Abstract 11840: Disease-Specific Induced Pluripotent Stem Cells Identify the Transcriptional Repression and Epigenetic Modification of NKX2-5, HAND1, and NOTCH1 During Cardiac Development of Hypoplastic Left Heart Syndrome
Backgrounds: Although heterozygous mutations have been reported in patients with hypoplastic left heart syndrome (HLHS), no genes have been found to specifically cause this syndrome. The aim of this study is to investigate the complex network of transcriptional regulation and epigenetic modification during the cardiac development of HLHS by using patient-specific induced pluripotent stem (iPS) cells.
Methods: Cardiac progenitor cells (CPCs) were isolated and four-independent iPS cell lines were generated from HLHS- and bi-ventricular (BV) heart-derived CPCs. Real-Time RT-PCR, exome sequencing, ChIP assay, and luciferase reporter assay were performed and compared with human embryonic stem and 201B7 iPS cells as controls.
Results: HLHS-derived iPS cells could give rise to cardiomyocytes with significantly lower expression of cardiac troponin-T (TNNT2), NKX2-5, HAND1/2, NOTCH1-HEY1/2 and TBX2 when compared with those from 201B7 and BV-derived iPS cells. To determine the target transcripts responsible for cardiac development of HLHS, luciferase reporter analyses of serum response element (SRE), TNNT2, and natriuretic peptide A (NPPA) were examined. We found that transcriptional activation of SRE, TNNT2, and NPPA was significantly suppressed in HLHS-derived CPCs and iPS cells compared with those from BV heart. No mutations in NKX2-5, HAND1, and NOTCH1 were identified in patients analyzed; however, repressed promoter activities of SRE and TNNT2 in HLHS-derived CPCs and iPS cells could be fully restored by transient transfection of these three transcripts by synergetic fashion. Notably, the transcriptional activation of NPPA was controlled by NKX2-5-dependent mechanism and loss-of-function studies by shRNA confirmed these observations. In addition, ChIP assay showed a marked decrease in histone enhancer marks such as H3K4me2 and acH3 but gain of repressive mark H3K27me3 on NKX2-5 promoter in differentiated HLHS-derived iPS cells.
Conclusions: These findings suggest that patient-specific iPS cells may provide molecular insights into complex transcriptional and epigenetic mechanisms, at least in part, through combinatorial expression of NKX2-5, HAND1, and NOTCH1 that coordinately contribute to cardiac malformations in HLHS.
Author Disclosures: J. Kobayashi: None. M. Yoshida: None. S. Tarui: None. S. Ishigami: None. M. Okuyama: None. Y. Nagai: None. S. Kasahara: None. K. Naruse: None. H. Ito: None. S. Sano: None. H. Oh: None.
- © 2014 by American Heart Association, Inc.