathways of systems in response to a biological challenge [24]. Previous transcriptomic studies have been performed on the DA, however this research has often focused on preterm and term DA gene expression in comparison with the aorta [[25], [26], [27], [28]]. A recent meta-analysis of the data from these studies compared differentially regulated genes between the human DA and aorta [29]. No global gene expression studies in either pre-clinical models or in human cells have examined changes in DA gene expression induced by oxygen. In this study we have purified and then cultured DASMCs from 10 human donors under hypoxic conditions that mimic fetal PaO2. After carefully ensuring a pure DASMC population we confirmed the DASMCs remained oxygen sensitive by demonstrating an acute rise in intracellular calcium upon exposure to normoxia which was accompanied by DASMC constriction. We then performed RNA sequencing, which revealed the transcriptomic signature of hypoxic and normoxic DA. The data show that the most oxygen-regulated biologic process involved generation of precursor metabolites and energy whilst the most oxygen regulated cellular component was mitochondrial matrix. This first examination of the effects of O2 on human DA transcriptomics supports an important role for mitochondria as oxygen sensors.

2. Materials and methods

2.1. Human studies

Ethics approval was obtained at each university where this research was performed. Human DA samples were isolated during the course of congenital heart surgery at either University of Chicago (IRB number A3523–01) or University of Nebraska (IRB number 100–11-EP). Ethics approval from Queen's University Health Sciences and Affiliated Teaching Hospitals Research Ethics Board (HSREB) was obtained for the continued use of the human cell lines in ongoing research (TRAQ #6007784).

2.2. Isolation and culture of human DASMCs (hDASMC)

Primary cell lines were previously established from the surgically harvested DAs following excision as previously described [21]. DAs were harvested and the endothelium and adventitia were removed. The DA media was then minced and cells were grown in culture. Ten hDASMC cell lines were used: five from the University of Chicago and five from the University of Nebraska. Demographics on sex, age at time of surgery, diagnosis, and treatment were collected where available. All cell culture reagents were obtained from Gibco (Carlsbad, California USA). hDASMCs were grown in M231 smooth muscle cell growth media supplemented with 10% FBS, 5% smooth muscle growth supplement, 1% l-glutamine, 1% penicillin/streptomycin, and ciprofloxacin HCl (10 μg/mL). hDASMCs were used within the first five passages in culture and were diligently maintained in hypoxia (2.5% O2, 5% CO2, balance N2) until protocols called for exposure to normoxia (19.6% O2, 5% CO2, balance N2).

2.3. Purification of hDASMC

To ensure that only DASMCs were present in culture we first performed a negative flow sorting experiment to exclude fibroblasts (CD90+) and endothelial cells (CD31+). Briefly, cultured cells were trypsinized (0.25% Trypsin-EDTA, Gibco), washed with PBS and resuspended in 100 μL hypoxic PBS with 4% FBS containing PE anti-CD90/Thy1 (Abcam ab33694 clone MRC OX-7, Cambridge, MA, USA; 5 μL per 106 cells) and APC anti-CD31 (BioLegend cat#102409 clone 390, San Diego, CA, USA; 1.25 μL per 106 cells [0.2 mg/mL]) or Brilliant Violet 421 anti-CD31 (BioLegend cat#10243, clone 390; 5 μL per 106 cells). Cells were incubated for 30 min at 37 °C in a hypoxic incubator (2.5% O2, 5% CO2, balance N2). Single staining and unstained controls were used to set up sorting and compensation parameters. Excess antibodies were washed away using warm hypoxic PBS, and cells were promptly sorted to only include CD90−CD31− cells using the SH800S cell sorter (Sony Biotechnology, San Jose, CA, USA). Sorted hDASMCs were collected in supplemented M231 smooth muscle cell growth media and immediately plated and cultured in hypoxic conditions. Purified cell lines were expanded and grown in hypoxic conditions prior to any further experiments.

