. 2020 Jul;98(7):1009-1020.

doi: 10.1007/s00109-020-01933-8. Epub 2020 Jun 12.

Gene-environment regulation of chamber-specific maturation during hypoxemic perinatal circulatory transition

Yan Zhao^#^{1

2}, Xuedong Kang^#^{1

2}, Alexander Barsegian^{1

2}, Jian He^{1

2}, Alejandra Guzman^{1

2}, Ryan P Lau³, Reshma Biniwale⁴, Madhuri Wadhra³, Brian Reemtsen⁴, Meena Garg¹, Nancy Halnon¹, Fabiola Quintero-Rivera³, Wayne W Grody³; UCLA Congenital Heart Defects BioCore Faculty; Glen Van Arsdell⁴, Stanley F Nelson^{1

5

6

7}, Marlin Touma^{8

9

10

11

12

13

14}

Affiliations

¹ Department of Pediatrics, David Geffen School of Medicine, University of California Los Angeles, Los Angeles 675 Charles E. Young Dr S, 3762 MacDonald Research Laboratories, Los Angeles, CA, 90024, USA.
² Neonatal/Congenital Heart Laboratory, Cardiovascular Research Laboratory, University of California Los Angeles, Los Angeles, CA, USA.
³ Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA.
⁴ Department of Cardiothoracic Surgery, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA.
⁵ Department of Neurology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA.
⁶ Department of Human Genetics, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA.
⁷ Institute of Precision Health, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA.
⁸ Department of Pediatrics, David Geffen School of Medicine, University of California Los Angeles, Los Angeles 675 Charles E. Young Dr S, 3762 MacDonald Research Laboratories, Los Angeles, CA, 90024, USA. mtouma@mednet.ucla.edu.
⁹ Neonatal/Congenital Heart Laboratory, Cardiovascular Research Laboratory, University of California Los Angeles, Los Angeles, CA, USA. mtouma@mednet.ucla.edu.
¹⁰ Department of Human Genetics, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA. mtouma@mednet.ucla.edu.
¹¹ Children's Discovery and Innovation Institute, Department of Pediatrics, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA. mtouma@mednet.ucla.edu.
¹² The Molecular Biology Institute, University of California Los Angeles, Los Angeles, CA, USA. mtouma@mednet.ucla.edu.
¹³ Eli and Edythe Stem Cell Institute, University of California Los Angeles, Los Angeles, CA, USA. mtouma@mednet.ucla.edu.
¹⁴ Institute of Precision Health, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA. mtouma@mednet.ucla.edu.

^# Contributed equally.

PMID: 32533200
PMCID: PMC7843236
DOI: 10.1007/s00109-020-01933-8

Gene-environment regulation of chamber-specific maturation during hypoxemic perinatal circulatory transition

Yan Zhao et al. J Mol Med (Berl). 2020 Jul.

. 2020 Jul;98(7):1009-1020.

doi: 10.1007/s00109-020-01933-8. Epub 2020 Jun 12.

Authors

Affiliations

¹ Department of Pediatrics, David Geffen School of Medicine, University of California Los Angeles, Los Angeles 675 Charles E. Young Dr S, 3762 MacDonald Research Laboratories, Los Angeles, CA, 90024, USA.
² Neonatal/Congenital Heart Laboratory, Cardiovascular Research Laboratory, University of California Los Angeles, Los Angeles, CA, USA.
³ Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA.
⁴ Department of Cardiothoracic Surgery, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA.
⁵ Department of Neurology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA.
⁶ Department of Human Genetics, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA.
⁷ Institute of Precision Health, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA.
⁸ Department of Pediatrics, David Geffen School of Medicine, University of California Los Angeles, Los Angeles 675 Charles E. Young Dr S, 3762 MacDonald Research Laboratories, Los Angeles, CA, 90024, USA. mtouma@mednet.ucla.edu.
⁹ Neonatal/Congenital Heart Laboratory, Cardiovascular Research Laboratory, University of California Los Angeles, Los Angeles, CA, USA. mtouma@mednet.ucla.edu.
¹⁰ Department of Human Genetics, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA. mtouma@mednet.ucla.edu.
¹¹ Children's Discovery and Innovation Institute, Department of Pediatrics, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA. mtouma@mednet.ucla.edu.
¹² The Molecular Biology Institute, University of California Los Angeles, Los Angeles, CA, USA. mtouma@mednet.ucla.edu.
¹³ Eli and Edythe Stem Cell Institute, University of California Los Angeles, Los Angeles, CA, USA. mtouma@mednet.ucla.edu.
¹⁴ Institute of Precision Health, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA. mtouma@mednet.ucla.edu.

