Fig 1: Molecular dysregulation in SHF underlies matHG-mediated risk of CHD.a–f Co-immunofluorescence staining shows Hand2 and Nkx2-5 (red, shown in insets) protein expression overlaying with GFP expression (green) driven by Isl1 in E9.5 hearts subjected to CNTRL and matHG conditions. The GFP+Hand2+ cells in E9.5 OFT in CNTRL and matHG-exposed embryos are shown in aii, bii (yellow arrowheads). GFP+Nkx2-5+ cells were shown in E9.5 OFT and RV-CM exposed to CNTRL and matHG environment (dii, diii, eii, eiii, yellow arrowheads). The relative fluorescence intensities of GFP+Hand2+ and GFP+Nkx2-5+ quantified in (c) (p value = 0.0005), and (f) (p value = 0.0121). Nuclei stained with DAPI are shown in blue. g, h Co-immunofluorescence staining of Mef2c (red) and Tnnt2 (green) protein expression in CNTRL vs. matHG-exposed E9.5 wt hearts. Yellow and white solid lines in (g) indicate distal and proximal OFT. White dotted lines in gii and hii indicates high magnification images shown in giii and hiii, respectively. Nuclei stained with DAPI are shown in blue. (i, j) Mef2c (red) and GFP (green) protein expression shown in E9.5 Isl1Cre+; RosamTmG/+ (mG) hearts exposed to CNTRL and matHG environment. Yellow arrowheads and dotted lines indicate the downregulation of Mef2c in the OFT. The relative fluorescence intensities of Mef2c+ and GFP+Mef2c+ cells quantified in (k) (p value = 0.0151 and p value = 0.003, respectively). n = 3 embryos/group in which multiple sections from each were used for quantification. Statistical comparisons made between CNTRL and matHG groups by unpaired t-test with Welch’s correction using GraphPad Prism 9. Data presented as mean ± SEM and * Indicates two-tailed p value <0.05. l The proposed “matHG induced CHD model” shows molecular dysregulation in Isl1-GRN components leads to impairments in CM differentiation and increases the risk of CHD (schematics created with BioRender.com). A yellow asterisk indicates ventricular septal defects observed in matPGDM exposed embryos. CNTRL control, matHG maternal hyperglycemia, OFT outflow tract, RV right ventricle, AS aortic sac, CM cardiomyocytes, CHD congenital heart defect, GRN gene regulatory network, PGDM pregestational diabetes mellitus. Scale bars: 100 µm (ai, bi), 20 µm (aii–iii, bii–iii, di–iii, ei–iii), and (gi–ji, gii–jii and giii–jiii): 50 µm.
Fig 2: CASZ1 is regulated by noradrenergic NB CRC components.A Results of analysis of publicly available ChIP-seq data of the CRC components show that the CRC components bind to the CASZ1 gene locus, with a relatively stronger signal of CRC components within the 2nd intron (red box) but a lower signal of H3K27ac. Bivalent mark of H3K27me3 and H3K4me3 is observed at the CASZ1 gene promoter in SY5Y cells (black box). B Realtime PCR shows that the siRNA knockdown of MYCN and CRC compoents (left panel), and the loss of HAND2 or TBX2 results in a decrease of CASZ1 mRNA levels (right panel). C ChIP-seq results show that decreasing expression of HAND2 in IMR32 cells results in a decrease of HAND2 signal within CASZ1 gene locus, which is accompanied by an increase of H3K27ac signal within the 2nd and 3rd intron (red and pink boxes), as well as an increase of H3K4me3 signal and a decrease of H3K27me3 signal within the CASZ1 promoter.
