High-Resolution Mapping of Chromatin Conformation in Cardiac Myocytes Reveals Structural Remodeling of the Epigenome in Heart Failure

Abstract

Background: Cardiovascular disease is associated with epigenomic changes in the heart; however, the endogenous structure of cardiac myocyte chromatin has never been determined.

Methods: To investigate the mechanisms of epigenomic function in the heart, genome-wide chromatin conformation capture (Hi-C) and DNA sequencing were performed in adult cardiac myocytes following development of pressure overload-induced hypertrophy. Mice with cardiac-specific deletion of CTCF (a ubiquitous chromatin structural protein) were generated to explore the role of this protein in chromatin structure and cardiac phenotype. Transcriptome analyses by RNA-seq were conducted as a functional readout of the epigenomic structural changes.

Results: Depletion of CTCF was sufficient to induce heart failure in mice, and human patients with heart failure receiving mechanical unloading via left ventricular assist devices show increased CTCF abundance. Chromatin structural analyses revealed interactions within the cardiac myocyte genome at 5-kb resolution, enabling examination of intra- and interchromosomal events, and providing a resource for future cardiac epigenomic investigations. Pressure overload or CTCF depletion selectively altered boundary strength between topologically associating domains and A/B compartmentalization, measurements of genome accessibility. Heart failure involved decreased stability of chromatin interactions around disease-causing genes. In addition, pressure overload or CTCF depletion remodeled long-range interactions of cardiac enhancers, resulting in a significant decrease in local chromatin interactions around these functional elements.

Conclusions: These findings provide a high-resolution chromatin architecture resource for cardiac epigenomic investigations and demonstrate that global structural remodeling of chromatin underpins heart failure. The newly identified principles of endogenous chromatin structure have key implications for epigenetic therapy.

Keywords: epigenomics; genomics; heart failure; hypertrophy.

Figure 1.

Loss of CTCF induces cardiac pathology. A, Echocardiography measurements of ejection fraction (EF) and left ventricular internal diameter at diastole or systole (LVIDd or LVIDs) demonstrate impairment of EF and chamber dilation in CTCF-KO (purple) and TAC (red) in comparison with control (CTRL; green). Heart weight to body weight ratio (HW/BW) indicates cardiac hypertrophy in CTCF-KO and TAC mice (n≥25/group; *P<0.01. Student’s t test). B, Picrosirius red staining shows fibrosis after CTCF depletion or TAC (n=3/group). C, Top, mean cardiomyocyte area (n=20 visual fields of wheat germ agglutinin–stained sections across 3 mice per condition); Bottom, quantitation of fibrosis from Picrosirius red sections (n=3/group; *P<0.01. Tukey HSD test). D, Stress response gene expression (log₁₀(FPKM+1)). E, Real-time qPCR measurements of CTCF levels in human myocardium before and after LVAD (before values normalized to 1 on a per-patient basis; n=4, *P<0.01 Student’s t test, bars SD). F, Western blots of CTCF levels in individual patients before and after LVAD (Left); quantitation of Western blots normalized to actin on a per-patient, per-sample basis (Right). G, Left ventricular end-diastolic dimension measurements before and after LVAD. Color-coding in E through G indicates separate patients. Lines shifted horizontally in G for ease of viewing. FPKM indicates fragments per kb of exon per million mapped reads; HSD, honest significant difference; KO, knockout; LVAD, left ventricular assist devices; qPCR, quantitative polymerase chain reaction; and TAC, transverse aortic constriction.

Figure 2.

High-resolution cardiac chromatin conformation analyses reveal changes in chromatin compartmentalization and gene expression in pressure overload and CTCF-KO mice. A, Structure of topological associating domains (TADs) is revealed from contact frequency heatmaps showing cis-interaction profile on example chromosome 5 for control, TAC, and CTCF-KO chromatin. B, Strength of boundaries between TADs are displayed for all chromosomes comparing control and TAC (red, higher; blue, lower; Insets, example region on chromosome 10 in control versus TAC and control versus CTCF-KO; Figure VIIA in the online-only Data Supplement shows control versus CTCF-KO for all chromosomes). C, Quantitation of insulation score differences in control versus KO (Left) or TAC (Right). Colored dots indicate significant changes (gray dots show range of variation between 2 control conditions: untreated wild-type mouse and untreated Cre^+/– mouse). D, A/B compartmentalization, an indicator of genome accessibility at individual loci, for an example region on chromosome 5 is plotted in blue (open, A) and yellow (closed, B): CTRL A/B status on Top, CTCF-KO in Middle, and TAC on Bottom. E, Quantification of the genome-wide changes in A/B compartment change with CTCF depletion (Left) or TAC (Right). Bottom highlight only bins that change compartment; dark and light colors represent up- or downregulated genes, respectively. F, Relationship of compartmentalization to gene expression is measured in TAC or CTCF-KO hearts. Log₂(fold-change) of FPKM for the differentially expressed genes that either remain in the same compartment or that change compartments with CTCF depletion (Top) and TAC (Bottom). P values via Wilcoxon test; whiskers indicate interquartile range. G, Heat matrix (Top) showing number of differentially expressed genes between CTRL, CTCF KO, and TAC (intensity indicates number of genes). Venn diagram (Bottom) showing overlap of differentially expressed genes between CTRL versus CTCF KO and CTRL versus TAC. H, Heatmap depicting log₂(KO/CTRL) and log₂(TAC/CTRL) FPKM for the differentially expressed genes with q<0.01 (Left). CTRL indicates control; FPKM, fragments per kb of exon per million mapped reads; KO, knockout; and TAC, transverse aortic constriction.

