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Tbx5 maintains atrial identity in postnatal cardiomyocytes by regulating an atrial-specific enhancer network

An Author Correction to this article was published on 31 October 2023

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Abstract

Understanding how the atrial and ventricular heart chambers maintain distinct identities is a prerequisite for treating chamber-specific diseases. In this study, we selectively knocked out the transcription factor Tbx5 in the atrial working myocardium to evaluate its requirement for atrial identity. Atrial Tbx5 inactivation downregulated atrial cardiomyocyte (aCM)-selective gene expression. Using concurrent single-nucleus transcriptome and open chromatin profiling, genomic accessibility differences were identified between control and Tbx5 knockout aCMs, revealing that 69% of the control-enriched ATAC regions were bound by TBX5. Genes associated with these regions were downregulated in knockout aCMs, suggesting that they function as TBX5-dependent enhancers. Comparing enhancer chromatin looping using H3K27ac HiChIP identified 510 chromatin loops sensitive to TBX5 dosage, and 74.8% of control-enriched loops contained anchors in control-enriched ATAC regions. Together, these data demonstrate that TBX5 maintains the atrial gene expression program by binding to and preserving the tissue-specific chromatin architecture of atrial enhancers.

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Fig. 1: Inactivation of Tbx5 in aCMs results in atrial remodeling.
Fig. 2: Inactivating Tbx5 alters the expression of aCM-selective genes.
Fig. 3: Concurrent scRNA-seq and scATAC-seq analysis of cell states in control and Tbx5AKO atria.
Fig. 4: TBX5 is required to promote the expression of aCM genes.
Fig. 5: Multiomics reveals a set of Tbx5-dependent CREs that promote aCM gene expression.
Fig. 6: TBX5 maintains local chromatin structure to regulate gene expression.
Fig. 7: TBX5 regulates enhancer activity, accessibility and looping at aCM-selective genes Myl7 and Bmp10.
Fig. 8: TBX5 promotes atrial identity.

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Data availability

High-throughput data used in this manuscript are available from the Gene Expression Omnibus, accession number GSE222970. Other datasets used were obtained from GSE215065 (ref. 3), GSE129503 (ref. 17) and GSE195905 (ref. 52). All other data supporting the findings in this study are included in the main article and associated files. Source data are provided with this manuscript.

Code availability

Analysis was performed using standard R packages. No custom analysis software was created to perform these analyses. Analyses were performed using published R packages that are cited within the text and the Methods section.

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Acknowledgements

M.E.S. and M.T. were supported by T32HL007572 and F32 F32HL163877. W.T.P. was supported by R01HL156503. Y.C., F.L. and P.W. were supported by AHA Postdoctoral Fellowships. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

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Contributions

M.E.S. and Y.C. contributed equally to this study. M.E.S. and Y.C. conceived of the study; designed and performed the experiments; and analyzed the data. X.Z., C.P.-C. and K.A. contributed to the data analysis. O.B.-T., B.N.A., Q.M., E.K., H.W., J.M.G., L.H., M.K.S., M.A.T., P.W., F.L., M.G., M.P. and R.H.B. contributed data, reagents and analyses. V.B., K.C., J.G.S., C.E.S., I.P.M. and W.T.P. oversaw the project and provided resources. M.E.S. and W.T.P. wrote the manuscript, with input from Y.C. and the other authors.

Corresponding author

Correspondence to William T. Pu.

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Nature Cardiovascular Research thanks Vincent Christoffels and the other, anonymous, reviewer(s), for their contribution to the peer review of this work. Primary Handling Editor: Vesna Todorovic, in collaboration with the Nature Cardiovascular Research team.

