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Open Access

Peer-reviewed

Research Article

Loss of heme oxygenase 2 causes reduced expression of genes in cardiac muscle development and contractility and leads to cardiomyopathy in mice

  • Rengul Cetin-Atalay ,

    Contributed equally to this work with: Rengul Cetin-Atalay, Angelo Y. Meliton

    Roles Conceptualization, Data curation, Formal analysis, Investigation, Writing – original draft, Writing – review & editing

    Affiliation Department of Medicine, Section of Pulmonary and Critical Care Medicine, University of Chicago, Chicago, Illinois, United States of America

    ⨯
  • Angelo Y. Meliton ,

    Contributed equally to this work with: Rengul Cetin-Atalay, Angelo Y. Meliton

    Roles Data curation, Formal analysis, Investigation, Writing – review & editing

    Affiliation Department of Medicine, Section of Pulmonary and Critical Care Medicine, University of Chicago, Chicago, Illinois, United States of America

    ⨯
  • Cevher Ozcan,

    Roles Formal analysis, Investigation, Resources, Visualization, Writing – original draft, Writing – review & editing

    Affiliation Department of Medicine, Section of Cardiology, University of Chicago, Chicago, Illinois, United States of America

    ⨯
  • Parker S. Woods,

    Roles Formal analysis, Investigation, Writing – review & editing

    Affiliation Department of Medicine, Section of Pulmonary and Critical Care Medicine, University of Chicago, Chicago, Illinois, United States of America

    ⨯
  • Kaitlyn A. Sun,

    Roles Data curation, Formal analysis, Investigation, Writing – review & editing

    Affiliation Department of Medicine, Section of Pulmonary and Critical Care Medicine, University of Chicago, Chicago, Illinois, United States of America

    ⨯
  • Yun Fang,

    Roles Formal analysis, Funding acquisition, Resources, Writing – review & editing

    Affiliation Department of Medicine, Section of Pulmonary and Critical Care Medicine, University of Chicago, Chicago, Illinois, United States of America

    ⨯
  • Robert B. Hamanaka,

    Roles Conceptualization, Formal analysis, Project administration, Writing – original draft, Writing – review & editing

    Affiliation Department of Medicine, Section of Pulmonary and Critical Care Medicine, University of Chicago, Chicago, Illinois, United States of America

    ⨯
  • Gökhan M. Mutlu

    Roles Conceptualization, Funding acquisition, Investigation, Resources, Writing – original draft, Writing – review & editing

    gmutlu@uchicago.edu

    Affiliation Department of Medicine, Section of Pulmonary and Critical Care Medicine, University of Chicago, Chicago, Illinois, United States of America

    ⨯

Abstract

Obstructive sleep apnea (OSA) is a common breathing disorder that affects a significant portion of the adult population. In addition to causing excessive daytime sleepiness and neurocognitive effects, OSA is an independent risk factor for cardiovascular disease; however, the underlying mechanisms are not completely understood. Using exposure to intermittent hypoxia (IH) to mimic OSA, we have recently reported that mice exposed to IH exhibit endothelial cell (EC) activation, which is an early process preceding the development of cardiovascular disease. Although widely used, IH models have several limitations such as the severity of hypoxia, which does not occur in most patients with OSA. Recent studies reported that mice with deletion of hemeoxygenase 2 (Hmox2-/-), which plays a key role in oxygen sensing in the carotid body, exhibit spontaneous apneas during sleep and elevated levels of catecholamines. Here, using RNA-sequencing we investigated the transcriptomic changes in aortic ECs and heart tissue to understand the changes that occur in Hmox2-/- mice. In addition, we evaluated cardiac structure, function, and electrical properties by using echocardiogram and electrocardiogram in these mice. We found that Hmox2-/- mice exhibited aortic EC activation. Transcriptomic analysis in aortic ECs showed differentially expressed genes enriched in blood coagulation, cell adhesion, cellular respiration and cardiac muscle development and contraction. Similarly, transcriptomic analysis in heart tissue showed a differentially expressed gene set enriched in mitochondrial translation, oxidative phosphorylation and cardiac muscle development. Analysis of transcriptomic data from aortic ECs and heart tissue showed loss of Hmox2 gene might have common cellular network footprints on aortic endothelial cells and heart tissue. Echocardiographic evaluation showed that Hmox2-/- mice develop progressive dilated cardiomyopathy and conduction abnormalities compared to Hmox2+/+ mice. In conclusion, we found that Hmox2-/- mice, which spontaneously develop apneas exhibit EC activation and transcriptomic and functional changes consistent with heart failure.