Figure 3.

Short- and long-range chromatin interactions, and stable chromatin looping, are altered after pressure overload or CTCF-KO. A, Schematic displaying that loops are demarcated by 2 anchors and contain regions of high-frequency interactions, indicated by the colored circles. B, The bioinformatic tool Juicebox is used to display an example loop that is lost with CTCF depletion (Middle) and TAC (Right). C, Quantitation of the phenomenon in B across genome, showing number of chromatin loops (Left), number of genes within loops (Middle), and loop sizes (Right; CTRL, green; CTCF-KO, purple; TAC, red). D, Overlap of loops only appearing in CTCF-KO or TAC in comparison with CTRL (Left); overlap of loops that disappear in CTCF-KO and TAC (Right). E, CTRL loops in which zero (green), one (gray), or both (yellow) anchors overlap with a CTCF peak. F, CTRL loops that lose ≥1 CTCF peak during CTCF depletion (blue) and CTRL loops that keep both CTCF peaks during CTCF depletion (red). Darker shade indicates loops that were preserved with CTCF depletion, whereas lighter color indicates loops that were lost. G, Schematic demonstrating types of alterations in looping architecture that can occur, including loss of loops mediating enhancer-promoter interactions (Top), no change (Middle), or formation (Bottom). Enhancers are orange, promoters green, and genes blue. H, Quantification of changes in enhancer-promoter loops. CTRL indicates control; KO, knockout; and TAC, transverse aortic constriction.

Figure 4.

Chromatin architecture is remodeled around cardiac genes during disease. A, As an example cardiac disease gene with changing long-range, intrachromosomal contacts, the sample-specific interactions emanating from Ppp3ca in control and after CTCF depletion or TAC are shown (q<0.01; 40-Kb resolution; outer circle, chromosome position; black, mm10 genes). B, Examining gene expression data for all 5443 genes with shared interaction behavior between CTCF-KO and TAC, 3651 genes were found to be differentially expressed in the same direction in perturbations in comparison with control. Most (86%) of these gene expression changes were associated with decreased chromatin interactions (green colors), with 1662 upregulated (dark green) and 1504 downregulated (light green). The remainder of the interaction changes (14%) were distributed between the other possible scenarios: increased interactions and expression (193, dark blue), increased interaction and decreased expression (154, light blue), no change in interactions and either an increase (71, dark orange) or decrease (67, light orange) in expression. C, Heatmap showing the number of significant (q<0.01) interactions overlapping with differentially expressed genes; top 40 shared differentially expressed genes shown, sorted by number of significant interactions in CTRL (gene name labeling shows direction of expression, where green is up and red is down). D, Higher-resolution mapping of local neighborhood interactions for the example genes Nppa/Nppb, Kcnd2, and Mef2c gene loci +/–1 Mb (q<0.01). Lines revealing precise contact sites are color coded by q-value significance, with red being the most significant. RNA-seq tracks depict gene expression for CTRL (green), CTCF-KO (purple), and TAC (red). CTRL indicates control; KO, knockout; and TAC, transverse aortic constriction.

Figure 5.

Three-dimensional interactions between enhancers and genes are restructured after pressure overload or CTCF-KO. A, Number of contact sites per cardiac enhancer are quantified (q<0.01; rows are top 50 enhancers sorted by number of interactions). B, Top, enhancers in which the number of significant (q<0.01) interactions (determined by the Fit-Hi-C tool) increases (blue), decreases (red), or remains the same (gray) with CTCF-KO (Left), TAC (Center), and with consistent changes with CTCF-KO or TAC (Right). Darker shading indicates enhancers that interact with genes; lighter colors are regions not annotated as coding. C, Number of genes that interact with enhancers from B, stratified by whether their expression is upregulated (green) or downregulated (red). Groupings are separated into enhancers whose number of overlapping interactions decreases (Left), increases (Middle), or remains the same (Right) after perturbation. D, Contact site mapping of interactions is shown for the example gene Rock2, which exhibits decreased interaction with enhancers in CTCF-KO and TAC, concomitant with decreased expression. CTRL indicates control; KO, knockout; and TAC, transverse aortic constriction.

Figure 6.

Model for epigenomic changes in development and disease. Development is accompanied by changes in chromatin to endow terminally differentiated cells with stable transcriptomes. Disease upsets this balance, transitioning select regions of the genome into more dynamic conformations through effects on chromatin structure, enhancer-gene looping, histone modifications, DNA methylation, and other factors. This model is based on findings from this article and previous publications^{–,,,–,–} as described in the text. NcRNA indicates noncoding RNA; TAD, topological associating domain; and TF, transcription factor.