Describing the Single-cell Multiomic Landscape of Human Heart Failure with Droplet Paired-Tag

By Stuart P. Atkinson

Single-cell multiomic, histone modification and Hi-C analyses uncover cell type specific transcriptome, epigenome and 3D genome maps of adult human hearts. G) Genome browser tracks reveal aggregated epigenetic profiles for all major cardiac cell types at selected marker gene loci. (I, J) Representative examples of cell type-specific Hi-C contact maps are shown for boFBN1 (I) and TBX5 (J) loci at 10kb resolution. -log2 insulation score (IS), compartment score and genome browser tracks for chromatin accessibility and histone modifications are shown below. vCM, ventricular cardiomyocytes; aCM, atrial cardiomyocytes; SM, smooth muscle cell.

Understanding the Epigenetic Underpinnings of Heart Failure

Heart failure due to acquired cardiovascular disease or myriad genetic causes remains a major global cause of human morbidity and mortality (Khan et al.). As the recent discovery and fine-mapping of heart failure-associated risk loci revealed that the vast majority resided within unannotated non-coding regions (Arvanitis et al., Garnier et al., Zheng et al., and Jurgens et al.), discovering how these loci impact cells critical to cardiac function could provide opportunities for the development of novel preventative or curative strategies. Additionally, while we understand the need for the complex coordinated function of diverse cardiac cell types to sustain cardiac function (Litviňukov et al.), we lack a comprehensive understanding of differences in function when comparing healthy patients with those suffering from ischemic/non-ischemic heart failure. Single-cell transcriptomics has begun to decipher such differences and identify critical cell types and interactions (Reichart et al.); however, we require a comprehensive investigation of the gene regulatory programs involved in cardiac cell type-specific responses to heart failure to better appreciate pathogenic mechanisms and develop preventive/curative strategies for heart failure and cardiac disease.

Analytical platforms that support multimodal transcriptomic and epigenetic profiling may offer deeper insights by describing gene regulatory networks and annotating heart failure-associated genetic variants within non-coding regions (Xie et al. and Chang et al.). Droplet Paired-Tag from Epigenome Technologies represents one such platform and represents the driving force of a new study headed by Allen Wang, Stavros G. Drakos, Kyle J. Gaulton Bing Ren, and Neil C. Chi. Their new MedRxiv preprint (Xie, Tucciarone, Farah, and Chang et al.) reports on the application of single-cell multiomic assays to generate transcriptomic, chromatin accessibility, histone modification, and chromatin conformation profiles of multiple cardiac cell types isolated from the cardiac chambers of 36 healthy and ischemic/non-ischemic human hearts to explore the regulatory underpinnings of heart failure at single-cell resolution. Their integrative analysis defines critical cell-specific gene regulatory and transcriptional programs associated with heart failure and helps define cell type-specific gene regulatory programs associated with disease-associated genetic variants that drive heart failure and cardiovascular risk.

Droplet Paired-Tag from Epigenome Technologies generates joint epigenetic and transcriptomic profiles at single-cell resolution and detects histone modifications and RNA transcripts in individual nuclei with comparable efficiency to single-nucleus RNA-seq/ChIP-seq assays while avoiding the need for cell sorting. Paired-Tag technology enables a more nuanced understanding of gene regulatory mechanisms and may improve disease management.

Cell-Specific Chromatin States and Enhancer Links Across Cardiac Cell Types — Comprehensive epigenomic profiling of human cardiac cell types reveals their gene regulation. (I) Representative genome browser tracks are shown for ChromHMM chromatin states across all major cell types on selected cell type-specific distal cCRE-gene links, including MYL2 (vCM-specific), KCNJ3 (aCM-specific), EGFLAM (Pericyte-specific), FBN1 (Fibroblast-specific), VWF (Endothelial-specific) and MARCO (Myeloid-specific).

Transcriptomic and Multimodal Epigenetic Profiling via Droplet Paired-Tag Describes the Failing Heart

This study analyzed heart tissue samples from 36 donor hearts (13 non-failing hearts; 13 ischemic- and 10 non-ischemic failing hearts) via single-cell multiome (assay for transposase-accessible chromatin and gene expression), simultaneous single-cell gene expression and H3K27ac/H3K27me3 profiling (Droplet Paired-Tag; Xie et al.) and single-cell chromatin conformation capture (Droplet Hi-C; Chang et al.) to generate a single-cell multimodal dataset spanning all cardiac chambers that provided a unique opportunity to explore the gene regulatory programs that control cell type-specific responses during homeostatic and pathological conditions of the heart. The multiomic single-cell data generated in this study, encompassing 776,479 cells, offer new insights into the transcriptomic and epigenomic landscapes of distinct cell types within the non-failing and failing human heart. This analysis describes how the chromatin organization and interactions underpinning the complex gene regulatory networks control cardiac cell type-specific enhancer-promoter interactions.