Citation: Cetin-Atalay R, Meliton AY, Ozcan C, Woods PS, Sun KA, Fang Y, et al. (2023) Loss of heme oxygenase 2 causes reduced expression of genes in cardiac muscle development and contractility and leads to cardiomyopathy in mice. PLoS ONE 18(10): e0292990. https://doi.org/10.1371/journal.pone.0292990

Editor: Chengming Fan, Second Xiangya Hospital, CHINA

Received: July 4, 2023; Accepted: October 3, 2023; Published: October 16, 2023

Copyright: © 2023 Cetin-Atalay et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: Source data for RNA-seq are accessible via GEO (GSE230725). https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE230725.

Funding: Funding: YF: National Institutes of Health R35HL161244, GMM: National Institutes of Health P01HL144454, R01ES015024 and RBH: National Institutes of Health 5R01HL151680. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Obstructive sleep apnea (OSA) is a common disorder with a prevalence of 24% in men and 9% in women (based on apnea-hypopnea index ≥5) [1]. Population based studies estimate that the prevalence of OSA with daytime sleepiness is 3–7% in men and 2–5% in women [2]. In addition to causing daytime sleepiness, OSA is an independent risk factor for hypertension, atherosclerotic cardiovascular disease and heart failure [1–15]. Despite improvements in our knowledge about the underlying molecular mechanisms by which OSA causes cardiovascular disease, our understanding is not complete.

Pathophysiologically, OSA is characterized by recurrent obstruction of upper airway leading to cessation of air flow resulting in intermittent hypoxia (IH), arousals, and activation of sympathetic nervous system [12, 13, 16, 17]. As a hallmark manifestation of OSA, IH plays an important role in the pathogenesis of OSA-related cardiovascular morbidity [18]. Therefore, exposure of rodents to IH is often used as a model of OSA to study the mechanisms by which OSA causes cardiovascular disease [19].

Using the IH exposure model in mice, we have recently studied the effect of IH on endothelial cell (EC) function [20]. EC activation is an early process in the pathogenesis of cardiovascular disease. Activated ECs express pro-inflammatory cytokines and cell adhesion molecules [21], which may then lead to leukocyte adhesion and activation, and platelet aggregation, all of which contribute to the development of cardiovascular disease [22–24]. We and others have previously linked EC activation with the blood flow dynamics in vasculature. ECs from aortic arch were activated while ECs from abdominal aorta were not [25–28]. Using IH exposure in mice as a model of OSA, we have found that IH causes EC activation [20]. However, this effect was not a direct effect of IH, but was indirectly mediated via the activation of sympathetic nervous system and release of catecholamines.

In addition to IH, recent studies suggest that mice with deletion of hemeoxygenase 2 (Hmox2-/-) may also be used as a model of OSA [29, 30]. HMOX2 is an enzyme that catalyzes the oxidative cleavage of heme leading to the generation of carbon monoxide [31]. In contrast to HMOX1, which is expressed at low levels in most tissues, HMOX2 is inducible and expressed in in the brain, testes, and gastrointestinal tract [32, 33]. HMOX2 has been shown to be important in oxygen sensing in carotid body [34] as Hmox2-/- mice showed a blunted hypoxic ventilatory response [34]. Peng and colleagues have reported that Hmox2-/- mice develop spontaneous apneas during sleep [29]. Furthermore, in a more recent study, they also showed that these mice have increased systemic levels of catecholamines [30]. These studies suggested that Hmox2-/- mice can be used as a model to mimic sleep apnea. However, how the cardiovascular system may be affected in the Hmox2-/- model of sleep apnea has not been studied. Here, we studied whether Hmox2-/- mice exhibit EC activation similar to the IH model of OSA. We also investigated the transcriptomic changes in aortic ECs and heart tissue as well as the functional changes in heart function in Hmox2-/- mice.

Materials and methods

Animals

All experiments and procedures involving animals were approved by the Institutional Animal Care and Use Committee at the University of Chicago (Protocol number 72573). We used mice with deletion of Hmox2 (Hmox2-/-) and their wildtype littermate controls (Hmox2+/+). All animal experiments were performed according to the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines [35]. Mice were euthanized using the euthanasia solution (Euthasol (pentobarbital sodium and phenytoin sodium)) followed by exsanguination and removal of vital organs. The method of euthanasia is consistent with the recommendations of the American Veterinary Medical Association Guidelines for the Euthanasia of Animals. After confirming anesthesia following the administration of euthanasia solution, we performed thoracotomy and first collected blood from the right ventricle for catecholamine levels. We then cut the inferior vena cava and flushed heart and vessels with phosphate buffered saline to remove remaining blood in the vasculature. We then harvested the hearts and aortas and placed them in cold plate containing phosphate buffered saline and dissected under microscope [20, 25, 36]. Heart tissue was frozen in liquid nitrogen for RNA isolation. Total RNA from aortic ECs was isolated by gentle and slow flushing with TRI Reagent inside of the aortic lumen with a blunt end needle insertion.

