Abstract 96: Epigenetic And Transcriptional Regulation Of Induced Pluripotent Stem Cells In Patients With Hypoplastic Left Heart Syndrome

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Abstract

Background: Although a number of studies have uncovered heterozygous mutations in cardiac regulatory genes caused hypoplastic left heart syndrome (HLHS), the identified genetic variants may not be directly correlated with the disease development. The aim of this study is to determine the epigenetic and transcriptional network responsible for myocardial patterning and morphogenesis during cardiac development in HLHS by using patient-derived induced pluripotent stem (iPS) cells.

Methods: Five-independent iPS cell lines were generated from HLHS and biventricle (BV) heart-derived cardiac progenitor cells (CPCs). Global gene expression analysis, real-time RT-PCR, mutation analysis, ChIP assay, and cardiac-specific gene promoter activities were examined during differentiation.

Results: We found one synonymous single nucleotide polymorphism in NKX2-5 and five in NOTCH1, respectively. Cardiac transcriptional factors such as NKX2-5, HAND1, and NOTCH/HEY, those are known to drive primary heart field and outflow tract development, were significantly reduced in HLHS-derived iPS cells after differentiation compared with BV- and control 201B7 iPS-derived cardiomyocytes. ChIP assay showed that a marked decrease in dimethylated histone H3-lysine 4 and acetylated histone H3 was found within the NKX2-5 promoter regions, whereas a significantly increased trimethylated H3-lysine 27 was identified in differentiated HLHS-derived iPS cells. To specify the target transcripts responsible for cardiac development of HLHS, cardiac troponin-T and natriuretic peptide A promoter analyses were performed. We found that both promoter activities were significantly suppressed in HLHS-derived CPCs and iPS cells compared with those from BV hearts. These repressed promoter activities could be fully restored by transient transfection of NKX2-5, HAND1, and NOTCH1 genes into these stem/progenitor cells by synergistic manner.

Conclusions: These findings suggest that patient-specific iPS cells provide a potential to dissecting the complex cardiac malformations in human. The epigenetic and transcriptional regulation of NKX2-5, HAND1, and NOTCH1 may mutually participate in the development of myocardial growth, patterning, and morphogenesis in HLHS.

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