Fig 3: MatHG impairs cardiomyocyte proliferation and lineage specifying transcription factors.a–h Immunofluorescent co-staining of PHH3 (red) and GFP (green) show Isl1-derived CM proliferation in CNTRL vs. matHG exposed E9.5 and E11.5 Cre- (mT) vs. Cre+ (mG) cardiac tissues (n = 4 embryos per timepoint/maternal condition). The GFP+PHH3+ cells are quantified in (i), presented on a log10 scale. Nuclei stained with DAPI are shown in blue. Data presented at mean ± SEM. Statistical comparisons made between CNTRL and matHG exposed E9.5 (p value = 0.072) and E11.5 (p value = 0.002) embryos by unpaired t-test using GraphPad Prism 9, ns non-significant and * indicates two-tailed p value < 0.05. j Cell count and regression analysis on CM subtypes (OFT, AVC, Atr and Ven-CM) using Seurat reveal count in cell-cycle phases (G1, S, and G2/M) in E9.5 and E11.5 embryonic hearts exposed to CNTRL vs. matHG. k–n Representative fluorescence images show the endogenous GFP expression in CNTRL and matHG exposed E9.5 and E11.5 Isl1Cre+; RosamTmG/+ (mG) embryos and in microdissected hearts. o Schematics show whole hearts dissociated into single cells by enzymatic digestion. GFP+ and GFP- cells were sorted using FACS and relative gene expression levels (Log10 fold change) measured by SYBR green-based qRT-PCR analysis. p Comparisons between CNTRL GFP+ vs. GFP- cells and matHG GFP+ vs. GFP- cells show enrichment of GFP and other SHF/CM markers (Isl1, Tbx1, Fgf10, Mef2c, Tbx20, Hand2, Nkx2.5, Myl2 and Cited2). Colors indicate gene names. Comparative gene expression analysis was performed in CNTRL GFP+ vs. matHG GFP+ cells (Isl1-derived cells) at the E9.5 and E11.5 stages. At least three independent embryos/timepoint/maternal diabetic status were used for qRT-PCR analysis. Data presented as Log10 (fold change) normalized to endogenous Gapdh. Statistical comparisons between groups were made in GraphPad Prism 9. CNTRL control, HG hyperglycemia, A atria, V ventricle, LV left ventricle, RV right ventricle, AVC atrioventricular canal, IVS interventricular septum, OFT outflow tract, FACS Fluorescence-Activated Cell Sorting, qRT-PCR quantitative real-time polymerase chain reaction, SHF second heart field, CM cardiomyocytes. Scale bar: 50 µm (ai–di, aii–dii), 100 µm (ei–hi, eii–hii), 100 µm (ki, li), 50 µm (kii–lii), 1 mm (mi, ni), and 200 µm (mii, nii).
Fig 4: Kaempferol and apigenin did not increase HAND2 expression in mouse uteri. Immunohistochemical staining against progesterone receptor target HAND2 on uterine cross sections of control- (A), progesterone- (B), kaempferol- (C,D) apigenin-treated mice. Scale bar = 100 µm; magnification 20×. (E) Quantification of HAND2 expression per treatment in representative images. One-way ANOVA test was used to determine significance (* p < 0.05).
Fig 5: HAND2-AS1 modulates DNA methylation in HAND2 promoter in human endometrial stromal cells. A, Human endometrial stromal cells were cultured in presence of 10 nM of E and 1 μM of P with 15 μM of 5-aza-2′-deoxycytidine (AZA+) or vehicle control (AZA-) for 3 days. Total RNAs were isolated and subjected to qRT-PCR to assess gene expression. The relative levels of HAND2, HAND2-AS1, and IL15 expression compared to AZA− controls after normalization to internal control genes are shown. *P < .001, n = 3. B, Human endometrial stromal cells were transfected with HAND2-AS1-specific siRNA oligo (HAND2-AS1 siRNA) or nonspecific siRNA control (NC-siRNA), and then cultured in presence of 10 nM of E and 1 μM of P for 3 days. Genomic DNAs were purified from these cells and subjected to digestion with DNA methylation sensitive and/or dependent enzymes, followed by qRT-PCR using primers specific to HAND2 bidirectional promoter. The unmethylated and methylated human genomic DNA were served as the negative (NC) and positive controls (PC), respectively. *, P < .01 (n = 3).
Supplier Page from Abcam for Anti-HAND2 antibody [EPR19451]