RNA isolation and sequencing

RNA isolation, sequencing, and analysis were done as we have previously described [37]. We isolated RNA from aortic ECs using TRI Reagent (Zymo Research, R2050-1-200) and Zymo Direct-zol RNA Miniprep Kit (Zymo Research, catalog number R2053) [20]. Heart tissue total RNA was isolated from fast-frozen whole heart. The tissue was pulverized under liquid nitrogen in a stainless-steel mortar and pestle and the frozen tissue powder quantitatively transferred to test tubes. Total RNA was isolated with RNeasy Plus Mini Kit (Qiagen, 74134). Total RNA from aortic ECs and heart tissues were submitted to the University of Chicago Genomics Core Facility for sequencing with the Illumina NovaSEQ6000 sequencer (100bp paired-end). Sequencing read (FASTQ) files were generated and assessed for per base sequence quality using FastQC. RNA-seq reads were pseudoaligned using Kallisto v.0.44.0 the at University of Chicago, CRI Gardner high performance computing cluster [38]. The Kallisto index was made with default parameters and the GENCODE (Mouse Release M32, GRCm39) and was run in quant mode with default parameters. Following pseudoalignment, we computed gene abundances using R package tximport v.1.18.0 [39]. Differential expression was calculated between the groups using R package edgeR [40]. edgeR performs read count filtering, normalization, estimating dispersion, and identification of differentially expressed genes. Differential gene expression was considered significant for genes with an FDR-adjusted p-value ≤ 0.05 and fold change (FC) > 2. All volcano plots were drawn using ggplot2 R package. All heatmaps were generated with Pretty heatmaps R package pheatmap package from Z-score normalized expression values. An absolute fold change ≥ 2 and false discovery rate (FDR) adjusted p-value ≤ 0.05 were used to select and classify the significant DEGs. Pathway enrichment analyses were performed, and plots were created using R clusterProfiler package [41] or using Enrichr search engine web interface on Enrichr database [42]. All packages were run on RStudio (2021.09.0 Build 351) with R version 4.0.3 Source data for RNA-seq are accessible via GEO (GSE230725). https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE230725

Transcription factor (TF)—target gene interactions gene regulatory network enrichment analysis was done with R DoRothEA package using mouse regulons [43]. DoRothEA contains 470,711 TF-target interactions for 1396 TFs for 20,238 unique genes.

Cardiac structure, function, and rhythm analysis

Transthoracic echocardiography (VisualSonics, Vevo 770–120, RMV707B probe, Toronto, Canada) was used to evaluate left ventricular (LV) systolic function and structure while mice were under light anesthesia (0.5–1% isoflurane). Two-dimensionally guided M-mode images of the LV were acquired in the parasternal long and short axes to measure LV cavity dimensions, anterior and posterior wall thicknesses, and fractional shortening. LV ejection fraction was calculated based on these measurements. Electrocardiogram (ECG) (PowerLab using LabChart software, AD Instruments) was performed to determine heart rate, rhythm and conduction intervals in mice. Electrodes were placed subcutaneously in the limbs for lead II position for continuous ECG monitoring up to 5 minutes in each session. RR interval, PR interval, QRS duration, and QT interval were recorded and analyzed from limb leads in lead II. AF, atrioventricular block (AVB), premature beats, pauses, supraventricular, and ventricular arrhythmias were documented on ECG recordings.

Statistics

The data were analyzed in Prism 9 (GraphPad Software Inc., La Jolla, CA). All data are shown as mean ± standard deviation. Significance was determined by unpaired, two-tailed Student’s t-test (for comparisons between two samples) or by one-way ANOVA using Bonferroni’s correction for multiple comparisons. Statistical significance was defined as *p<0.05, **p<0.005, ***p<0.001, ****p<0.0001.

Results

Hmox2-/- mice exhibit EC activation in aorta

Increasing evidence suggest that Hmox2 plays an important role in oxygen sensing [31, 44]. Furthermore, recent studies by Peng and colleagues have reported that Hmox2-/- mice exhibit spontaneous apneas during sleep [29, 30]. They also showed that these mice have increased systemic levels of catecholamines [30]. These results suggested the suitability of Hmox2-/- as a model of sleep apnea; however, the cardiovascular changes in Hmox2-/- are not completely understood.

