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. Author manuscript; available in PMC: 2021 Oct 1.
Published in final edited form as: Cell Stem Cell. 2020 Aug 17;27(4):574–589.e8. doi: 10.1016/j.stem.2020.07.015

Figure 6. HLHS DNMs altered endocardial and myocardial functions through transcriptional regulation of endocardial genes and FN1.

Figure 6.

(A) qPCR detection of HLHS DNM genes in iEECs from control (n=3) and HLHS patients (n=3). Red denotes high expression, blue minimal expression. Genes not shown mean non-detectable in iEECs. Genes highlighted by red are transcription factors or chromatin modifiers. qPCR validation of the endocardial gene expression (B) and FN1 (C) in primary human fetal endocardial cells after ETS1 or CHD7 knock-down, respectively. (D) Left: RNAPII and histone marker ChIP-seq tracks of the NPR3 gene in HUVECs from the ENCODE. The black box and red shade indicate the designated location of primers for ChIP-qPCR. The red arrow indicates the transcriptional start site (TSS) and direction of transcription. Right: ChIP-qPCR of ETS1 or CHD7 binding to the NPR3 promoter region in control and HLHS iEECs. (E) Left: Histone ChIP-seq tracks of the FN1 gene in HUVECs from the ENCODE. Right: ChIP-qPCR for ETS1 or CHD7 to the FN1 promoter (a) and enhancer (b) regions. *p<0.05, **p<0.01, control (n=4) vs HLHS (n=3). Data are represented as mean ± SEM. (F) EndoMT-related gene expression in endocardial cells after ETS1 knock-down. (G) iPSC-CMs were co-cultured with isolated fetal left heart endocardial cells with or without ETS1 knock-down. Expression of genes related to cardiomyocyte maturation (G) and ion channel (H) in iPSC-CMs were examined by qPCR. (I) Immunostaining of Ki67 (green) and TNNT2 (red) in iPSC-CMs from (G). DAPI: nucleus. (J) qPCR for proliferative genes in iPSC-CMs from (G). (K) Illustration of control iPSC-CM and iEEC co-culture experiments with ETS1 overexpression (5 multiplicity of infection-MOI, 48 hr) in HLHS iEECs (HLHS+ETS1). Contraction velocity measurement (L) and sarcomere organization (M) on day 30 iPSC-CMs from (K). (O) Ki67 positive iPSC-CMs on day 15 iPSC-CMs from (K). DAPI: nucleus; Green: Ki67 positive nucleus; Red: TNNT2 positive cardiomyocytes. qPCR quantification of gene expressions related to myocardial maturation (N) and proliferation (P). n=3 patients in each treatment group. Data shown as the mean ± SEM. *p<0.05, **p<0.01, scramble vs. siRNA knockdown or HLHS vs. HLHS+ETS1. See also Figure S6.