Transcriptional and Epigenetic Landscape of Cardiac Pacemaker Cells: Insights Into Cellular Specialization in the Sinoatrial Node

Abstract

Cardiac pacemaker cells differentiate and functionally specialize early in embryonic development through activation of critical gene regulatory networks. In general, cellular specification and differentiation require that combinations of cell type-specific transcriptional regulators activate expression of key effector genes by binding to DNA regulatory elements including enhancers and promoters. However, because genomic DNA is tightly packaged by histones that must be covalently modified in order to render DNA regulatory elements and promoters accessible for transcription, the process of development and differentiation is intimately connected to the epigenetic regulation of chromatin accessibility. Although the difficulty of obtaining sufficient quantities of pure populations of pacemaker cells has limited progress in this field, the advent of low-input genomic technologies has the potential to catalyze a rapid growth of knowledge in this important area. The goal of this review is to outline the key transcriptional networks that control pacemaker cell development, with particular attention to our emerging understanding of how chromatin accessibility is modified and regulated during pacemaker cell differentiation. In addition, we will discuss the relevance of these findings to adult sinus node function, sinus node diseases, and origins of genetic variation in heart rhythm. Lastly, we will outline the current challenges facing this field and promising directions for future investigation.

Keywords: ATAC-seq and chromatin accessibility; cardiac pacemaker cell; enhancer; heart rate; sinoatrial node; sinus node dysfunction; transcriptional regulation.

FIGURE 1

Differences between pacemaker cells and working cardiomyocytes. (A) Characteristics of typical pacemaker cells: (top panel) micrograph of an isolated pacemaker cell taken from an adult Hcn4-GFP mouse demonstrates an elongated spindle-like morphology; (middle panel) a typical pacemaker cell action potential with elevated resting membrane potential and diastolic depolarization; (bottom panel), a list of key functional proteins enriched in pacemaker cells. (B) For comparison, typical ventricular cardiomyocyte characteristics are shown, including (top panel) a micrograph of an adult mouse ventricular cardiomyocyte stained for alpha-actinin (green) to demonstrate sarcomeres and connexin-43 (red) to demonstrate gap junctions; (middle panel) a typical working cardiomyocyte is quiescent with a negative resting potential, rapid upstroke, and plateau phase; (bottom panel) key functional genes expressed in working cardiomyocytes that are reduced in pacemaker cells.

FIGURE 2

Genome organization, chromatin, and mechanisms of transcription. (A) Within the nucleus, chromosomes occupy distinct territories and within a given territory, compacted, non-transcribed DNA is clustered into lamina-associated domains (LADs) adjacent to the nuclear periphery. DNA that is actively transcribed is located in the interior of the nucleus, where is it organized into topologically associated domains (TADs). (B) Enhancers and promoters are brought into proximity through loop extrusion by the ring-like Cohesion complex bound to Ctcf (red circle). (C) Chromatin is tightly wound around histone complexes. At sites of active enhancers, histone proteins are modified, allowing access of transcription factors (purple) to bind to DNA. (D) An example of processed ATAC-seq data aligned with the cartoon in (C), expressed as a graph of read count versus genomic location, showing a “peak” or region of open chromatin, where histones are modified to make DNA accessible to a transposase. (E) An example of processed Hi-C data expressed as a heatmap, where the horizontal axis reflects genomic location. Pixel color represents the number of read counts, corresponding to the extent of contact frequency in cis between two regions of genomic DNA positioned along a chromosome. Topologically associated domains, visualized as discrete triangular shaped structures on the heatmap, are defined by regions that are permissive for long-range contacts.

FIGURE 3

Pacemaker cell-specific chromatin accessibility profiles are deeply conserved. (A) Alignment of ATAC-seq data at the Hcn4 genomic locus from mouse neonatal pacemaker cells with human induced pacemaker-like cells demonstrate regions of open chromatin that are present in both species, highlighted in cyan. (B) Motif enrichment analysis of differentially accessible regions between SAN and non-SAN myocytes demonstrated that enrichment of similar transcription factor motifs are present in mouse and human pacemaker cells, suggesting a conserved mammalian pacemaker cell transcriptional program. SANLPC, sinoatrial node pacemaker cells; RACM, right atrial cardiomyocytes; SANLPC, sinoatrial node-like pacemaker cells; VLCM, ventricular-like cardiomyocytes.

FIGURE 4

Sinoatrial node hypoplasia and dysfunction after deletion of a key pacemaker cell-specific enhancer. (A) Three-dimensional reconstruction of light sheet fluorescence image of Hcn4 + sinoatrial node (san, green), right atrium (pink, ra) and superior vena cava (gray, svc) from a WT control adult heart (left) in comparison to (B) a heart obtained from a littermate lacking both copies of an SAN-specific enhancer (ISE) for the transcription factor Isl1. Hearts were stained in whole mount with Hcn4 antibody and san, ra, and svc were manually segmented. Note the markedly reduced size of the Hcn4 + SAN in the Isl1^ΔISE/ΔISE heart. (C) Electrocardiograms recorded from a WT control adult mouse (left) and (D) an Isl1^ΔISE/ΔISE littermate demonstrate an example of sinus arrhythmias in animals lacking the Isl1 enhancer.

FIGURE 5

Working model for transcriptional regulation in pacemaker cells. Transcriptional control of the SAN gene program and right atrial cardiomyocyte gene program are contrasted. Both tissues require the cardiac factors Mef2, Gata4, and Tbx5 to active cardiac gene expression. However, reciprocal repression of SAN genes by Nkx2.5 and atrial genes by Tbx3, Tbx18, and Shox2, along with activation of SAN genes by Isl1 (with possible interacting partners) allows for cell type specific gene expression patterns. Question marks indicate a hypothesized interaction that has not been tested yet. Solid lines represent direct promotion/inhibition and dashed lines represent either direct or indirect promotion/inhibition.

FIGURE 6

Common genetic variants in pacemaker cell enhancers affect sinoatrial node function in human populations. (A) Aligned SAN ATAC-seq data from human iPSC-derived pacemaker-like cells (SANLPC) and ventricular like cells (VLCM) with syntenic genomic region from mouse sinoatrial node pacemaker cells (SANPC) and right atrial cardiomyocytes (RACM) demonstrate differentially accessible chromatin regions shared by both sets of pacemaker cells (highlighted in cyan). (B) shows alignment of a resting heart rate GWAS at the TBX3 locus, with highly significant associations seen in the region with differentially accessible ATAC-seq peaks, underscoring the relevance of SAN enhancers to human sinoatrial node function. GWAS results were downloaded from http://www.nealelab.is/uk-biobank/.