We have recently reported that exposure to IH as a model of sleep apnea in mice is associated with aortic endothelial cell (EC) activation [20]. To determine whether Hmox2 deficiency is associated with EC activation, we isolated aortic ECs from both Hmox2+/+ and Hmox2-/- mice to measure expression of genes associated with EC activation. As previously reported [29, 30], compared to control Hmox2+/+ mice, Hmox2-/- mice had spontaneous apneas and increased systemic levels of catecholamines (S1 Fig and S1 Text). We also confirmed the loss of Hmox2 gene in aortic ECs isolated from Hmox2-/- mice (S2 Fig and S1 Text). We found that compared to ECs from Hmox2+/+ mice, ECs from Hmox2-/- mice exhibited increased expression of il6, but there was no increase in the expression of Kc, Icam1 or Vcam1 as we have previously reported in ECs isolated from wild-type mice exposed to IH [20] (Fig 1A). We also evaluated the expression of EC activation-associated genes selp, and nampt. Selp encodes P-selectin, which mediates the rolling of leukocytes on the surface of the endothelium and initiates their attachment to ECs [45]. Nicotinamide phosphoribosyltransferase (Nampt) is the rate-limiting enzyme of nicotinamide adenine dinucleotide salvage biosynthesis. Nampt has been shown to be associated with EC activation/dysfunction, vascular inflammation and progression of atherosclerosis [46, 47]. Consistent with EC activation in sleep apnea, we found that ECs from Hmox2-/- mice showed increased expression of selp and nampt (Fig 1B). Collectively, these results suggested that aortic ECs from Hmox2-/- mice exhibit gene expression changes consistent with EC activation.

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Fig 1. Loss of Hmox2 is associated with endothelial cell activation.

We isolated aortic endothelial cells from Hmox2-/- and littermate control Hmox2+/+ mice and performed qPCR to measure expression of (A) cytokines and adhesion molecules (il6, kc, icam1, vcam1) and (B) other EC activation markers (selp, nampt) (n = 5/strain). *p<0.05.

https://doi.org/10.1371/journal.pone.0292990.g001

Loss of Hmox2 is associated with DEGs enriched in blood coagulation, cell adhesion, cellular respiration, and cardiac muscle development and contraction in endothelial cells

To better understand the effect of sleep apnea on ECs beyond their activation in the Hmox2-/- model, we performed RNA-sequencing in ECs isolated from Hmox2-/- and Hmox2+/+ mice. The t-sne plot showed good separation between samples from Hmox2-/- and Hmox2+/+ mice based on differentially expressed genes (DEGs) (Fig 2A). Compared to ECs from Hmox2+/+ mice, the expression of 255 genes was upregulated, and 200 genes were downregulated in ECs from Hmox2-/- mice (Fig 2B). Fig 2C shows the top 50 differentially expressed genes in ECs between Hmox2-/- and Hmox2+/+ mice. Gene ontology biological processes (GO_BP) enrichment analysis showed that blood coagulation, hemostasis, positive regulation of cytokine production, activation of immune response and positive regulation of cell adhesion were activated in ECs from Hmox2-/- mice (Fig 2D). In contrast, oxidative phosphorylation and cellular respiration were processes that were downregulated in ECs from Hmox2-/- mice. Interestingly, muscle contraction and heart contraction were also processes that were downregulated. Similar processes and pathways were identified using the gene set enrichment analysis (GSEA) of DEGs in ECs from Hmox2-/- mice including cellular respiration, oxidative phosphorylation, blood circulation, cardiac muscle cell development and muscle contraction, and ribonucleotide metabolic processes. Additionally, enrichment analysis of DEGs across GO, KEGG and MGI_phenotype multiple datasets by Enrichr-KG tool also significantly identified cardiomyopathy and oxidative phosphorylation KEGG pathways and abnormal glucose homeostasis MGI-Mouse phenotype along with the above stated GO terms (S3 Fig). These results suggested that ECs from Hmox2-/- mice, a model of sleep apnea, have a significant number of induced DEGs enriched in coagulation and cell adhesion, further supporting EC activation, and repressed DEGs enriched in cellular respiration and, oxidative phosphorylation. Surprisingly, ECs from Hmox2-/- mice have a significant number of DEGs enriched in cardiac muscle development and contraction.

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Fig 2. Transcriptional changes in aortic endothelial cells in Hmox2-/- and Hmox2+/+ mice.

RNA-sequencing was performed on aortic ECs collected from Hmox2-/- and Hmox2+/+ mice (n = 3/strain). (A) Similarity level of high dimensional data of differentially expressed genes (DEGs) in aortic ECs from Hmox2-/- and Hmox2+/+ mice visualized by t-sne plot of Log2 fold (LogFC) changes. (B) Volcano plot of DEGs (6151) and top significantly differentially regulated (255 up and 200 down) genes in Hmox2-/-. (C) Heat map of the top 50 significantly DEGs in aortic ECs. (D) Significantly activated and suppressed enriched gene sets of Gene ontology biological processes (GO_BP) terms enriched based on the significantly activated and suppressed genes in heart tissue. (E) Gene network associated with the identified biological processes in heart tissue. Gene set enrichment analysis (GSEA) parameters for significance were set to absolute fold change ≥2 and FDR adjusted p-value≤0.05 between Hmox2+/+ and Hmox2-/-. Source data for RNA-seq are accessible via GEO (GSE230725). https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE230725.