2.4. Flow cytometric assessment of SMC phenotype

Putative purified hDASMCs (CD90−CD31− cells) were trypsinized, washed, resuspended in PBS and counted. 106 cells were washed with PBS and incubated with a viability stain (cat#L34975; ThermoFisher Scientific, Mississauga, ON, Canada; 1:1000 in 500 μL) for 30 min at 4 °C, then washed with PBS. Samples were fixed in 4% paraformaldehyde for 15 min at 4 °C, washed using PBS with 0.05% Tween® 20 (PBS-T; Fisher Scientific cat#BP337-500, Whitby, ON, Canada) and 1% Bovine Serum Albumin (BSA; Sigma-Aldrich, Oakville ON, Canada), permeabilized with Triton® X-100 (0.5%, Fisher Scientific cat#BP151-100) for 15 min at 4 °C, then washed with the PBS-T/BSA. Nonspecific binding was blocked using PBS with 1% BSA and 2% FBS for 30 min at room temperature. hDASMCs were washed, resuspended in 100 μL of the PBS-T/BSA containing Alexa-Fluor 488 anti-α-smooth muscle actin (clone 1A4, cat#53–9760-82; ThermoFisher Scientific, Mississauga, ON, Canada; 1 μL [0.5 mg/mL]), human smoothelin B Alexa-Fluor® 405-conjugated antibody (cat#IC8278Vl; R&D systems, Bio-Techne, Oakville, ON, Canada; 3 μL), and PE-conjugated human Caldesmon antibody (clone REA1120, cat#130–119-344; Miltenyi Biotech, Auburn, CA, USA; 2 μL), incubating for 1 h at 4 °C. Excess antibody was washed with PBS-T, and the expression of alpha-smooth muscle actin, smoothelin B, and caldesmon were measured using the SH800S cell sorter/cytometer. Debris was excluded using side and forward scatter and laser settings were defined using unstained samples. Fluorescence minus one (FMO) controls were used to ensure accurate gating for analysis. Cells were analyzed using FlowJo software (FlowJo ™, Becton, Dickinson & Company, USA).

2.5. Proliferation of cells grown in hypoxia and normoxia

Proliferation of hDASMCs was measured using the xCELLigence® Real-Time Cell Analysis (RTCA) DP Instrument (Agilent, Santa Clara, CA, USA). Five randomly selected hDASMC cell lines were grown in hypoxia prior to the proliferation experiment. The cells were then trypsinized, resuspended in M231 supplemented media, counted and plated in duplicate at a density of 5000 cells per well in a total volume of 200 μL in an RTCA CIM Plate 16 (ref#5665817001; Agilent, Santa Clara, CA, USA). The instrument was kept in a cell incubator and a plate with M231 supplemented media was used to calibrate the instrument. Recordings were taken every 15 min for 96 h, pausing recording approximately two days after plating to change the media. The experiment was repeated with the same cell lines, freshly plated in either hypoxia (2.5% O2, 5% CO2, balance N2) or normoxia (19.6% O2, 5% CO2, balance N2). Duplicate wells of each cell line in each oxygen condition were averaged and the hDASMC proliferation is demonstrated by the change in cell index every 24 h, where cell index is a unitless measure reporting the impedance of electron flow caused by adherent cells and is calculated as: cell index = (impedance at time n – impedance in the absence of cells)/nominal impedance value.

