Understanding Single-Cell Gene-Regulatory Mechanisms in Heart Failure

Heart failure - a leading cause of morbidity and mortality (Khan et al.) - represents an all-too-common multifactorial problem facing patients across the globe; however, the gene-regulatory mechanisms that underpin disease development remain somewhat unexplored. Heart failure-associated gene variants identified to date tend to affect non-coding regions of the genome (Arvanitis et al., Garnier et al., Zheng et al., and Jurgens et al.), suggesting they may impact cis-regulatory regions and, therefore, drive pathogenesis by altering gene regulatory programs. The heterogeneous cell types that comprise the human heart (Litviňuková et al.) and their distinct transcriptional programs and pathological responses pose a challenge to the application of conventional bulk epigenetic and transcriptomic assays that aim to understand disease-associated gene-regulatory mechanisms; however, single-cell assays represent an exciting alternative means of delineating how non-coding variants affect specific cardiac cell types (Reichart et al. and Miranda et al.) and, as such, provide an opportunity to develop novel and highly specific therapeutic or disease-prevention strategies in the realm of heart failure.

A team of researchers led by Allen Wang, Stavros G. Drakos, Kyle J. Gaulton, Bing Ren, and Neil C. Chi recently applied complementary single-cell approaches to cells from all cardiac chambers of 13 non-failing and 23 failing human adult hearts to explore how gene regulatory programs become altered during heart failure. In their recent Science study (Xie, Tucciarone, and Farah et al.), the authors applied 10x Multiome for the joint profiling of transcription and chromatin accessibility, Droplet Hi-C for single-cell 3D chromatin conformation mapping, and, perhaps most importantly, Droplet Paired-Tag from Epigenome Technologies to simultaneously measure transcription and histone modifications in the same single cells. The combination of these approaches uncovered alterations in cell-type composition, gene regulatory programs, and chromatin organization (with cardiomyocytes and fibroblasts as central players) and enabled the identification of causal genetic contributors to heart failure by mapping cell-type-specific enhancer-gene interactions. This exciting advance now provides a cell-type-resolved atlas of alterations in the regulatory landscape of the human heart in health and disease, which may enable the design of cell-type-targeted therapies to treat heart failure.

[! Multi-panel image showing embedding, cell types, and heatmaps associated with ventral cardiomyocytes](img/image0.png Panels A–C present single-cell analyses of ventricular cardiomyocyte (vCM) subpopulations from failing (HF) and non-failing human hearts. Panel A displays UMAP plots of vCM subpopulations with RNA velocity trajectories and condition contributions, while Panel B features bar graphs and heatmaps showing subpopulation-specific cellular contributions, gene expression, and chromatin accessibility. Panel C utilizes SCENIC+ to display transcription factor gene regulatory networks (GRNs) for each vCM subpopulation, highlighting regulatory specificities across health and heart failure and transcription factors potentially resposible for mediatig vCM dysfunction. From (Xie, Tucciarone, and Farah et al.).

The Power of Droplet Paired-Tag: Single-cell Multimodal Data

In brief, the authors revealed that heart failure-associated chromatin remodeling at gene regulatory elements occurred alongside coordinated changes in transcription factor activity and target gene expression. While the study highlighted the general implication of all cardiac cell types, the team highlighted extensive rewiring of gene regulatory programs in cardiomyocytes and fibroblasts specifically. They also demonstrated the existence of intermediate cell states between healthy and diseased conditions characterized by the activation of stress-responsive transcription factors, suggesting their critical role in driving disease progression. The subsequent analysis of the single-cell chromatin conformation also described a link between heart failure development and the cell-type-specific reorganization of genome architecture; the altered gene regulation observed in cardiomyocytes during heart disease occurred in a background of disrupted chromatin compartments and increased long-range chromatin interactions, linking coordinated dysregulation at the level of cis-regulatory elements and higher-order genome organization with heart failure. Finally, the integration of genetic association data revealed enrichment of heart failure-associated genetic risk variants within cell-type-specific active regulatory elements (present in all cells, but especially in cardiomyocytes) and highlighted the exertion of these effects through long-range chromatin interactions. Overall, the integration of cardiac cell type-regulatory programs with human genome-wide association study data highlighted cell types, gene networks, cis-regulatory elements, and target genes that may provide potential therapeutic targets for the treatment of heart failure patients.