https://doi.org/10.1371/journal.pone.0292990.g002

Loss of Hmox2 is associated with DEGs enriched in mitochondrial translation, oxidative phosphorylation and cardiac muscle development in heart tissue

Surprisingly, the transcriptomic analysis of ECs showed reduced expression of genes in cardiac muscle development and contraction in Hmox2-/- mice. Consistent with our data, a recent study in which the investigators performed single cell RNA-sequencing on mouse aorta reported the presence of a cluster of aortic ECs which express high levels of troponin and other myocyte markers [48]. These findings led us to perform RNA-sequencing in heart tissue from Hmox2-/- and Hmox2+/+ mice. We confirmed the loss of Hmox2 gene in heart tissue isolated from Hmox2-/- mice (S2 Fig and S1 Text). The t-sne plot showed good separation between samples from Hmox2-/- and Hmox2+/+ mice based on differentially expressed genes (DEGs) (Fig 3A). Compared to heart tissue from Hmox2+/+ mice, the expression of 479 genes was upregulated, and 332 genes were downregulated in heart tissue from Hmox2-/- mice (Fig 3B). Fig 3C shows the top 50 DEGs in heart tissue between Hmox2-/- and Hmox2+/+ mice. GO_BP enrichment analysis showed that mitochondrial translation, mitochondrial gene expression, aerobic respiration as well as nucleoside triphosphate biosynthetic process were activated in heart tissue from Hmox2-/- mice (Fig 3D). Similar to ECs, cardiac muscle development was one of the processes downregulated in heart tissue from Hmox2-/- mice (Fig 3D). GSEA of DEGs showed again mitochondrial translation, oxidative phosphorylation, and cardiac muscle development as pathways enriched in heart tissues from Hmox2-/- mice. MGI-Mouse Phenotype terms of decreased skeletal muscle fiber diameter, abnormal muscle physiology and decreased cardiac muscle contractility were notably associated with the loss of Hmox2 by the enrichment analysis with Enrichr-KG tool (S4 Fig), These results suggested that heart from Hmox2-/- mice have a significant number of DEGs enriched in mitochondrial translation, oxidative phosphorylation and cardiac muscle development, which are similar to those we found in ECs.

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Fig 3. Transcriptional changes in heart tissue from Hmox2-/- and Hmox2+/+ mice.

RNA-sequencing was performed on heart tissues that were collected from Hmox2-/- and Hmox2+/+ mice (n = 3/strain). (A) Similarity level of high dimensional data of DEGs in heart tissues from Hmox2-/- and Hmox2+/+ mice visualized by t-sne plot of Log2 fold (LogFC) changes. (B) Volcano plot of DEGs (4422) and top significantly differentially regulated (479 up and 332 down) genes in Hmox2-/-. (C) Heat map of top 50 significant DEGs in heart tissue. (D) Gene ontology biological processes (GO_BP) terms enriched based on the significantly activated and suppressed genes in heart tissue. (E) Gene network associated with the identified biological processes in heart tissue. Gene set enrichment analysis (GSEA) parameters for significance were set to absolute fold change ≥2 and FDR adjusted p-value≤0.05 between Hmox2+/+ and Hmox2-/-. Source data for RNA-seq are accessible via GEO (GSE230725). https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE230725.

https://doi.org/10.1371/journal.pone.0292990.g003

Shared pathway footprints and transcriptional regulation of gene expression in aortic ECs and heart tissue in Hmox2-/- mice

Transcriptome profiles from aortic ECs and heart tissue from Hmox2-/- mice in comparison with their wild-type control littermates were very similar in terms of their GO_BP enrichments (Figs 2D and 3D). In addition, there were common genes in top 50 DEG sets between ECs and heart (Figs 2C and 3C). These findings suggested that these two RNA expression data from two different sources of the cardiovascular system might share common cellular network footprints in Hmox2-/- mice. Therefore, we analyzed aortic ECs and heart tissue RNA expression profiles in combination. t-SNE analysis of RNAseq read counts from ECs and heart tissue from Hmox2+/+ and Hmox2-/- mice showed that gene expression profiles were linearly separable for all 4 groups (Fig 4A). 206 DEGs were common in ECs and heart tissue when Hmox2-/- compared to their Hmox2+/+ littermates (Fig 4B). The majority of the top 50 common DEGs had parallel expression regulation (Fig 4C). These findings suggest that deletion of Hmox2 might have common cellular network footprints on aortic ECs and heart tissue.

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Fig 4. Combined gene expression analysis of aortic ECs and heart tissue from Hmox2-/- and Hmox2+/+ mice.

