Cardiovascular Disease and the Epigenetic Status of Gene Regulatory Elements

The onset of cardiovascular diseases occurs alongside widespread epigenetic alterations to the distal cis-regulatory elements (CREs, including gene enhancers) that control the gene expression programs underpinning normal heart development, leading to altered gene expression and cell dysfunction. While we generally lack knowledge regarding CRE activity and function in specific cell types or states, a previously reported comparative analysis of mouse cardiac cells (which included cardiomyocytes) described the cell-type-specific responses of CREs to cardiovascular disease (Lother et al.). At the epigenetic level, a joint analysis of histone modifications and DNA methylation in human cardiomyocytes identified over 100,000 CREs, many of which displayed an enrichment for cardiovascular disease-associated non-coding variants (Gilsbach et al., Tan et al., and Hocker et al.). Genetic variants within CREs may exert cell-type-specific effects, with cardiac arrhythmia and hypertrophy known to be associated with CRE variants in distinct cardiomyocyte populations (Kanemaru et al.). These non-coding variants can alter transcription factor binding to CREs, thereby disrupting enhancer-promoter interactions and altering gene expression ([Ameen et al.](https://doi.org/10.1016/j.cell.2022.11.028

With this research in mind, the laboratory of Ralf Gilsbach recently set out to create a high-resolution genome-wide map of chromatin interactions between CREs and target gene promoters in human adult cardiomyocytes from distinct heart chambers, thereby linking them to gene expression programs and genetic risk factors. Their analyses involved high-resolution high-throughput chromosome conformation capture sequencing (Hi-C; Rao et al.) of healthy atrial and ventricular cardiomyocytes and failing ventricular cardiomyocytes and the integration of additional epigenetic and transcriptomic datasets. Fascinatingly, their new Nature Communications study now provides deeper insights into the chamber-specific regulatory mechanisms controlling human cardiomyocyte function and the interpretation of non-coding variants that affect cardiovascular diseases (Haydar et al.).

Paired-Tag technology from Epigenome Technologies generates joint epigenetic and transcriptomic profiles at single-cell resolution and detects histone modifications and RNA transcripts in individual nuclei with efficiency comparable to single-nucleus RNA-seq/ChIP-seq assays. Could integrating Paired-Tag datasets have provided additional robustness to this study, furthering our understanding of chamber- and cell-specific CREs and their links to cardiovascular disease?

Epigenetic Analyses of Chamber-specific Human Cardiomyocytes Link CREs to Cardiovascular Disease

The authors initially isolated bulk cardiac tissue or cardiomyocyte nuclei from three non-failing left ventricles, generated Hi-C data, constructed genome-wide chromatin state models using ChIP-seq data and ChromHMM (Ernst & Kellis), and identified CREs as regions with low CpG methylation (Gilsbach et al., Stadler et al., and Burger et al.). Analysis of this data revealed the highly cell-type-specific nature of chromatin interactions and 3D genome organization in the human heart, thereby suggesting a requirement for chamber-specific cardiomyocyte analysis to accurately “decode” the distinct regulatory architectures present. They then undertook a similar analysis in human cardiomyocytes from non-failing left atria and left ventricles, and from terminally failing ventricles, to support differential chromatin interaction analysis in healthy vs. diseased chamber-specific cardiomyocytes. An initial analysis indicated the highly conserved nature of higher-order genome structure (topologically associating domains and A/B compartments) across human cardiomyocyte subtypes, despite transcriptional and epigenetic differences.

The next team focused on higher-order chromatin structures to study interactions between gene promoters and CREs (typically enhancer elements), and also integrated chromatin state annotations, DNA methylation profiles, and RNA-seq data. This analysis supported the identification of promoter-interacting CREs in chamber-specific cardiomyocytes, which the team functionally validated using CRISPR interference-mediated perturbation (Qi et al.), a method that enables targeted epigenetic silencing without altering the underlying genomic sequence. The study highlighted links between chamber-specific promoter-CRE interactions and gene expression in cardiomyocytes, and a comparative analysis revealed significant alterations in promoter interactions in diseased cardiomyocytes.

Cardiac-specific CREs generally display the enrichment of cardiac disease-associated genetic variants (see the original study for a range of references). To address the contribution of non-coding genetic variants to atrial- and ventricular-specific cardiovascular diseases, the authors integrated chamber-specific promoter-CRE interaction data, thereby enhancing variant-to-gene mapping and supporting the discovery of additional disease-associated regulatory loci. This analysis underscored the chamber-specific enrichment of variants associated with heart disease traits in active CREs; for example, the study discovered a more significant prevalence of atrial fibrillation-associated variants in promoter-interacting CREs in atrial as compared to ventricular cardiomyocytes. As such, they highlighted the KCNJ2 gene locus (which encodes a potassium channel associated with ventricular arrhythmia susceptibility) and identified uncharacterized CREs harboring variants that affected QT duration. This study (which also included functional epigenetic silencing) identified enhancer elements harboring QT-duration-associated genetic risk factors that modulated gene expression, altered KCNJ2-dependent channel current, and affected repolarization kinetics in cardiomyocytes, thereby providing mechanistic insights into the genetic basis of QT syndrome (a heart rhythm disorder that causes fast, chaotic heartbeats). Of note, additional CREs highlighted included those interacting with the SCN5A, KCNH219, and KCNH2 gene loci; the authors emphasize that only cell-type-specific epigenome and chromatin-interaction profiling can detect promoter interactions in cardiac tissues and, as such, decode the disease relevance of non-coding regulatory elements.

The Next Steps Towards Decoding Cardiovascular Disease

This paper underscored the general utility of combining CRISPR-based gene silencing with cell- and chamber-type-specific chromatin interaction mapping to identify and explore the function of CREs, exemplified by KCNJ2-associated CREs, which harbor genetic variants associated with the onset of ventricular arrhythmias and whose activity controls gene expression and cellular electrophysiological outcomes. The team now hopes that their maps will help to uncover novel genetic variants driving cardiovascular disease.

The implementation of Paired-Tag technology from Epigenome Technologies, which generates joint epigenetic and transcriptomic profiles at single-cell resolution and detects histone modifications and RNA transcripts in individual nuclei with efficiency comparable to single-nucleus RNA-seq/ChIP-seq assays, has the potential to provide deeper insight into such research aims. What more could the simultaneous single-cell analysis of histone modification and transcriptomic profiles tell us about chamber- and cell-specific CREs and their links to cardiovascular disease?