Concentrating on Droplet Paired-Tag analysis, the authors simultaneously evaluated RNA expression and either the permissive histone modification H3K27ac (67,453 nuclei) or the repressive histone modification H3K27me3 (62,127 nuclei). Assessing H3K27ac levels helped identify cell-type-specific genes and active enhancer elements, while H3K27me3 levels marked regions silenced in specific cell types. The integration of Droplet Paired-Tag data with Droplet Hi-C/10x Multiome data enabled the creation of a single-cell multimodal dataset that systematically identified the gene-regulatory programs underlying cell-type-specific responses in heart failure.

[! Multi-panel image showing enrichment results for genetic variation across traits, genomic state, and cell type](img/image1.png Panels A–C present fine-mapping analyses integrating genetic traits with the epigenetic landscapes of cardiac cell types. Panel A uses LD score regression (LDSC) to show enrichment of cardiovascular and heart failure trait-associated variants across total, open, and active cCREs in distinct cardiac cell types. Panel B quantifies the number of fine-mapped variants and signals overlapping these cCRE categories in enriched lineages, while Panel C displays cumulative posterior probability association (PPA) values across cardiac cell types to pinpoint dilated cardiomyopathy (DCM) risk loci. Notably, active chromatin shows consistently stronger enrichment than merely open chromatin. From (Xie, Tucciarone, and Farah et al.).

The integrative exploration of these data helped identify cell-type-specific gene-regulatory programs in adult human hearts, revealing that the cis-regulatory elements identified likely functioned as enhancers that help regulate cell-type-specific genes in distinct cardiac lineages and, overall, highlighting how cell-type-specific epigenetic landscapes orchestrate gene regulation across cardiac cell types. The data also permitted a global view of gene-regulatory and chromatin structural changes of cardiac cell types during heart failure, highlighting how distal cis-regulatory elements regulate target gene activities in a cell type-specific manner, uncovering transcriptional programs across cardiac cell types (further highlighting the distinct gene-regulatory changes underlying disease progression), and, overall, demonstrating a coordinated level of transcriptional, epigenetic, and chromatin structural remodeling across cardiac cell types during disease development.

A subsequent focus on cardiomyocytes revealed a broad spectrum of cellular states in control and diseased hearts; indeed, the data defined the existence of a spectrum of cardiomyocyte subpopulations in ventricular and atrial cardiac chambers that may describe a transition between healthy and diseased states and so may provide actionable pathways/mechanisms to slow or even reverse disease progression. A similar focus on cardiac fibroblasts, as the second highlighted cardiac cell type, revealed a hierarchy of gene regulatory networks that control how these cells transition between healthy and pathologic states during heart failure. The gene-regulatory programs and transcription factors driving the transition from a healthy to a disease state included the reactivation of fetal cardiac programs that promote pathologic cardiac hypertrophy and remodeling, but not the reparative cardiomyocyte proliferation characteristic of fetal hearts, and fibroblast activation programs that direct cells from quiescent to activated pathologic states.

Droplet Paired-Tag: Helping to Provide Deep Insight and Highlighting Therapeutic Targets

Overall, the integration of single-cell epigenetic assays – including Droplet Paired-Tag - revealed profound insight into the gene-regulatory mechanisms present in cardiac cells associated with heart failure. The data highlight pronounced alterations in the transcriptional, epigenomic, and chromatin architecture levels in cardiomyocytes and fibroblasts, which may reflect disease-associated changes in nuclear organization and function. Importantly, these data may now guide therapeutic strategies that reprogram specific cardiac cell types from pathologic to reparative states and facilitate the interpretation of non-coding variants associated with heart disease in a cell-type-specific manner. Has this study piqued your interest? If so, check out all the related Paired-Tag products and services on our site and get in touch to find out how we can accelerate your single-cell epigenetics research forward!