Read counts both from aortic ECs and heart tissue from Hmox2+/+ and Hmox2-/- mice were analyzed in combination. (A) Similarity level of high dimensional data of differentially expressed genes (DEGs) in aortic ECs from Hmox2-/- and Hmox2+/+ mice and heart tissues from Hmox2-/- and Hmox2+/+ mice visualized by t-sne plot of Log2 fold (LogFC) changes. (B) Venn diagram of significant DEGs (logFC > = 0.5 p-value<0.05) in aortic ECs and heart tissue from Hmox2+/+ vs. Hmox2-/- mice. (C) Heat map of top 50 genes from shared 206 DEGs between aortic ECs and heart tissue.

https://doi.org/10.1371/journal.pone.0292990.g004

We used the functional genomics tool PROGENy to analyze the transcriptional regulation of signaling pathways. PROGENy prioritizes the most responsive genes upon corresponding pathway perturbation as “footprint gene sets” rather than the number of genes involved in the pathway [49, 50]. While PI3K, Estrogen, Trail, VEGF and EGFR, cellular survival pathway footprints were activated, Hypoxia, p53, WINT cellular stress response pathway footprints were downregulated with the deletion of Hmox2 (Fig 5A). These findings obtained from mice on room air and not under oxygen-related stress support a molecular sensory function of Hmox2 and a role for this gene in sensing environmental stress.

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Fig 5. Shared gene alteration dependent gene regulatory network analysis of aortic ECs and heart tissue from Hmox2-/- and Hmox2+/+ mice.

(A) Significantly upregulated and downregulated PROGENy pathways. The color legend indicates the degree of enrichment. Normalized enrichment score: NES (B) Transcription factor (TF)-target gene interactions computed with DoRothEA R tool shown as TF enrichment. (C) Negatively and positively enriched TFs. The color legend indicates TF activity. (D) Volcano plots of Hif1a regulated targets for both aortic ECs and heart tissue in Hmox2-/- mice.

https://doi.org/10.1371/journal.pone.0292990.g005

Transcription factor (TF) activities of Hmox2-/- mice were investigated to support cellular network footprints. Transcription factor (TF) activities were analyzed with DoRothEA TF-target interactions (regulons) tool and TF activities were inferred in parallel with pathway footprints (Fig 5B). Consistent with downregulation of hypoxia-associated pathways, transcription factor activity of hypoxia response gene Hif1a was significantly reduced in Hmox2-/- tissues (Fig 5C). Hif1a target genes were significantly downregulated while TFs target genes involved in cell survival were upregulated when both Hmox2-/- mice ECs and heart tissue transcriptome profiles analyzed in combination (Fig 5D).

Hmox2-/- mice develop progressive cardiomyopathy

Since heart tissue from Hmox2-/- mice showed decreased expression of genes involved in cardiac muscle development, we evaluated cardiac structure and function of these mice by using echocardiographic images. Left ventricle (LV) chamber size, wall thickness and systolic function were measured. Young (6–8 weeks old) or aged (6 months old) Hmox2-/- mice developed significant LV dilation as shown by increased LV end diastolic diameter and LV end systolic diameter compared to Hmox2+/+ heart (n = 3 in each group, p<0.05 for all groups) (Fig 6A). This was associated with LV systolic dysfunction. As a marker of cardiac contractility, LV ejection fraction was significantly reduced in hearts of young (70.08±1.76%) and aged (29.46±2.59%) Hmox2-/- mice versus hearts in Hmox2+/+ mice (86.43±2.03% in young and 61.06±2.95) (n = 3 in each group, p<0.05 for all groups). In parallel, we found that LV posterior wall thickness and interventricular septum thickness in young and aged Hmox2-/- hearts were decreased. LV ejection fraction was significantly worsened through aging process of Hmox2-/- mice. Also, LV dilation (systolic and diastolic) and wall thickness were deteriorated with aging of Hmox2-/- mice. Thus, these morphological changes in Hmox2-/- hearts were consistent with dilated cardiomyopathy and heart failure with reduced ejection fraction. We also studied cardiac electrical properties in all mice by analyzing ECG characteristics. Young Hmox2-/- mice showed similar heart rate compared to Hmox2+/+ mice; however, ECG recordings showed higher heart rate in aged Hmox2-/- mice. This increase in heart rate was likely due to reduced LV ejection fraction and cardiomyopathy. There was atrioventricular conduction delay in young and aged Hmox2-/- mice as reflected by prolonged PR interval; however, QRS duration was comparable in Hmox2-/- and Hmox2+/+. Hearts from young and aged Hmox2-/- mice showed a repolarization abnormality with shorter QT interval compared to hearts from control mice. These results are consistent with the development of dilated cardiomyopathy in Hmox2-/- mice and are also in agreement with the transcriptomic data showing DEGs enriched in cardiac muscle development.

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Fig 6. Hmox2-/- mice develop dilated cardiomyopathy and conduction delay.

