Disruption of spatiotemporal hypoxic signaling causes congenital heart disease in mice
Xuejun Yuan, Hui Qi, Xiang Li, Fan Wu, Jian Fang, Eva Bober, Gergana Dobreva, Yonggang Zhou, Thomas Braun
Xuejun Yuan, Hui Qi, Xiang Li, Fan Wu, Jian Fang, Eva Bober, Gergana Dobreva, Yonggang Zhou, Thomas Braun
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Research Article Cardiology

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 6

ISL1 represses Nkx2.5 transcription by recruitment of HDACs.

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ISL1 represses Nkx2.5 transcription by recruitment of HDACs. (A) ChIP an...
(A) ChIP analysis of ISL1 binding to the Nkx2.5 promoter. ***P < 0.001, t test (amplicons a–c, n = 4; amplicon d and ISL1, n = 3). Both the HRE motif and the putative ISL1-binding site are located at –9040 to –8859 (amplicon b). (B) Quantification of NKX2.5hi cells following in vitro differentiation of ISL1+ cells after treatment with HDAC inhibitors. *P < 0.05; ***P < 0.001, ANOVA with Dunnett’s post hoc correction (control, n = 6; Ex527, n = 5; Ms275 and MC1568, n = 3; TMP269, n = 5). (C) Co-IP analysis of the interaction of ISL1 with different HDACs in HEK293T cells after transfection. Two independent experiments were performed, generating similar results. (D) Sequential Co-IP analysis of the interaction of ISL1 with HDAC1 and HDAC5 in HEK293T cells. (E) ChIP analysis of HDAC1 and HDAC5 binding to the ISL1 site in Nkx2.5 promoter. **P < 0.01, ANOVA with Dunnett’s post hoc correction (amplicons a, c, d, ISL1, n = 3; amplicon b, n = 6). (F) ChIP analysis showing HDAC1 and HDAC5 binding to the Nkx2.5 promoter (positions –9040 to –8859) after lentiviral-mediated expression of ISL1 shRNA. **P < 0.01, t test (n = 3). (G) ChIP analysis showing ISL1 and HIF1α binding to the Nkx2.5 promoter (positions –9040 to –8859) after inhibition of SIRT1 enzymatic activity by Ex527 (1 μM). Note that enzymatic inhibition of SIRT1 enhances binding of ISL1, but reduces binding of HIF1α. *P < 0.05; ****P < 0.0001, t test (n = 3).