Epigenetic Keys Unlock Secrets of the Heart
February 4, 2022
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Of all the organs in vertebrates, development of the heart occurs first, and our hearts beat on throughout our lives without ever resting (ideally!). Ironically, heart disease is the leading cause of deaths both in the US and worldwide. So, it’s of utmost importance that we take care of our hearts, which can be augmented by a greater understanding what is good and bad for the heart. It’s important to understand the molecular mechanisms behind normal heart development, as well as those that contribute to cardiovascular diseases and heart failure. The list of factors that impact cardiac health is long and is growing faster with the new discoveries in the field of epigenetics, where several epigenetic factors and signatures are found to regulate heart development, function, and disease.
Epigenetics comprises the study of heritable changes in gene expression which are not due to changes in the sequence of bases in DNA. A key group of epigenetic factors include enzymes responsible for post-translational modifications of histones, those proteins around which DNA wraps around such that a loose conformation is conducive to transcription and a condensed organization makes DNA inaccessible to transcription. The organization of DNA around histones collectively gives rise to chromatin structure whose dynamic remodeling regulates and impacts gene expression. Other epigenetic factors include enzymes which regulate modification of DNA methylation, non-coding RNA, prions, etc.
Applying Epigenetic Tools to Cardiac Samples
Several studies have shown that DNA methylation, histone post-translational modifications (PTMs), chromatin remodeling enzymes, and non-coding RNA can all epigenetically regulate cardiac development, function, and disease. However, the field is largely unexplored and numerous labs around the world are dedicated to this growing research area. Even after the discovery of the importance of epigenetics in health, there were early challenges to exploring the epigenetics of cardiac biology due to technological limitations and scarce sample sizes. With advancement in technology, methods have evolved to become high-throughput, provide extensive high resolution analysis at single-cell levels, and to be more compatible with limited starting material.
A Multiomic Approach to Cardiovascular Disease
Five elegant methods have been employed in a single recent study by Chapski et al., which investigates chromatin remodeling events in the failing heart. The group takes a multiomic approach including reduced representation bisulfite sequencing (RRBS) for DNA methylation analysis, RNA sequencing (RNA-Seq) for transcriptomic analysis, ATAC-Seq for chromatin accessibility analysis, ChIP-Seq for chromatin occupancy by proteins of interest, and Hi-C for 3-dimensional chromatin conformation.
Cardiac hypertrophy and heart failure are typically investigated using a variety of model systems, among which the surgical model of transverse aortic constriction (TAC) is widely used to study how pressure overload induces these conditions (Richards et al.). In this model system, a permanent constriction around the transverse aorta is placed to limit outflow from the left ventricle, leading to pressure overload in the region. What results is a range of outcomes including cardiac hypertrophy, fibrosis, dysfunction, and heart failure.
Here we look at the 2021 publication by Chapski, which presents an epigenetic investigation of the TAC model, showing that where pathologic stimuli to the heart reorganizes chromatin, early compensation of pressure overload may also affect chromatin accessibility and DNA methylation. Further, this remodeling persists during subsequent decompensatory phases of cardiac failure. Overall, the study shows the relationships of various layers of epigenetic phenomenon like DNA methylation, histone modifications, transcription factor localization with chromatin structure, supported by analysis of the transcriptome and chromatin accessibility. Global analysis of multiomic, high-throughput data integration reveals how transcription is impacted by local epigenetic landscapes. Of interest are these potential chromatin reorganizational processes that might be therapeutically targeted to prevent heart failure – the primary global assassin.