2.6. Intracellular calcium indicator Cal-520 AM imaging

One day prior to imaging, hDASMCs were plated into 35 mm glass bottom dishes (No. 1.5 uncoated γ-irradiated, P35G-1.5-14-C MatTek Corporation, Ashland, MA, USA) in M231 supplemented media at a density of 5 × 105 cells per dish. Imaging experiments were conducted in an oxygen-sensing assay buffer (modified Krebs solution: 115 mM NaCl, 5.4 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 25 mM NaHCO3, 10 mM HEPES, 10 mM d-glucose). hDASMCs were loaded with 10 μM Cal-520 AM (Abcam ab171868) mixed with 0.02% Pluronic F-127 (Invitrogen P6866) in O2-sensing assay buffer and incubated in the dark at 37 °C and 2.5% oxygen (5% CO2, balance nitrogen) for 30 min. Following dye loading, cells were washed with assay buffer and incubated at 37 °C and 2.5% oxygen for 15 min prior to imaging. The assay was then conducted in 1 mL O2-sensing assay buffer, modulating pO2 using an OkoLab stage-top microscope incubator (OkoLab Bold Line, Pozzuoli, Italy). Live cell imaging was performed using a Leica TCS SP8 X confocal microscope (Leica Microsystems, Wetzlar, Germany). Cells were imaged during hypoxia (3% O2, 5% CO2, balance N2) or normoxia (room air). Care was taken to avoid oxygen except when a normoxic challenge was administered. pO2 and pH were confirmed using an ABL90 FLEX blood-gas analyzer (Radiometer, Mississauga, ON, Canada). Cells were imaged for 15 min in hypoxia, followed by 15 min in normoxia, and a further 15 min returning to hypoxia, capturing one frame every 15 s. The dynamic range of the dye (positive control) was established using 10 μM ionomycin and 20 mM EGTA to establish maximum and minimum intensity values. The oxygen-induced calcium response was measured as a percentage increase from hypoxic baseline. Cell constriction was also measured to determine oxygen-responsiveness. The length of ten randomly selected cells were measured during the hypoxic baseline and during the 15 min of normoxia. The cell constriction is reported as percentage decrease in cell length relative to the hypoxic baseline.

2.7. RNA isolation and sequencing of purified hDASMC cells under hypoxic or normoxic environmental conditions

Ten oxygen-responsive hDASMC cell lines were grown for 96 h in either hypoxia (2.5% O2, 5% CO2, balance N2) or normoxia (19.6% O2, 5% CO2, balance N2). The cells were then collected and stored at −80 °C prior to RNA isolation. Cells were lysed using 600–1000 μL TRI Reagent® (Sigma-Aldrich, cat# T9424-100ML, Lot # MKCK9023). RNA extraction was processed in bulk using Zymo Direct-zol RNA MiniPrep (cat# R2050, Zymo Research, California, USA), according to manufacturer's protocols, and then quantified using Qubit spectrophotometer (Thermo Fisher Scientific, Waitham, Massachusetts, USA). Libraries were prepared using a QuantSeq 3′ FWD mRNA-Seq Library Prep Kit for Illumina (Lexogen, Austria) with an input concentration of 450 ng. We used 16 cycles of final PCR amplification. Individual libraries were pooled at a concentration of 35fM each and the pooled samples cleaned using Lexogen bead purification ready for sequencing using the Illumina NextSeq 550 using the Mid V2.5 chemistry.

2.8. RNAsequencing bioinformatics

All bioinformatics were performed either on the Centre for Advanced Computing (CAC) Frontenac server, or on local machines running R (R Foundation for Statistical Computing, Vienna, Austria). Briefly, Fastq files were assembled from bcl2 output without lane split, and subject to quality control per-sample with fastqc and across samples using multiqc. Low quality reads and adapters were trimmed from reads using BBDUK (https://jgi.doe.gov/) [30], and aligned to the human genome (HG38) using STAR [31]. Reads were indexed prior to counting with HTSeq-count [32]. Within the R environment, count files from each sample were assembled into condition-wise groups and analyzed using DESeq2 [33]. Functional analysis was performed using Cluster Profiler [34] on a list of differentially regulated genes that satisfied corrected p-value (padj) <0.05. Functional groups were resolved in terms of GO domains: Cellular Component (CC), Biological Process (BP) and Molecular Function (MF), with enriched terms satisfying a Benjamini Hochberg corrected p-value cutoff <0.05.