^# Contributed equally.

PMID: 32533200
PMCID: PMC7843236
DOI: 10.1007/s00109-020-01933-8

Abstract

Chamber-specific and temporally regulated perinatal cardiac growth and maturation is critical for functional adaptation of the heart and may be altered significantly in response to perinatal stress, such as systemic hypoxia (hypoxemia), leading to significant pathology, even mortality. Understanding transcriptome regulation of neonatal heart chambers in response to hypoxemia is necessary to develop chamber-specific therapies for infants with cyanotic congenital heart defects (CHDs). We sought to determine chamber-specific transcriptome programming during hypoxemic perinatal circulatory transition. We performed transcriptome-wide analysis on right ventricle (RV) and left ventricle (LV) of postnatal day 3 (P3) mouse hearts exposed to perinatal hypoxemia. Hypoxemia decreased baseline differences between RV and LV leading to significant attenuation of ventricular patterning (AVP), which involved several molecular pathways, including Wnt signaling suppression and cell cycle induction. Notably, robust changes in RV transcriptome in hypoxemic condition contributed significantly to the AVP. Remarkably, suppression of epithelial mesenchymal transition (EMT) and dysregulation of the TP53 signaling were prominent hallmarks of the AVP genes in neonatal mouse heart. Furthermore, members of the TP53-related gene family were dysregulated in the hypoxemic RVs of neonatal mouse and cyanotic Tetralogy of Fallot hearts. Integrated analysis of chamber-specific transcriptome revealed hypoxemia-specific changes that were more robust in RVs compared with LVs, leading to previously uncharacterized AVP induced by perinatal hypoxemia. Remarkably, reprogramming of EMT process and dysregulation of the TP53 network contributed to transcriptome remodeling of neonatal heart during hypoxemic circulatory transition. These insights may enhance our understanding of hypoxemia-induced pathogenesis in newborn infants with cyanotic CHD phenotypes. KEY MESSAGES: During perinatal circulatory transition, transcriptome programming is a major driving force of cardiac chamber-specific maturation and adaptation to hemodynamic load and external environment. During hypoxemic perinatal transition, transcriptome reprogramming may affect chamber-specific growth and development, particularly in newborns with congenital heart defects (CHDs). Chamber-specific transcriptome changes during hypoxemic perinatal transition are yet to be fully elucidated. Systems-based analysis of hypoxemic neonatal hearts at postnatal day 3 reveals chamber-specific transcriptome signatures during hypoxemic perinatal transition, which involve attenuation of ventricular patterning (AVP) and repression of epithelial mesenchymal transition (EMT). Key regulatory circuits involved in hypoxemia response were identified including suppression of Wnt signaling, induction of cellular proliferation and dysregulation of TP53 network.

Keywords: Congenital heart defects; Hypoxia; Neonatal heart maturation; Tetralogy of Fallot; Transcriptome.

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Conflict of interest statement

CONFLICT OF INTEREST:

None.

Figures

**Figure 1.. Perinatal Induction of Systemic Hypoxia (Hypoxemia) in Neonatal Mouse Heart.**
A. Schematic illustration of experimental design for perinatal systemic hypoxia exposure. The dams carrying the experimental group (hypoxia) were acclimatized in the hypoxia chamber by decreasing FIO2 by 2% daily for at least 5 days preceding the experiment starting at E15.5 to reach 10% at E20.5. Neonatal pups were reared with their dams in either normoxia or hypoxia (10% FIO2) and maintained until postnatal day 3 (P3). B. Hif1a immunohistochemistry (IHC) staining of normoxia-treated or hypoxia-treated P3 neonatal mouse hearts demonstrates nuclear Hif1a stabilization in response to perinatal hypoxia. C. Quantitative expression analysis (qRT-PCR) of Hif1a homeostasis genes, Cited2 and EP300, in RV and LV of P3 neonatal mouse hearts in normoxia and hypoxia conditions. Error bars represent standard error of means (SEM). *P ≤0.05, **P≤0.01, ***P≤0.005; two tailed Student’s t test; n=3 (Cited2), n=4 (Ep300).