Chromatin accessibility in heart disease - by ATAC-Seq
To investigate changes in chromatin accessibility in the failing heart, the authors performed ATAC-Seq, a fairly new epigenetic assay which delivers genome-wide profiles of open and accessible regions of chromatin – indicative of active regulatory genes. This assay was applied within the TAC model simultaneously with depletion of the chromatin structural protein CTCF, a transcription factor important in maintenance of cardiac chromatin architecture. Depletion of CTCF causes heart failure, with a majority of phenotypes also shared with the TAC model. TAC was performed on 8-week-old mice where a vascular clamp with a band of silastic tubing was placed around the aorta. Left ventricles were obtained from mice which had 3 days TAC, 3 weeks TAC, and CTCF depleted. Nuclei were isolated from the tissue and tagmented, which is simultaneous DNA fragmentation and ligation with sequencing adapters. The libraries were sequenced, and reads were mapped to mouse genome mm10. Among the various observations from ATAC-Seq were notable ones like large-scale alterations in chromatin accessibility, trends for reducing accessibility with time, novel changes in accessibility with time, and higher accessibility induced by TAC which suggests that progression of heart disease by pressure overload opens up chromatin.
Histone PTMs & Transcription Factors in the Failing Heart – by ChIP-qPCR and ChIP-Seq
To test whether the observed changes in chromatin accessibility impacted the association of transcription factors or histone modification with chromatin, ChIP (Chromatin ImmunoPrecipitation) was also performed. Cardiomyocytes isolated from left ventricles were subjected to ChIP to map genomic loci where H3K27acetylation, GATA, and NKX2.5 are localized. The rationale was that H3K27ac enriched regions help to probe for enhancers in the mouse heart, while GATA and NKX2.5 are relevant cardiac transcription factors. The results showed that transcription factors and histone marks of transcriptional activation are enriched at enhancer regions and that enhancer activity, in conjunction with other epigenetic processes, regulate gene expression. ChIP also revealed association of GATA4 and NKX2.5 at enhancers and promoters of cardiac developmental genes like Itga9 and Nppa, which support their transcriptional upregulation in the failing heart.
Cardiomyocyte DNA methylation - by reduced representation bisulfite sequencing (RRBS)
Analysis of DNA methylation in adult cardiomyocytes isolated from left ventricle of the above mice models showed that this epigenetic status was also retained at 3 days and up to 3 weeks post TAC. DNA methylation occurs at cytosine residues of CpG islands, and it epigenetically regulates gene expression. It is studied by RRBS which differentiates between methylated vs. unmethylated cytosines. This method involves bisulfite conversion of only unmethylated cytosines, but not methylated cytosines, to uracil. Using RRBS, the study showed that alterations in DNA methylation, in response to early compensation for pressure overload, were preferentially in intergenic and intronic regions. Further, a majority of the alterations in DNA methylation at enhancers and promoters 3 days post TAC, prior to the onset of heart failure, persist for 3 weeks post TAC. Interestingly, similar phenotypes were seen for chromatin accessibility.
In addition to the above epigenetic processes, the study employed transcriptomic analysis and chromosome conformation capture (Hi-C) to investigate multiple layers of epigenetics associated with heart disease. RNA-sequencing of adult cardiomyocytes isolated from left ventricle, and data analysis from Hi-C assay using adult mouse cardiac myocytes from a previous study (Rosa-Garrido et al.) by the authors, showed how the transcriptome and chromatin conformation, respectively, are impacted by epigenetic changes during progression towards heart failure.
Overall, individual epigenetics assays and multiomic data integration in this work reveal that epigenetic mechanisms associated with cardiac pathology get established during early stages of the disease. Therefore, it may be that certain epigenetic alterations that are detected early may serve as biomarkers to predict heart disease. Interventions to reverse epigenetic remodeling associated with heart disease, as seen in this study, may be instrumental in prevention of heart disease and inhibition of pathological progression.
View our Webinar: Cardio Epigenetics Chromatin Meeting, 2021
About the author
Rwik Sen, Ph.D.
Rwik is from Kolkata in eastern India, a city of history, multiple cultures and food. Kolkata is in the state of West Bengal which hosts 2 UNESCO World Heritages, and has part of the Himalayan mountains to its north and the Bay of Bengal to its south. Love of the natural world and mystery novels made Rwik passionate about scientific discovery. Hence, after his undergraduate in biotechnology, Rwik went to Southern Illinois University for a Ph.D., followed by postdoctoral training at the University of Colorado. Interacting with people, traveling, promoting STEM outreach and inclusion, are some of the things Rwik enjoys in addition to the ocean and dancing.
Contact Rwik on LinkedIn with any questions