2.9. Quantitative PCR (qPCR) validation

Six randomly selected hDASMC cell lines were woken from storage in liquid nitrogen and grown in hypoxia until confluent. Cells were then trypsinized, resuspended in M231 media, and counted. Each cell line was plated at a density of 150,000 cells per well in duplicate per oxygen condition in a 6-well cell culture dish. Cells were grown for 96 h in either hypoxia (2.5% O2, 5% CO2, balance N2) or normoxia (19.6% O2, 5% CO2, balance N2), following which they were collected and lysed in 700 μL TRI Reagent® (Sigma-Aldrich, cat# T9424-100ML, Lot # MKCK9023). RNA was isolated using Zymo Direct-zol RNA MiniPrep (cat# R2050, Zymo Research, California, USA), according to manufacturer's protocols, and quantified using the DropSense 16 (Trinean, Pleasanton, CA, USA) according to manufacturer's protocols. Complementary DNA (cDNA) was synthesized using qScript™ cDNA Supermix (Quantabio, Beverly, MA, USA) according to manufacturer's protocol, with 240 ng RNA input per sample. cDNA was then quantified using the DropSense 16, and all samples were adjusted to the lowest concentration.

qPCR was conducted using TaqMan™ probes (ThermoFisher Scientific, Mississauga, ON, CA) and PerfeCTa Fastmix II (Quantabio, Beverly, MA, USA) according to manufacturer's protocol, using the QuantStudio™ 3–96-well 0.2 mL Block (cat#A28567, ThermoFisher Scientific, Mississauga, ON, CA). We validated a selection of genes that were differentially regulated on our transcriptomic analysis. These genes were chosen either because they were the most differentially regulated or were differentially regulated genes that were considered to be of functional importance to the proposed mechanism of DA constriction. All probes for target genes used FAM as the reporter and NFQ-MGB as the quencher. All samples were also run with Eukaryotic 18S rRNA Endogenous Control (cat#4319413E, ThermoFisher Scientific, Mississauga, ON, CA) with the reporter VIC and quencher NFQ-MGB, a housekeeping gene used as an internal control in all experiments. The genes tested were the following: NADH-ubiquinone oxidoreductase (NDUF), NDUFS2 (Hs00190020_m1), NDUFS5 (Hs02578754_g1), NDUFS7 (Hs01086219_m1), NDUFS8 (Hs00159597_m1), NDUFA9 (Hs00245308_m1), NDUFA13 (Hs00363071_m1), HIF1A (Hs00153153_m1), PSAT1 (Hs01107691_g1), EGLN3 (Hs00222966_m1), and APLN (Hs00175572_m1). Samples were run in triplicate, averaging the CT values, with a no template control well. The delta CT (ΔCT), difference between the target gene and the housekeeping gene (18S rRNA), was calculated for all targets and differences between ΔCT in hypoxia and normoxia were compared using two-tailed unpaired t-tests. Gene expression data from qPCR validation is all reported as log2 fold change [35].

2.10. Statistics and data analysis

Data are presented as mean ± standard error mean (SEM) unless otherwise stated. Descriptive statistics were performed to check for equal variance between groups and the Shapiro-Wilk test was used to test for normal distribution. All data analysis was conducted using Prism 9 (GraphPad Software, LLC, San Diego, CA, USA). The Kruskal-Wallis test with Dunn's correction for multiple comparisons was used to compare the percent increase in calcium signal across all cell lines. Paired t-tests with false-discovery rate correction method of two-stage step-up Benjamini, Krieger and Yekutieli was used to test the calcium response of each cell line, comparing the Cal-520 signal in normoxia vs hypoxia. The same tests were used to compare cell length in hypoxia vs normoxia to examine cell constriction. Multiple unpaired t-tests with Welch correction and false-discovery rate correction method of two-stage step-up Benjamini, Krieger, and Yekutieli was used to compare proliferation data of hDASMCs grown in hypoxia vs normoxia. To examine the proliferation of cells grown in hypoxia and normoxia, RM one-way