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Extended Data

Extended Data Fig. 1 Characterization of the cardiac expression domain of AAV9:Nppa-EGFP.

a-b, Hcn4CreERT2/+; Rosa26LSL-Tomato pups were injected with 2 X 1011 viral genomes per gram bodyweight (VG/g) AAV9:Nppa-EGFP at P8 and injected with tamoxifen at P21 and P22 to activate tomato expression in the conduction system including the sinoatrial and atrioventricular nodes. Hearts were harvested in PBS and brightfield, GFP, and Tomato fluorescent images were acquired. Mutually exclusive GFP and tomato signals were observed in the atrium and the SA node, respectively. A similar result was observed with administration at P2. c, Hearts were sectioned and stained with WGA. Tomato signal marked the sinoatrial node (SAN) and atrioventricular node (AVN) but not the working myocardium. GFP was restricted to the working myocardium. A similar staining pattern was observed in 4 other littermates.

Extended Data Fig. 2 Phenotypic characterization of Tbx5AKO mice.

a, Trichrome staining of Tbx5Flox/Flox and Tbx5AKO hearts. b, RT-qPCR quantification of Postn (periostin) and Col1a1 (collagen type 1 alpha 1 chain), two indicators of fibrosis (n = 3 control and 4 KO mice). Unpaired two-sided t-test. Error bars represent mean values +/-SEM. c-d, Immunostaining for sarcomeric α-actinin (SAA) or FSD2, markers of the Z line and the junctional Sarcoplasmic reticulum, respectively, in the left atrium of the indicated genotypes. Localization of both proteins is disrupted in Tbx5AKO atria. e, Pattern of SAA signal intensity. SAA intensity along the long axis of cardiomyocytes demonstrated a periodic signal in control, consistent with regular position of sarcomere Z-lines, and loss of periodicity in Tbx5AKO. f, Preserved ventricular function of Tbx5AKO mice. Tbx5Flox/Flox mice were treated with AAV:Nppa-EGFP (control) or AAV:Nppa-Cre (Tbx5AKO) at P2. Echocardiography was performed at P20. LVID;d, left ventricular internal diameter at end diastole. EF, ejection fraction. n = 5 control and 4 KO mice. Graphs show mean ± SEM. g, Surface EKG recordings. Orange and black bars highlight successive RR intervals. h, Poincaré plots. The RR interval of greater than 1500 beats on EKG recordings is plotted versus the RR interval of the subsequent beat (RR[ + 1]). to visualize the dispersion of interbeat intervals in Tbx5AKO, consistent with atrial fibrillation. i, Standard deviation of the RR interval, a measure of heart rate irregularity, was calculated for at least 1500 beats for each group at P21. n = 4 control and 9 KO mice. Unpaired two-sided t-test: ***, P = 0.005. Graphs show mean ± SEM. j, Time course of heart rate irregularity. Serial EKGs were acquired from control or Tbx5AKO mice at the indicated time points. SDRR was measured and compared between groups at each time point using a two-way ANOVA with Sidek’s multiple comparison test. **, P < 0.01. ***, P < 0.001. For P14 and P17 timepoints, n = 3 control and 3 KO mice. For P8 and P21 timepoints, n = 5 control and 8 KO mice. Graphs show mean ± SEM. k, Simultaneous intracardiac and surface ECG recordings demonstrate normal synchronous atrial-ventricular rhythm in Tbx5Flox/+ mice injected with AAV-Nppa-Cre. l, In contrast, animals with complete atrial ablation of Tbx5 (AAV-Nppa-Cre + Tbx5flox/flox) demonstratee nearly continuous low-amplitude atrial activity and irregularly irregular ventricular response consistent with atrial fibrillation.

Extended Data Fig. 3 Tbx5 overexpression atrializes ventricular myocytes.

a, Strategy to generate TBX5-OE ventricles. A full length Tbx5 cDNA downstream of the ubiquitous CAG promoter was activated by the cardiomyocyte specific Myh6-Cre transgene. b-c, Volcano plot comparing the change in gene expression of mouse left ventricle overexpressing Tbx5 compared to control LV. aCM genes are marked in red in (a) and vCM genes are denoted in blue in (b). b-c, Wald’s test followed by Benjamini Hochberg correction. d-e, Fisher’s exact test (two tailed) was performed to determine if changes in chamber selective gene expression downstream of Tbx5 overexpression were significant. f-h, Staining for MYL4, MYL7, and MYL2 in atria and ventricles of control and TBX5-OE hearts. Staining for each marker was performed using 3 control hearts and 2 TBX5-OE hearts, and representative images are shown.