An integrated analysis enabled the interrogation of chromatin states in regulatory elements across distinct cardiac cell types, the prediction of distal enhancer elements for genes of interest, and the definition of cell-type-specific enhancer-promoter interactions. The data expanded the annotation of cardiac cis-regulatory sequences tenfold and precisely mapped cell-type-specific enhancer-gene interactions. The authors identified dynamically regulated genes under the tight control of a diverse set of distal enhancer elements that exhibited differing chromatin states according to cardiac cell type and health status. The activity of unique combinations of transcription factors at these distal enhancers controlled the regulation of target genes in a cardiac cell type-specific manner, thus enabling precisely regulated gene expression across the distinct cardiac cell lineages. Cardiomyocytes and cardiac fibroblasts suffered the most significant alterations at the transcriptomic and epigenetic (chromatin accessibility and chromatin conformation) levels in the failing heart, which agrees with studies describing heart failure-associated nuclear remodeling (Wohlschlaeger et al. and Ninh et al.). The team noted that the heart failure-associated alterations in chromatin conformation align well with those observed in cancer and developmental disorders (Lupiez et al. and Dixon et al.) and, as such, may reflect specific regulatory mechanisms that alter nuclear organization and function in cardiomyocytes and other cardiac cell types.

A deeper examination of cardiac cell types revealed multiple distinct intermediate and pathological cellular states that may direct responses during heart failure. Network analyses of the integrated single-cell data identified crucial gene regulatory programs and transcription factors governing the transition of cell types from non-disease to intermediate-stage and end-stage disease states, revealing the activation of pathologic gene programs and the suppression of homeostatic gene programs in a cell-type-specific manner during heart failure. As an example, the authors highlighted the molecular mechanisms driving the reactivation of fetal cardiac genes during heart failure (promoting pathologic cardiac hypertrophy and remodeling) but not reparative cardiomyocyte proliferation (Porrello et al.).

Finally, this comprehensive atlas of human cardiac cell type-specific epigenomes and regulatory interactomes also enabled the interrogation of how non-coding variants influence gene expression, enhancer activity, and disease susceptibility in a cell-type-specific manner. The data provided an exploratory resource that permits the prioritization of efforts to identify candidate cardiovascular-disease-associated genetic variants, which will aid the functional examination and identification of causal genetic variants via high-throughput genetic screening assays (Fulco et al., Chardon et al., and Agarwal et al.).

Fig. 3. Cell type-specific epigenetic changes direct gene regulatory responses during heart failure. (A) Dot plot shows the number of genomic regions and genes identified from differential analysis between heart failure (HF) and non-heart failure (Non-HF) conditions across cell types. (B) Sankey plots show the chromatin state dynamics for differentially accessible cCREs in HF and Non-HF hearts. Percentage was calculated as the averaged percentage of chromatin states across cell types. (C) Scatter plots compare the log2 fold changes of gene expression (GEX) and chromatin accessibility (ATAC) for genes and cCREs from putative ABC links between HF and Non-HF conditions. Links are colored based on whether the associated gene and/or cCRE is classified as differentially expressed (DEG) or differentially accessible (DAR). (D) Normalized aggregate peak analysis (APA) scores of Droplet Hi-C signals in vCM and myeloid cells are shown for vCM down-regulated and myeloid up-regulated ABC links. APA scores are represented as P2LL (peak-to-lower-left) values. (E) Genome browser tracks show an example of a differential ABC link targeting PM20D2 in vCM. Pseudobulk chromatin accessibility tracks across selected cardiac cell types (top) as well as differential epigenetic signals and chromatin states in HF versus Non-HF vCMs (bottom) are shown.

Droplet Paired-Tag: Creating an Improved Understanding of Heart Failure

With the help of Droplet Paired-Tag, this fascinating study has created an atlas that provides insight into the chromatin dynamics that establish gene regulatory programs associated with heart failure, a leading cause of morbidity and mortality worldwid e. This new understanding will support ongoing efforts to develop cell-type-targeted preventive and curative therapeutic strategies that can direct specific cardiac cell types from a pathologic to a reparative state. Notably, the authors also state that their data may hold relevance to a broader range of heart failure representations represented in this study due to significant pathophysiological overlaps (Simonson et al.).

Droplet Paired-Tag from Epigenome Technologies generates joint epigenetic and transcriptomic profiles at the single-cell resolution and detects histone modifications and RNA transcripts in individual nuclei with an efficiency comparable to single-nucleus RNA-seq/ChIP-seq assays. Furthermore, Epigenome Technologies offers a range of single-cell products and services tailored to meet the majority of research requirements. As such, applying Paired-Tag technology may enable giant leaps forward in understanding gene regulation and complement the findings of this exciting study.

For more information on how Droplet Paired-Tag helped to describe the single-cell multiomic landscape of human heart failure, see medRxiv, May 2025.