Disruption of spatiotemporal hypoxic signaling causes congenital heart disease in mice
Xuejun Yuan, … , Yonggang Zhou, Thomas Braun
Xuejun Yuan, … , Yonggang Zhou, Thomas Braun
Published April 24, 2017
Citation Information: J Clin Invest. 2017;127(6):2235-2248. https://doi.org/10.1172/JCI88725.
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Disruption of spatiotemporal hypoxic signaling causes congenital heart disease in mice

Abstract

Congenital heart disease (CHD) represents the most prevalent inborn anomaly. Only a minority of CHD cases are attributed to genetic causes, suggesting a major role of environmental factors. Nonphysiological hypoxia during early pregnancy induces CHD, but the underlying reasons are unknown. Here, we have demonstrated that cells in the mouse heart tube are hypoxic, while cardiac progenitor cells (CPCs) expressing islet 1 (ISL1) in the secondary heart field (SHF) are normoxic. In ISL1+ CPCs, induction of hypoxic responses caused CHD by repressing Isl1 and activating NK2 homeobox 5 (Nkx2.5), resulting in decreased cell proliferation and enhanced cardiomyocyte specification. We found that HIF1α formed a complex with the Notch effector hes family bHLH transcription factor 1 (HES1) and the protein deacetylase sirtuin 1 (SIRT1) at the Isl1 gene. This complex repressed Isl1 in the hypoxic heart tube or following induction of ectopic hypoxic responses. Subsequently, reduced Isl1 expression abrogated ISL1-dependent recruitment of histone deacetylases HDAC1/5, inhibiting Nkx2.5 expression. Inactivation of Sirt1 in ISL1+ CPCs blocked Isl1 suppression via the HIF1α/HES1/SIRT1 complex and prevented CHDs induced by pathological hypoxia. Our results indicate that spatial differences in oxygenation of the developing heart serve as signals to control CPC expansion and cardiac morphogenesis. We propose that physiological hypoxia coordinates homeostasis of CPCs, providing mechanistic explanations for some nongenetic causes of CHD.

Authors

Xuejun Yuan, Hui Qi, Xiang Li, Fan Wu, Jian Fang, Eva Bober, Gergana Dobreva, Yonggang Zhou, Thomas Braun

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Figure 3

A SIRT1-HES1–containing complex represses Isl1 expression in a hypoxia-dependent manner.

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A SIRT1-HES1–containing complex represses Isl1 expression in a hypoxia-d...
(A) ChIP analysis of HIF1α at Isl1 (positions –468 to –285) (n = 4) and Nkx2.5 (positions –9040 to –8859) (n = 3) promoters after hypoxia treatment of day-6 EBs (1% O2, 16 hours). *P < 0.05; **P < 0.01; ***P < 0.001, ANOVA with Tukey’s post hoc correction. Enrichment of HIF1α was normalized to input DNA. (B) Co-IP assay of SIRT1 with HIF1α or HES1 under hypoxia. 5% input was used as loading control. 2 independent experiments were performed, generating similar results. (C) ChIP analyses of SIRT1 and HES1 binding to the Isl1 promoter (positions –468 to –285) under hypoxia (1% O2, 12 hours) and normoxia (21% O2) in E8.5 embryos. Enrichment of SIRT1 or HES1 was normalized to input DNA. ***P < 0.001, ANOVA with Tukey’s post hoc correction (n = 3). (D) ChIP analyses of SIRT1 (n = 8) and HES1 (n = 6) binding to the Isl1 promoter (positions –468 to –285) after lentivirus-mediated Hif1a knockdown in CoCl2–treated differentiating ESCs. Enrichment of SIRT1 or HES1 was normalized to input DNA. *P < 0.05, ANOVA with Tukey’s post hoc correction. (E) ChIP analysis of SIRT1 binding to the promoter of Isl1 (positions –468 to –285) in CPCs after Hes1 knockdown. *P < 0.05; **P < 0.01, ANOVA with Tukey’s post hoc correction (n = 3). (F) RT-qPCR analysis of Isl1, Nkx2.5, Flk1 (EB at E6), a-SMA, and Myh7 (EB at E8) expression in differentiating ES cells after treatment with the SIRT1 inhibitor (1 μM EX527, 24 hours; Isl1, Nkx2.5, a-SMA, Myh7: n = 3; Flk1: n = 5). The m34b4 gene was used as a reference for normalization. *P < 0.05; ***P < 0.001, t test. SIRT1 inhibitor treatment increases Isl1, but decreases Nkx2.5 and Flk1 expression. (G) FACS analyses of ISL1+ cells in EBs 6 days after Sirt1 knockdown. Left panels, histograms of ISL1+ cells. Right panel, quantification of ISL1+ cells. *P < 0.05, t test (n = 3). (H) ChIP analysis of SIRT1 binding to Isl1 (positions –468 to –285) and Nkx2.5 promoters (positions –9040 to –8859) in embryonic hearts after induction of hypoxia responses. *P < 0.05, t test (n = 3). (I) Luciferase reporter assays of the proximal Isl1 promoter with WT and H633Y mutant SIRT1. *P < 0.05; **P < 0.01, ANOVA with Dunnett’s post hoc correction (n = 3). (J) ChIP analysis of H3K9ac and H4K16ac at the Isl1 promoter in NKX2.5–EmGFP+ cells under hypoxia conditions. Enrichment of H3K9ac and histone H4K16ac was normalized to histone H3. **P < 0.01; ***P < 0.001, t test (n = 3).