Extended Data Fig. 4 Single cell dataset metrics.

a, Parameters from single nucleus datasets. Mean values per nucleus: nCount_ATAC, number of ATAC fragments; nFeature_ATAC, number of ATAC peaks with at least one read; nCount_RNA, number of RNA fragments; and nFeature_RNA, number of genes. b, Transcription start site (TSS) aggregation plots for the scATAC multiome datasets showing the expected enrichment of ATAC fragments. c, WNN UMAP of the dataset split by original sample. Each of the KO and control replicates have a high degree of overlap, demonstrating high reproducibility of cell state changes in Tbx5AKO atria.

Extended Data Fig. 5 Differentially expressed genes between myocyte clusters.

a, Volcano plot of differentially expressed genes (DEGs) between early and late pseudotime clusters in the control trajectory (Myocyte_4 vs. Myocyte_1, left) and the KO trajectory (Myocyte_5 vs. Myocyte_6; right). Wilcoxon rank sum test, Bonferroni correction. b. MA plot of RNA-seq experiment comparing P0 and P28 aCMs. c-d. Volcano plots shown in (a) overlayed with genes enriched in P28 and P0 aCMs. Wilcoxon rank sum test, Bonferroni correction. e. The proportion of DEGs from comparisons in (a) that overlap genes selectively expressed in aCMs at P0 and P28. We did not observe enrichment of P0 or P28 selective aCM genes in early or late pseudotime clusters, respectively. This suggested that pseudotime trajectories did not correspond to chronological time. f-g. GO biological process terms enriched for DEGs for the comparisons shown in (a). The top 10 terms enriched by genes upregulated in the indicated cluster are shown. Functional terms related to cardiomyocyte function were enriched in the late pseudotime clusters, Myo_1 (control) and Myo_6 (AKO). Two-tailed Fisher’s exact test, Bonferroni correction. h, WNN UMAP plot colored by a ‘functional cardiac gene’ index, which was calculated based on the aggregate expression of the six indicated genes, which are required for the efficient pumping function of aCMs.

Extended Data Fig. 6 A comparison of TBX5 RNA-seq datasets.

We compared snRNAseq data from Tbx5AKO aCMs to three other bulk RNA-seq datasets involving Tbx5 gain- or loss- of function. Control_aCM indicates Myocyte_1 and KO_aCM indicates Myocyte_6. a, c, e: Scatter plot of fold-change in Tbx5AKO aCMs compared to the other three datasets. Left, points are colored by significance in each dataset (Padj < 0.05). Right, genes with significant differential expression in both datasets are colored by chamber selectivity. Pie charts summarize the proportion of each class of genes within each quadrant. b, d, f: Enrichment of aCM or vCM genes in the indicated quadrants of the scatter plots. Fisher’s exact test, two-tailed. a-b. Comparison to LA tissue with ubiquitous, adult-induced inactivation of Tbx5 (Tbx5iKO; Nadadur et al., 2016). c-d. Comparison to LA tissue with mild Tbx5 upregulation due to deletion of an intronic regulatory element (Tbx5Re(int)KO; Bosada et al., 2023). e-f. Comparison to LV tissue with Tbx5 overexpression in cardiomyocytes (Tbx5-OE; this study). g. Comparison of changes in expression of aCM- and vCM-selective genes across all four datasets. Fold-change was calculated between condition with higher Tbx5 (numerator) to condition with lower Tbx5 (denominator). Genes were ordered by ascending Log2(WT/TBX5iKO). Genes not detected (ND) for a given experiment are colored white. Normalized enrichment score (NES) and false discovery rate (FDR) values from GSEA using the aCM-selective or vCM-selective gene lists are shown. Negative NES indicates enrichment in the genes upregulated in the lower Tbx5 condition, whereas positive NES indicates enrichment in the genes upregulated in the higher Tbx5 condition.

Extended Data Fig. 7 Characterization of myocyte differentially accessible regions.