**Figure 2.. Hypoxia-Induced Attenuation of Ventricular Patterning in Neonatal Mouse Heart.**
A. Unsupervised Hierarchical clustering of all transcripts from RV and LV of normoxia-treated and hypoxia-treated neonatal mouse hearts. B. Expression heat map of significant differentially expressed genes (DEGs). C. Volcano Plots display pair-wise comparison analysis (RV vs LV) in normoxia and hypoxia conditions. D. Pathway correlation plot of normoxia-treated RV (X Axis) compared to hypoxia-treated RV (Y Axis) depicts significant inverse correlation between cell cycle processes and Wnt signaling. E. Heat map depicts pathway enrichment signature in RV compared to LV in normoxia and hypoxia conditions. The chamber-specific signature is lost in hypoxia-treated hearts more significantly in RV. Blue: negative score. Orange: positive score. F. Volcano blots of Wnt signaling genes display reduced DGE in RV vs LV in neonatal mouse heart in hypoxia-treated neonatal mouse hearts compared to normoxia condition. G. Pathway enrichment analysis demonstrates trend changes of pathway scores (Bonferroni-adjusted P value) in RV (normoxia vs hypoxia) and in LV (normoxia vs hypoxia).

**Figure 3.. Perinatal Hypoxemia Regulates Major Signaling Pathways in Neonatal Mouse Heart.**
Box plots depict global changes of major signaling pathways scores in RV (normoxia vs hypoxia) and in LV (normoxia vs hypoxia).

**Figure 4.. Hypoxemia-Induced Attenuation of Ventricular Patterning (AVP)**
**A and B.** P value distribution (A) and magnitude of expression differences [Log2 Fold Change (FC)] (B) of top 20 differentially expressed genes (RV vs LV) that exhibited attenuation of ventricular patterning (AVP) in P3 neonatal mouse heart in response to systemic hypoxia (hypoxemia) treatment compared to normoxia condition (FDR P Value ≤ 0.05). C. Top GO terms enriched in hypoxia-induced AVP genes in neonatal mouse (Bonferroni-adjusted p value). D. Quantitative expression analysis (qRT-PCR) of hypoxia homeostasis genes (EP300 and Cited2) and Wnt related genes (Wif1 and Axin2) in LV and RV of WT P3 mouse demonstrates significant attenuation of ventricular patterning (fold change and P Values) in hypoxia-treated hearts compared to normoxia condition. ^$P ≤ 0.05, ^$$P ≤ 0.01 (hypoxia-treated RV compared to RV normoxia); two-tailed Student’s t test; n=4 per group per condition. E. Correlation analysis of top twenty AVP genes reveals significant concordance in mouse RV and LV. F. Correlation analysis of top genes with inversed attenuation of ventricular patterning (IVP) in hypoxia compared to baseline normoxia. G. Venn diagram depicting the distribution of DEGs in RV vs LV in hypoxia (HP) vs Normoxia (NM) conditions.

**Figure 5.. TP53 is a Potential Key Regulator of Hypoxia-Induced Transcriptome Reprogramming.**
A. Schematic representation of P53 transcriptional network. B. Volcano blots of TP53_regulated genes display reduced DGE in RV vs LV in neonatal mouse heart in hypoxia-treated neonatal mouse hearts compared to normoxia condition. C. IPA-based upstream analysis reveals TP53 as a key regulator of the DGE genes involved in epithelial-mesenchymal transition and extracellular matrix remodeling. D. Correlation plot depicts significant concordance of TP53 -target gene expression in hypoxemic RVs from mouse and human TOF cases. E. Quantitative expression analysis (qRT-PCR) of TP53, TP53INP2, TP53RK in RV and LV myocardium of P3 neonatal mouse depicts robust induction in hypoxia-treated RVs compared to normoxia condition; n=3 per group per condition. *P ≤ 0.05, **P ≤ 0.01, (hypoxia compared to normoxia condition).

See this image and copyright information in PMC

References

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Gene-environment regulation of chamber-specific maturation during hypoxemic perinatal circulatory transition

Affiliations

Gene-environment regulation of chamber-specific maturation during hypoxemic perinatal circulatory transition

Authors

Affiliations

Abstract

Conflict of interest statement

Figures

References

Publication types

MeSH terms

Grants and funding

LinkOut - more resources

Full Text Sources

Medical

Research Materials

Miscellaneous