We performed echocardiography on young (6–8 weeks old) and aged (6-months old) Hmox2-/- and Hmox2+/+ mice. (A) Table shows LV end-diastolic, and systolic diameter, LV ejection, posterior wall and interventricular thickness and graph for LV ejection fraction (n = 3/group, *p<0.05). (B) We also performed electrocardiography on these mice. Heart rate, PR interval, QRS duration and QT interval are shown.

https://doi.org/10.1371/journal.pone.0292990.g006

Discussion

OSA is a highly prevalent condition affecting up to 20% of the adult population in the US [1]. Recent estimates show that OSA is present in approximately 1 billion people in the world [51]. Clinically, OSA may present with neurocognitive changes including excessive daytime sleepiness. In addition, OSA is an independent risk factor for cardiovascular disease including hypertension, coronary artery disease and heart failure [1, 2, 7–15]. The pathophysiology of OSA includes repetitive upper airway collapse resulting in IH, intrathoracic pressure swings, arousals and activation of the sympathetic nervous system. Although the mechanisms of cardiovascular disease in OSA are not completely understood, both IH and increased catecholamines due to activation of the sympathetic nervous system have been implicated in the pathogenesis of cardiovascular disease. EC activation is an early process in the pathogenesis of atherosclerosis. We have recently reported that IH induces EC activation [20]; however, we found that IH-induced EC activation was not a direct effect of IH but required sympathetic nervous system activation and catecholamine release [20].

Exposure to IH is often used as a model to study the mechanisms of OSA. Although it is widely used, the IH model also has limitations [52]. IH does not reproduce all the physiological changes that occur in patients with OSA, such as increased respiratory efforts, intrathoracic pressure swings and hypercapnia. Importantly, IH models induce severe hypoxemia that does not occur in most patients with OSA. Peng and colleagues have recently described Hmox2-/- mice as a spontaneous model of sleep apnea [29]. They also reported that Hmox2-/- mice not only develop spontaneous apneas but also exhibit elevated levels of catecholamines [29, 30].

In this study, we investigated the transcriptional changes that occur in aortic ECs in Hmox2-/- mice to better understand the impact of OSA and increased catecholamines on EC function. We found that aortic ECs from Hmox2-/- mice exhibit hallmarks of activation. RNA-sequencing demonstrated that compared to aortic ECs from Hmox2+/+ mice, ECs from Hmox2-/- mice had a significant number of DEGs enriched in blood coagulation, hemostasis, positive regulation of cytokine production, activation of immune response and cell adhesion, which were also consistent with EC activation and dysfunction and vascular inflammation. In addition, oxidative phosphorylation and cellular respiration were processes that were downregulated in ECs from Hmox2-/- mice. These results are consistent with the published data showing EC activation with other models of OSA including IH [20, 53, 54]. They are also in agreement with Bellner et al. who showed that aortic ECs from Hmox2-/- mice exhibit increased expression of pro-inflammatory cytokines and nuclear factor κB activation, which are consistent with EC activation [55].

Analysis of transcriptomic data also showed enrichment of DEGs in cardiac muscle development and contraction, which were downregulated in ECs from Hmox2-/- mice. These surprising findings were consistent with a recently published data using single cell RNA-sequencing in mouse aortic ECs [48]. Lukowski and colleagues identified 4 different clusters of ECs, one of which was a cluster of aortic ECs which had high expression of cardiomyocyte genes such as troponin [48]. It is possible that these genes may be expressed in a group of ECs close to heart in the aortic arch. These findings led us to perform RNA-sequencing in heart tissue from Hmox2-/- and Hmox2+/+ mice. Compared to control, heart tissue from Hmox2-/- mice had DEGs enriched in mitochondrial gene expression and translation, and aerobic respiration. Similar to what we found in ECs, cardiac muscle development was one of the processes downregulated in heart tissue from Hmox2-/- mice. Furthermore, MGI-Mouse Phenotype terms of decreased skeletal muscle fiber diameter, abnormal muscle physiology and decreased cardiac muscle contractility were associated with the loss of Hmox2. The gene expression changes that we observed in heart tissue from Hmox2-/- mice are in agreement with the changes that occur in cardiomyocytes exposed to IH in vitro [56–59]. Exposure to IH causes cardiac inflammation and injury and decreases the viability of cardiomyocytes [56–59].

Given that genes that were involved in cardiomyocyte development and contractility were downregulated in Hmox2-/- mice, we then evaluated whether Hmox2-/- mice had any functional limitations. Echocardiographic evaluation showed structural and functional changes consistent with dilated cardiomyopathy, which progressed with aging. Collectively, we found that Hmox2-/- mice, which spontaneously develop apneas and increased systemic level of catecholamines have EC activation, and cardiac dysfunction.

