Figure 3. Hyperglycemia alters chromatin accessibility, TF occupancy, and NO production.

(A) Loss of ATAC-seq peaks in HG- (25 mM) compared with NG-treated (5.5 mM) atrioventricular mesenchymal (AVM) cells. Heatmap generated from HG normalized to NG input, with peaks centered, including 1.5 kb upstream and downstream of the transcriptional start site. Red and blue represent low and high signal intensity, respectively. (B) Distribution of 49,035 differential peaks. Pie chart highlights distribution of peaks within promoter (33%), inside gene (17%), upstream (31%), and downstream (19%) regions. Histogram displays frequency of peaks at a given distance from TSS. (C) Volcano plot highlights fold change and significance of identified peaks as plotted by –log10(P value) versus fold change. (D) Integrative genome viewer tracks of normalized merged data sets, highlighting peak loss at Nos3 loci in HG. Square boxes highlight 3 regions (R1–R3) with relative chromatin closure in HG. (E) ChIP-qPCR with H3K27ac showed significant downregulation of activation marks at Nos3R1–R3 (n = 4). (F and G) Decreased expression of Nos3 in HG at mRNA and protein levels (n ≥ 5) by IF respectively. Measurement of NO (H and I) and superoxide (J and K) production in AVM cells maintained in NG and HG for 48 hours, probed with diaminorhodamine (DAR-4M AM) and dihydroethidium (DHE), respectively. (L and M) DAR-4M AM and (N and O) DHE staining of E13.5 murine hearts exposed to nondiabetic and diabetic environments demonstrates increased ROS and reduced NO with diabetes (n = 3 per group). Nuclear DAPI stain, blue; atrioventricular cushions (yellow arrows); LV, left ventricle; RV, right ventricle; IVS, interventricular septum. Mean ± SEM; *P < 0.05, 2-tailed Student’s t test in F and G, with Holm-Bonferroni correction in E. Scale bars: 50 μm (G–K); 100 μm (L–O).