Heatmap (a) shows the patterns of differential accessibility between myocyte clusters. The rows contain the union of regions with differential accessibility in the four pairwise comparisons shown in Fig. 5a. ATAC signal in each region is shown for myocyte clusters 1 (control_aCM cluster), 4, 6 (KO_aCM cluster), and 5. The regions are grouped (groups a-f) by their pattern of accessibility change in the four pairwise comparisons. Please also refer to Supp. Table 4. Arrows denote significant enrichment in one cluster compared to another. These six groups fit two predominant patterns: those with and those without predominant accessibility in the control aCM cluster (Myocyte_1). Most regions with predominant accessibility in the control aCM cluster were occupied by TBX5 and had GO terms related to cardiac cell development or striated muscle contraction (b). In contrast, a minority of regions without predominant accessibility in the control aCM cluster were occupied by TBX5 and had GO terms that were atypical for cardiomyocytes (c). Groups with less than 200 regions are not shown in the heatmap.

Extended Data Fig. 8 Proximity and predicted gene linkages demonstrate the regulation of the atrial GRN by control peaks.

a, The different types of association of a genomic region with a gene: (1) Linkage (L). Region-to-gene linkages are predicted based on co-variance of accessibility and expression on a nucleus-by-nucleus basis in the multiome data. (2) Proximity (P). Region-to-gene relationships are inferred by proximity of the region to the gene’s transcriptional start site. (3) Proximity and linkage (P + L). A region-to-gene association can be supported by both proximity and linkage. b, Genes were associated with control and KO regions by linkage (n = 2 control and 2 KO multiome biological replicates). Ratio of gene expression between the control and KO aCM clusters was plotted and compared between groups by the two-sided Mann-Whitney test. P = 9.1E-48. c, Association of control and KO ATAC regions with aCM and vCM genes. Associations were made based on proximity, linkage, or both. d, aCM and vCM genes were interrogated for associations with control and KO ATAC regions by proximity, linkage, or both.

Extended Data Fig. 9 Motif analysis of control and KO ATAC regions.

a, Aggregation plots for H3K27Ac at control and KO regions. b, Heatmaps of the H3K27Ac signal at control and KO peaks. c, Top-enriched motifs identified in control regions. The TBOX motif was the most enriched, followed by MEF2. An extended table of non-redundant motifs showing the top 21 most enriched motifs in control regions. d, Transcription factor footprinting analysis demonstrates footprints at TBOX and MEF2 motifs in control clusters (Myocyte_1 and Myocyte_4) compared to KO clusters (Myocyte_5 and Myocyte_6). e, Top-enriched motifs identified in KO regions and extended table of the top non-redundant motifs. f. Occupancy of control and KO ATAC regions by cardiac TFs in aCMs. Occupancy data is from GEO GSE2150653.

Extended Data Fig. 10 Chromatin loops link TBX5 dependent enhancers with atrial genes.

a, Contact maps of Myl7 or Bmp10. Black boxed regions are loops called in each sample and blue boxed regions mark differential loops that are significantly stronger in control samples. b, Genes near control anchors grouped by adjacency to aCM-selective, vCM-selective, or non-chamber selective expression. Control anchors were present near 62 aCM-selective genes and of these, 46 were expressed at greater levels in control Myocyte_1 compared to KO Myocyte_6. Notable genes from previous figures include Nppa, Bmp10, Sbk2 and Myl7. 38 non-chamber selective genes were also upregulated in control samples and linked to enhancers by TBX5-dependent looping. These included Gja1 and Tead1. Only 5 vCM-selective genes were found near control anchors. c, 50 genes neighbored KO anchors. These genes included 5 aCM-selective genes and 3 vCM-selective genes. Most of the differentially expressed genes near KO anchors were more highly expressed in Myocyte_6 (KO) compared to Myocyte_1 (Ctrl).

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diffloop HiChIP results.

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Sweat, M.E., Cao, Y., Zhang, X. et al. Tbx5 maintains atrial identity in postnatal cardiomyocytes by regulating an atrial-specific enhancer network. Nat Cardiovasc Res 2, 881–898 (2023). https://doi.org/10.1038/s44161-023-00334-7

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