Integrative analysis of the transcriptomes from aortic ECs and heart tissue showed that they share common network footprints. Using functional genomics tools, we found that PI3K, Estrogen, Trail, VEGF and EGFR, cellular survival pathway footprints were activated, while hypoxia, p53, WINT cellular stress response pathway footprints were downregulated with the deletion of Hmox2. Consistent with the downregulation of the hypoxia pathway, analysis of transcription factors showed reduced activity of HIF1α in samples from Hmox2-/- mice.

Our study had several limitations. First, we did not have another model of OSA such as IH exposure in wild-type mice as a control group limiting the translation of our findings in Hmox2-/- mice to OSA. Since Hmox2 is deleted in ECs and heart tissue, the effect of loss of Hmox2 on ECs and heart tissue may not be solely due to increased apneas but may also be caused by the loss of Hmox2 gene in these cells/tissues. Further studies are warranted to determine whether the effects of IH are similar to those observed in Hmox2-/- mice. Second, we also used bulk RNA-sequencing in heart tissue instead of single cell RNA-sequencing. Since the majority of the cells in heart tissue are cardiomyocytes, the bulk RNA-sequencing likely provided information of cardiomyocytes but the signal from other cells in the heart tissue including coronary ECs, macrophages, and fibroblasts were likely lost. Third, while we showed progression of dilated cardiomyopathy in aged mice, our sequencing data were obtained from young mice. Finally, we did not study the mechanisms underlying the cardiac changes we observed in Hmox2-/- mice. HMOX2 has been implicated in the regulation of inflammation, redox sensing, oxidative stress and wound healing [33, 60–64]. Loss of Hmox2 leads to increased leukocyte infiltration and inflammatory cytokines in injury models [33, 60–63]. It is not completely understood how Hmox2 may regulate inflammation and oxidative stress; however, it may be due to the toxic effects of heme that could not be catabolized or buffered by Hmox2 [65].

In conclusion, our data in Hmox2-/- mice, a spontaneous OSA model, showed expression of genes consistent with EC activation, downregulation of genes involved in cardiac muscle development and contractility, and developed progressive cardiomyopathy. While enrichment of DEGs in aerobic respiration suggested a change in mitochondrial function as a mechanism for the cardiac changes in Hmox2-/- mice, further studies will be needed to better understand the mechanisms leading to heart failure in these mice.

Supporting information

S1 Fig. Hmox2-/- mice exhibit spontaneous apneas and have increased systemic levels of catecholamines.

We measured (A) apnea index using body plethysmography and (B) catecholamine levels in plasma in Hmox2+/+ and Hmox2-/- mice (n = 5 for Hmox2+/+ and n = 10 for Hmox2-/-). *p<0.05. NE: norepinephrine, Epi: epinephrine.

https://doi.org/10.1371/journal.pone.0292990.s001

(TIF)

S2 Fig. Confirmation of loss of Hmox2 expression in aortic ECs and heart tissue from Hmox2-/- mice.

We measured mRNA expression of Hmox2 using qPCR in (A) aortic ECs and (B) heart tissue from Hmox2+/+ and Hmox2-/- mice.

https://doi.org/10.1371/journal.pone.0292990.s002

(TIF)

S3 Fig. Enrichment analysis of DEGs from mouse aortic ECs.

Enrichment analysis of significantly (LogFC ≥1 and p≤0.05) DEGs from RNA-sequencing data from aortic ECs isolated from Hmox2-/- and Hmox2+/+ mice (n = 3/strain), across GO, KEGG and MGI-Mouse phenotype datasets by Enrichr-KG tool. Nodes are genes (green) and functional terms, edges connect genes to their enriched terms in the enrichment graph.

https://doi.org/10.1371/journal.pone.0292990.s003

(TIF)

S4 Fig. Enrichment analysis of DEGs from mouse heart tissue.

Enrichment analysis of significantly (LogFC ≥1 and p≤0.05) DEGs from RNA-sequencing data from mouse heart tissue from Hmox2-/- and Hmox2+/+ mice (n = 3/strain), across GO, KEGG and MGI-Mouse phenotype datasets by Enrichr-KG tool. Nodes are genes (green) and functional terms, edges connect genes to their enriched terms in the enrichment graph.

https://doi.org/10.1371/journal.pone.0292990.s004

(TIF)

S1 Text. Additional methods and results.

We provide methods on the measurement of apnea index, plasma catecholamines and qPCR for Hmox2. We also show the results about the apnea index, plasma catecholamine levels and Hmox2 expression in aortic ECs and heart tissue from Hmox2-/- mice.

https://doi.org/10.1371/journal.pone.0292990.s005

(DOCX)

Acknowledgments

The authors thank Dr. Nanduri Prabhakar, University of Chicago for providing Hmox2-/- mice and their wildtype littermate controls (Hmox2+/+) as well as the data on apnea index and systemic catecholamine levels.

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